616459204-ASH-2022-Education-Book-1

Dr Prakash Singh Shekhawat Hematology 2022 AMERICAN SOCIETY OF HEMATOLOGY EDUCATION PROGRAM 64th ASH® Annual Meeting and Exposition Editors Hanny Al-Samkari, MD Executive Editor, Hematology 2022 Massachusetts General Hospital Boston, Massachusetts Alison R. Walker, MD, MPH, MBA Deputy Editor, Hematology 2022 H. Lee Moffitt Cancer Center Tampa, Florida Rakhi P. Naik, MD, MHS Deputy Editor, Hematology 2022 Johns Hopkins Medicine Baltimore, Maryland Alex F. Herrera, MD Deputy Editor, Hematology 2022 City of Hope Duarte, California Ang Li, MD, MS Special Section Editor, Hematology 2022 Baylor College of Medicine Houston, Texas AMERICAN SOCIETY OF HEMATOLOGY • WASHINGTON, DC Dr Prakash Singh Shekhawat © 2022 by The American Society of Hematology ISSN 1520–4383 (online) Hematology, the ASH Education Program, is published annually by The American Society of Hematology (ASH) in one volume per year. Committee on Educational Affairs All business correspondence and purchase and reprint requests should be addressed to The American Society of Hematology, 2021 L Street, NW, Suite 900, Washington, DC 20036, USA; phone: 202–776–0544; fax: 202–776–0545; e-mail: ASH@hematology.org. Marc J. Braunstein, MD, PhD (‘25) Nicholas R. Burwick, MD (‘25) Shruti Chaturvedi, MBBS, MS (‘25) Faith Davies, MD (‘24) Amy E. DeZern, MD (‘26) James M. Foran, MD (‘22) Nobuko Hijiya, MD (‘24) Margaret Kasner, MD (‘26) Kah Poh (Melissa) Loh, MD (‘23) Martha P. Mims, MD, PhD (‘23) Casey L. O’Connell, MD (‘23) Heather A. O’Leary, PhD (‘23) Nicki Panoskaltsis, MD, PhD (‘25) Hematology, the ASH Education Program, is available on the Internet at https://ashpublications.org/hematology. © 2022 by The American Society of Hematology. All rights reserved. Copyright is not claimed in any works of the United States Government. Except as expressly permitted in this statement, no part of this publication may be used (as here in after defined) in any form or by any means now or hereafter known, electronic or mechanical, without permission in writing from the Publisher, The American Society of Hematology. For purposes of this notice, the term “use” includes but is not limited to reproduction, photocopying, storage in a retrieval system, translation, and transmittal. The Publisher hereby consents to the use of this publication or any part hereof for any noncommercial educational purpose within the health field such as classroom instruction and clinical and residency training. This publication or any part thereof may be used for educational purposes at conferences, continuing education courses, and other educational activity, provided no fee or other compensation is charged therefor. All materials so used must acknowledge the Publisher’s copyright therein as “© 2022 by The American Society of Hematology.” When requesting the Publisher’s permission to use this publication or any part thereof, please contact the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923, USA; phone: 978–750–8400; fax: 978–750–4470; or The American Society of Hematology Publications Department at 2021 L Street, NW, Suite 900, Washington, DC 20036, USA; phone: 202–776–0544; fax: 202–776–0545. Article Citations Cite articles in this volume by listing Author(s), Title, Hematology Am Soc Hematol Educ Program. 2022;2022: beginning page number-ending page number. Hematology Rights and Permission To request permission to reprint or reuse Hematology articles, figures, or tables, please visit the Hematology page at the Copyright Clearance Center (https://marketplace. copyright.com/rs-ui-web/mp/details/journal/123161909). Jennifer R. Brown, MD, PhD (‘23) (Chair) Anjali Advani, MD (‘23) (Vice Chair) Ex Officio Members Jane N. Winter, MD (‘22) – President Robert A. Brodsky, MD (‘23) – President-Elect Mohandas Narla, DSc (‘24) – Vice President Liaisons Hanny Al-Samkari, MD (‘24) – Executive Editor, Hematology ASH Education Program Jessica Altman, MD (‘21) – Senior Executive Editor, ASH-SAP 8 Laura M. De Castro, MD (‘23) – Member, Committee on Practice Cynthia E. Dunbar, MD (‘24) – Secretary David Garcia, MD (‘22) – 2022 Education Program Co-Chair Molly Weidner Mandernach, MD, MPH (‘22) – Junior Executive Editor, ASH-SAP 8 Olatoyosi Odenike, MD (‘22) – 2022 Education Program Co-Chair Cover: Illustration of a blood clot with red blood cells (red), white blood cells (blue), and activated platelets (purple). Source: Thom Leach/Science Photo Library. This activity is supported by educational grants from AstraZeneca; GlaxoSmithKline; Incyte Corp.; Jazz Pharmaceuticals, Inc.; Merck Sharp & Dohme Corp.; and Novartis Pharmaceuticals Corp. The Publisher disclaims responsibility for opinions expressed by the authors. ii Dr Prakash Singh Shekhawat Contents ix x xi Editors’ Message Reviewers Continuing Medical Education Information Ham-Wasserman Lecture 1 Leukemogenesis in infants and young children with trisomy 21 IRENE ROBERTS Acute Myeloid Leukemia: Improving Outcomes in Challenging Subsets 9 Achieving MRD negativity in AML: how important is this and how do we get there? CHRISTOPHER S. HOURIGAN 15 Novel investigational approaches for high-risk genetic subsets of AML: TP53, KMT2A, FLT3 KIERAN D. SAHASRABUDHE AND ALICE S. MIMS 23 Optimizing outcomes in secondary AML ANDREW MATTHEWS AND KEITH W. PRATZ 30 EVIDENCE-BASED MINIREVIEW Clinical utilization of panel-based molecular testing for patients with AML BRANDON J. AUBREY AND ANDREW BRUNNER Anxiety Provoking Consultations: Mast Cells and Eosinophils 34 Available and emerging therapies for bona fide advanced systemic mastocytosis and primary eosinophilic neoplasms JASON GOTLIB 47 Approach to the patient with suspected hypereosinophilic syndrome AMY D. KLION 55 How to evaluate the patient with a suspected mast cell disorder and how/when to manage symptoms CEM AKIN Are Alternative Donors Now Mainstream in Allogeneic Transplant? 64 In 2022, which is preferred: haploidentical or cord transplant? ARNON NAGLER AND MOHAMAD MOHTY 74 New strategies for mismatched unrelated donor (MMUD) hematopoietic cell transplant (HCT) SHUKAIB ARSLAN AND MONZR M. AL MALKI 83 Hematology 2022—what is complete HLA match in 2022? STEPHEN R. SPELLMAN Dr Prakash Singh Shekhawat Contents | iii Autoimmune Hemolytic Anemias 90 Cold AIHA and the best treatment strategies JENNY MCDADE DESPOTOVIC AND TAYLOR OLMSTED KIM 96 Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies SUSAN T. JOHNSON AND KATHLEEN E. PUCA 105 Warm autoimmune hemolytic anemia and the best treatment strategies DAVID J. KUTER Beyond Routine Frontline Therapy of CML 114 Transplantation in CML in the TKI era: who, when, and how? CHRISTIAN NIEDERWIESER AND NICOLAUS KRÖGER 123 Treatment of CML in pregnancy HARRY F. ROBERTSON AND JANE F. APPERLEY 129 Management of TKI-resistant chronic phase CML TIMOTHY P. HUGHES AND NARANIE SHANMUGANATHAN Controversies in Aggressive NHL 138 CNS prophylaxis in aggressive B-cell lymphoma MATTHEW R. WILSON, SABELA BOBILLO, AND KATE CWYNARSKI 146 Sequencing therapy in relapsed DLBCL CHRISTOPHER R. FLOWERS AND OREOFE O. ODEJIDE 155 What is the role of up-front autologous stem cell transplantation in mantle cell lymphoma? ANITA KUMAR Immunotherapy in Multiple Myeloma 163 Antibodies and bispecifics for multiple myeloma: effective effector therapy CHRISTOPHER CIPKAR, CHRISTINE CHEN, AND SUZANNE TRUDEL 173 Beyond the cell: novel noncellular immunotherapy approaches to multiple myeloma SARAH A. HOLSTEIN 180 Cellular therapy for multiple myeloma: what’s now and what’s next PAULA RODRIGUEZ-OTERO AND JESÚS F. SAN-MIGUEL Improving Outcomes in ALL 190 New developments in ALL in AYA NICOLAS BOISSEL 197 Optimal approach to T-cell ALL KRISTEN M. O’DWYER 206 Ph+ ALL in 2022: is there an optimal approach? MATTHEW J. WIEDUWILT 213 EVIDENCE-BASED MINIREVIEW What is the optimal tyrosine kinase inhibitor for adults with newly diagnosed Philadelphia chromosome–positive acute lymphoblastic leukemia? FADI G. HADDAD AND NICHOLAS J. SHORT JAK/STAT Inhibition and Beyond in Ph-Negative MPNs 218 Hitting the brakes on accelerated and blast-phase myeloproliferative neoplasms: current and emerging concepts JAN PHILIPP BEWERSDORF AND RAAJIT K. RAMPAL iv | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat 225 Molecular prognostication in Ph-negative MPNs in 2022 ALESSANDRO MARIA VANNUCCHI AND PAOLA GUGLIELMELLI 235 New approaches to tackle cytopenic myelofibrosis SAMUEL B. REYNOLDS AND KRISTEN PETTIT Long-Term Effects Monitoring for Survivors of Pediatric Hematologic Malignancies 245 Germline risk factors for second malignant neoplasms after treatment for pediatric hematologic malignancies SMITA BHATIA 251 Mitigating, monitoring, and managing long-term chemotherapy- and radiation-induced cardiac toxicity WENDY BOTTINOR AND ERIC J. CHOW 259 Risk factors and screening for neurocognitive impacts of therapy KEVIN R. KRULL Long-Term Health Effects of Curative Therapy for Sickle Cell Disease 266 Knowledge to date on secondary malignancy following hematopoietic cell transplantation for sickle cell disease COURTNEY D. FITZHUGH 272 Long-term health outcomes following curative therapies for sickle cell disease ROHINI CHAKRAVARTHY AND DEBRA L. FRIEDMAN 277 Organ function indications and potential improvements following curative therapy for sickle cell disease MONICA L. HULBERT, ALLISON A. KING, AND SHALINI SHENOY 283 EVIDENCE-BASED MINIREVIEW How to utilize new therapies for sickle cell disease STEPHANIE GUARINO AND SOPHIE LANZKRON Managing Thrombocytopenia in Challenging Situations 286 Thrombopoietin receptor agonists for chemotherapy-induced thrombocytopenia: a new solution for an old problem HANNY AL-SAMKARI 296 Thrombocytopenia and liver disease: pathophysiology and periprocedural management HANA I. LIM AND ADAM CUKER 303 Thrombocytopenia in pregnancy ALLYSON M. PISHKO AND ARIELA L. MARSHALL 312 EVIDENCE-BASED MINIREVIEW Full dose, modified dose, or no anticoagulation for patients with cancer and acute VTE and thrombocytopenia RUSHAD PATELL AND JEFFREY I. ZWICKER Maximizing Outcomes in CLL 316 Novel therapies and combinations in CLL refractory to BTK inhibitors and venetoclax LYDIA SCARFÒ 323 Selecting initial therapy in CLL INHYE E. AHN AND JENNIFER R. BROWN 329 Treatment of Richter’s syndrome PHILIP A. THOMPSON AND TANYA SIDDIQI Multiple Myeloma: Assessing the Patient and the Disease 337 Fitness and frailty in myeloma CHARLOTTE PAWLYN, ABDULLAH M. KHAN, AND CIARA L. FREEMAN Dr Prakash Singh Shekhawat Contents | v 349 High or low? Assessing disease risk in multiple myeloma TIMOTHY MARTIN SCHMIDT 356 The burden of myeloma: novel approaches to disease assessment MATTHEW HO AND TAXIARCHIS KOURELIS 363 Mitigating the risk of venous thromboembolism in patients with multiple myeloma receiving immunomodulatory-based therapy FAHRETTIN COVUT AND KRISTEN M. SANFILIPPO Novel Approaches in MDS 368 New investigational combinations for higher-risk MDS KRISTIN L. KOENIG AND UMA BORATE 375 Risk stratifying MDS in the time of precision medicine MARIO CAZZOLA 382 Targeting inflammation in lower-risk MDS* JESUS D. GONZALEZ-LUGO AND AMIT VERMA Obstetric Management and Complications in Sickle Cell Disease in High- and Low-Income Countries 388 Acute pain episodes, acute chest syndrome, and pulmonary thromboembolism in pregnancy EUGENIA VICKY ASARE, MICHAEL R. DEBAUN, EDEGHONGHON OLAYEMI, THEODORE BOAFOR, AND SAMUEL A. OPPONG 408 Evidence-based management of pregnant women with sickle cell disease in high-income countries EUGENE OTENG-NTIM AND PANICOS SHANGARIS 414 Evidence-based obstetric management of women with sickle cell disease in low-income countries BOSEDE B. AFOLABI, OCHUWA A. BABAH, AND TITILOPE A. ADEYEMO Prophylactic Platelet Transfusions 421 Novel platelet products including cold-stored platelets DANA V. DEVINE 424 Expanding the platelet inventory to mitigate the impact of severe shortages JAMES R. STUBBS, BETH H. SHAZ, RALPH R. VASSALLO, AND JOHN D. ROBACK 430 Platelet components and bacterial contamination: hospital perspective 2022 ZBIGNIEW M. SZCZEPIORKOWSKI AND MONICA B. PAGANO 437 EVIDENCE-BASED MINIREVIEW Strategies to manage a severely HLA-alloimmunized patient with refractory thrombocytopenia DEBBIE JIANG AND SANDHYA R. PANCH Reproductive and Sexual Health in Sickle Cell Disease 442 Incorporating gonadal health counseling into pediatric care of sickle cell patients LILLIAN R. MEACHAM, LYDIA H. PECKER, BEATRICE GEE, AND ADRIENNE MISHKIN 450 Epidemiology and treatment of priapism in sickle cell disease IBRAHIM M. IDRIS, ARTHUR L. BURNETT, AND MICHAEL R. DEBAUN 459 No crystal stair: supporting fertility care and the pursuit of pregnancy in women with sickle cell disease LYDIA H. PECKER, ALECIA NERO, AND MINDY CHRISTIANSON Thrombosis and Anticoagulation: Clinical Considerations in Selected Populations 467 Anticoagulant therapy for women: implications for menstruation, pregnancy, and lactation EMMA DELOUGHERY AND BETHANY SAMUELSON BANNOW 474 Thrombosis and anticoagulation: clinical issues of special importance to hematologists who practice in Asia KOCHAWAN BOONYAWAT AND PANTEP ANGCHAISUKSIRI vi | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat 481 Thrombosis questions from the inpatient wards GEORGE GOSHUA, PAVAN K. BENDAPUDI, AND ALFRED IAN LEE 491 EVIDENCE-BASED MINIREVIEW Should caplacizumab be used routinely in unselected patients with immune thrombotic thrombocytopenic purpura? GEORGE GOSHUA AND PAVAN K. BENDAPUDI Thrombosis Prevention and Treatment 495 Coming soon to a pharmacy near you? FXI and FXII inhibitors to prevent or treat thromboembolism OMRI COHEN AND WALTER AGENO 506 To prophylax or not, and how much and how long? Controversies in VTE prevention for medical inpatients, including COVID-19 inpatients ALEX C. SPYROPOULOS 515 The 5 most frequently asked questions about factor Xa inhibitors TZU-FEI WANG AND MARC CARRIER To Transplant or Not to Transplant in Active or High-Risk Myeloid Disease 522 Transplant for TP53-mutated MDS and AML: because we can or because we should? JURJEN VERSLUIS AND R. COLEMAN LINDSLEY 528 Transplant in AML with measurable residual disease: proceed or defer? CHARLES CRADDOCK 534 Allogeneic transplantation for advanced acute leukemia DANIEL WEISDORF Treatment Approaches for the Multiple Myeloma Patient in 2022 539 Newly diagnosed multiple myeloma: making sense of the menu CAITLIN L. COSTELLO 551 The consultant’s guide to smoldering multiple myeloma SIGRUN THORSTEINSDOTTIR AND SIGURDUR YNGVI KRISTINSSON 560 The first relapse in multiple myeloma: how to pick the next best thing SRINIVAS DEVARAKONDA, NIDHI SHARMA, AND YVONNE EFEBERA Update in Hemophilia 569 Gene therapy for hemophilia AMIT C. NATHWANI 579 Long-term prophylaxis: what are our options and how to define success? MARILYN JEAN MANCO-JOHNSON AND BETH BOULDEN WARREN 586 Perioperative hemostasis for patients with hemophilia JACQUELINE N POSTON AND REBECCA KRUSE-JARRES Updates in Targeted Therapy in Pediatric Leukemia 594 Clinical screening for Ph-like ALL and the developing role of TKIs THAI HOA TRAN AND SARAH K. TASIAN 603 The evolution of targeted therapy in pediatric AML: gemtuzumab ozogamicin, FLT3/IDH/BCL2 inhibitors, and other therapies LAUREN POMMERT AND KATHERINE TARLOCK 611 Updates in infant acute lymphoblastic leukemia and the potential for targeted therapy RISHI S. KOTECHA Dr Prakash Singh Shekhawat Contents | vii Von Willebrand Disease 618 Diagnostic pitfalls and conundrums in type 1 von Willebrand disease ROBERT F. SIDONIO AND MICHELLE LAVIN 624 Special considerations in GI bleeding in VWD patients NICHOLAS L.J. CHORNENKI, EDWIN OCRAN, AND PAULA D. JAMES 631 What have we learned about the patient’s experience of von Willebrand disease? A focus on women HEATHER VANDERMEULEN, SUMEDHA ARYA, SARAH NERSESIAN, NATALIE PHILBERT, AND MICHELLE SHOLZBERG What is New in Classical Bone Marrow Failure Syndromes? (Focus on Management) 637 Dyskeratosis congenita and telomere biology disorders SHARON A. SAVAGE 649 Modern management of Fanconi anemia CARLO DUFOUR AND FILOMENA PIERRI 658 Genetics of severe congenital neutropenia as a gateway to personalized therapy JEAN DONADIEU AND CHRISTINE BELLANNÉ-CHANTELOT What’s New in Indolent Lymphoma 666 Divergent paths: management of early relapsed follicular lymphoma RADHIKA TAKIAR, YASMIN KARIMI, AND TYCEL J. PHILLIPS 676 Management of marginal zone lymphomas MICHELE MERLI AND LUCA ARCAINI 688 Biology of follicular lymphoma: insights and windows of clinical opportunity MEGAN PERRETT, CARINA EDMONDSON, AND JESSICA OKOSUN 695 EVIDENCE-BASED MINIREVIEW When should autologous transplant or cellular therapy be considered for follicular lymphoma? DAVID A. BOND AND AJAY K. GOPAL Where Are We Headed in Hodgkin Lymphoma? 699 Do allpatients with primary refractory/first relapse of HL need autologous stem cell transplant? ALISON J. MOSKOWITZ 706 Incorporating novel agents into frontline treatment of Hodgkin lymphoma SWETHA KAMBHAMPATI AND ALEX F. HERRERA 717 Individualized patient care in nodular lymphocyte-predominant Hodgkin lymphoma SVEN BORCHMANN Errata 723 Perioperative consultative hematology: can you clear my patient for a procedure? Hematology Am Soc Hematol Educ Program. 2021;2021:521-528. BURNETT AE, RAGHEB B, KAATZ S. 724 Noninfectious complications of hematopoietic cell transplantation. Hematology Am Soc Hematol Educ Program. 2021;2021:578-586. WILLIAMS KM. 725 COVID-19 and thrombosis: searching for evidence. Hematology Am Soc Hematol Educ Program. 2021;2021:621-627. THILAGAR B, BEIDOUN M, RHOADES R, KAATZ S. *Note The article “Targeting inflammation in lower-risk MDS” beginning on page 382 is not eligible for continuing medical education (CME) credit. viii | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Editors’ Message Welcome to the 64th Annual Meeting of the American Society of Hematology. This year marks the second year of the Annual Meeting as a hybrid meeting, with many attendees thankful to reconnect in person with colleagues from all over the world and others happy to attend virtually. Whichever option you have chosen, you will hear the many striking scientific and clinical advances in hematology over the past year and find a remarkable wealth of educational content covering all major areas within both classical and malignant hematology. To this end, we have developed the Hematology ASH Education Program to be your primary and indispensable resource. Your authors, with help from our invaluable peer reviewers and guidance from us, have worked to develop concise, readable, and informative articles filled with useful tables and figures and headlined by a visual abstract. All of the articles either are based directly on a talk given by the author at the meeting’s education program (using a case-based format) or take the form of an evidence-based minireview exploring the current data for some of the most challenging questions in modern hematology practice. This year marks the first year of a new team of Hematology editors, and we are thrilled to lead this venerable, over-5-decade-old ASH publication for the next 3 years. Of course, Hematology is possible only thanks to the state-of-the-art education program developed this year by Education Program Co-Chairs Dr. David Garcia and Dr. Olatoyosi Odenike, as well as the tireless efforts of ASH staff, including Sheehan Misko, Erin Roberts, Jeremiah Murphy, Brian Cannon, Glenn Landis, and Kenneth April. We are so thankful for all their efforts. Happy reading, from your Hematology editors! Hanny Al-Samkari, MD Alison R. Walker, MD, MPH, MBA Rakhi P. Naik, MD, MHS Dr Prakash Singh Shekhawat Alex F. Herrera, MD Ang Li, MD, MS ix Reviewers The editors thank the session chairs and the reviewers who worked hard to ensure the reliability of the material presented in Hematology 2022: Anjali S. Advani Ranjana Advani Cem Akin Monzr M. Al Malki Aref Al-Kali Frederick Appelbaum Eugenia Vicky Asare K. Scott Baker Paul M. Barr Nancy L. Bartlett Renato Bassan Shannon Bates Lisa Baumann Kreuziger Rafael Bejar Jesus G. Berdeja Smita Bhatia Andrea Biondi Larissa Bornikova Jennifer R. Brown Natalie S. Callander Gunnar Cario Shannon Carpenter Marc Carrier Giancarlo Castaman Eric J. Chow Adam D. Cohen Claudia S. Cohn Nathan T. Connell Jorge Cortes Charles Craddock Stacy E. Croteau Adam Cuker Corey S. Cutler Agnieszka Czechowicz Anita D’Souza Alexey V. Danilov Faith E. Davies Amy E. DeZern Sunny H. Dzik x Yvonne A. Efebera Matthew Ehrhardt Toby A. Eyre Joshua Field Courtney D. Fitzhugh Christopher R. Flowers Annemarie E. Fogerty Jacqueline S. Garcia James N. George Tracy I. George Irene M. Ghobrial Gerald J. Gleich Victor R. Gordeuk Jed B. Gorlin Jason Gotlib Erin M. Guest Sumit Gupta Sandra L. Haberichter Claire N. Harrison Andreas Hochhaus Timothy P. Hughes Ibrahim M. Idris Tania Jain Andra H. James Paula D. James Jill M. Johnsen Moonjung Jung Brad S. Kahl Julie Kanter Raj S. Kasthuri Richard Kaufman Alok A. Khorana Adam S. Kittai Jan-Henning Klusmann Rami S. Komrokji John Koreth Johanna A. Kremer Hovinga Amrita Y. Krishnan Michael H. Kroll Rebecca Kruse-Jarres Roshni Kulkarni Kevin H. M. Kuo John Kuruvilla David J. Kuter Andrew T. Kuykendall Ola Landgren Alfred I. Lee Lai Heng Lee Howard A. Liebman R. Coleman Lindsley Sagar Lonial Izidore S. Lossos Kami Maddocks Paul Mansfield John O. Mascarenhas Michael J. Mauro Eileen Merriman Reid W. Merryman Joseph Mikhael Alice S. Mims Alison J. Moskowitz Kasiani C. Myers Loretta J. Nastoupil Robert S. Nickel Omar Niss Kristen M. O’Dwyer Adam Jan Olszewski Monica B. Pagano Lydia H. Pecker Kristen M. Pettit Tycel J. Phillips Allyson Pishko Jerald P. Radich Delphine Rea Josep-Maria Ribera Paul G. Richardson Jacob M. Rowe Dr Prakash Singh Shekhawat Jeffrey E. Rubnitz Sana Saif Ur Rehman David A. Sallman Kristen Sanfilippo Santosh L. Saraf Sharon A. Savage Farzana Sayani Mikkael A. Sekeres Nina Shah Urvi A. Shah Joseph J. Shatzel Bronwen E. Shaw Shalini Shenoy Nicholas J. Short Sonali M. Smith Gerald A. Soff Stephen R. Spellman Sarah K. Tasian Jeffrey W. Taub Philip A. Thompson Darrell J. Triulzi Suzanne Trudel Lucie M. Turcotte Geoffrey L. Uy Emile Van Schaftingen Sumithira Vasu Tzu-Fei Wang Jonathan Webster Oliver Weigert Matthew J. Wieduwilt Tanya Wildes Raymond Siu Ming Wong Jennifer C. Yui Continuing Medical Education Information The Hematology ASH Education Program is an annual publication that provides practicing hematologists with invaluable information on the most important areas of clinical progress. Hematology 2022 is a peer-reviewed collection of articles written by the 2022 ASH Education Program speakers and the Ham-Wasserman Lecturer. The papers showcase groundbreaking advances and new concepts in 29 different fields. Every year, the periodical provides an updated and comprehensive review of each of the topics covered in the annual meeting education sessions. ABIM Maintenance of Certification Successful completion of this CME activity enables the participant to earn up to 40 Knowledge Points in the American Board of Internal Medicine’s (ABIM) Maintenance of Certification (MOC) program. Participants will earn MOC points equivalent to the amount of CME credits claimed for the activity. It is the CME activity provider’s responsibility to submit participant completion information to ACCME for the purpose of granting ABIM MOC credit. Claiming CME credits and ABIM points Educational objectives 1. Employ the knowledge gained regarding the diagnosis and treatment of malignant and nonmalignant hematologic disorders to improve patient care. 2. Discuss the state-of-the-art therapeutics in hematology. 3. Analyze the potential contribution of novel, not-yet-approved modalities of therapy to current evidence-based management of malignant and nonmalignant hematologic disorders. Date of release December 2022 Date of expiration On December 31, 2023, the ability to earn Continuing Medical Education credit and American Board of Internal Medicine Maintenance of Certification Medical Knowledge points for this product expires. This is the last date for users to claim credit for this product. For questions about credit, please contact the ASH Education Department at cme@hematology.org or call tollfree 866-828-1231 within the United States only; 1-202-776-0544 internationally. The estimated time to complete this educational activity is 40 hours. To claim CME credit, users must complete an evaluation of the product and a test of medical knowledge, both of which are accessed through ASH Academy On Demand (academy.hema tology.org). There is a one-time processing fee for claiming CME/ MOC credit. Users with scores of 80% or better on these selfassessment modules are eligible to claim credit for the activity. To facilitate claiming of credit, the test for this product is divided into two subtests, one of which focuses on malignant hematology content with the other focusing on nonmalignant content. Successful completion of each test earns the user 20 AMA Category 1 PRA CreditsTM. Users claim CME and/or ABIM MOC credit for each test individually. You can take one or both tests, depending on your CME and MOC needs. The malignant hematology test consists of 31 questions, and the nonmalignant hematology test consists of 21 questions. The questions in each test can be answered in one sitting, or a user can save their progress and return to complete the test at a later time. The malignant hematology test covers information presented in the following sections: • Accreditation and Credit Designation The American Society of Hematology is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The American Society of Hematology designates this enduring material for a maximum of 40 AMA PRA Category 1 CreditsTM. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who participate in this CME activity but are not licensed in the United States are also eligible for AMA Category 1 PRA CreditTM. To earn these credits, readers must pass two online tests (malignant and nonmalignant) based on chapters from the book. • • • • • • • • • • Acute Myeloid Leukemia: Improving Outcomes in Challenging Subsets Are Alternative Donors Now Mainstream in Allogeneic Transplant? Beyond Routine Frontline Therapy of CML Controversies in Aggressive NHL Ham-Wasserman Lecture Immunotherapy in Multiple Myeloma Improving Outcomes in ALL JAK/STAT Inhibition and Beyond in Ph-Negative MPNs Long-Term Effects Monitoring for Survivors of Pediatric Hematologic Malignancies Maximizing Outcomes in CLL Multiple Myeloma: Assessing the Patient and the Disease Dr Prakash Singh Shekhawat xi • • • • • Novel Approaches in MDS To Transplant or Not to Transplant in Active or High-Risk Myeloid Disease Treatment Approaches for the Multiple Myeloma Patient in 2022 Updates in Targeted Therapy in Pediatric Leukemia Where Are We Headed in Hodgkin Lymphoma? The nonmalignant hematology test covers information presented in the following sections: • • xii Anxiety-Provoking Consultations: Mast Cells and Eosinophils Autoimmune Hemolytic Anemias • • • • • • • • • Long-Term Effects of Curative Therapy for Sickle Cell Disease Managing Thrombocytopenia in Challenging Situations Obstetric Management and Complications in Sickle Cell Disease in High- and Low-Income Countries Prophylactic Platelet Transfusions Reproductive and Sexual Health in Sickle Cell Disease Thrombosis Prevention and Treatment Update in Hemophilia Von Willebrand Disease What Is New in Classical Bone Marrow Failure Syndromes? (Focus on Management) Dr Prakash Singh Shekhawat HAM - WASSERMAN LECTURE Leukemogenesis in infants and young children with trisomy 21 Department of Paediatrics and MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford, UK Children with Down syndrome (DS) have a greater than 100-fold increased risk of developing acute myeloid leukemia (ML) and an approximately 30-fold increased risk of acute lymphoblastic leukemia (ALL) before their fifth birthday. ML-DS originates in utero and typically presents with a self-limiting, neonatal leukemic syndrome known as transient abnormal myelopoiesis (TAM) that is caused by cooperation between trisomy 21–associated abnormalities of fetal hematopoiesis and somatic N-terminal mutations in the transcription factor GATA1. Around 10% of neonates with DS have clinical signs of TAM, although the frequency of hematologically silent GATA1 mutations in DS neonates is much higher (~25%). While most cases of TAM/silent TAM resolve without treatment within 3 to 4 months, in 10% to 20% of cases transformation to full-blown leukemia occurs within the first 4 years of life when cells harboring GATA1 mutations persist and acquire secondary mutations, most often in cohesin genes. By contrast, DS-ALL, which is almost always B-lineage, presents after the first few months of life and is characterized by a high frequency of rearrangement of the CRLF2 gene (60%), often co-occurring with activating mutations in JAK2 or RAS genes. While treatment of ML-DS achieves long-term survival in approximately 90% of children, the outcome of DS-ALL is inferior to ALL in children without DS. Ongoing studies in primary cells and model systems indicate that the role of trisomy 21 in DS leukemogenesis is complex and cell context dependent but show promise in improving management and the treatment of relapse, in which the outcome of both ML-DS and DS-ALL remains poor. LEARNING OBJECTIVES • Understand the risk of leukemia developing in children with Down syndrome • Understand the natural history of myeloid leukemia in young children with Down syndrome Background Children with Down syndrome (DS) due to constitutional trisomy 21 (T21) have a more than 50-fold increased risk of developing acute leukemia before their fifth birthday compared to children without DS.1,2 Few genetic disorders show such a strong link to leukemia and have offered so many clues to clinicians and scientists about why this should be so. While it is clear that the hematologic problems in DS must be underpinned by the presence of a supernumerary copy of chromosome 21 (Hsa21), the more we learn about potentially relevant mechanisms, the more questions remain and the more enigmatic the answers become. Nevertheless, clinical and mechanistic studies have together significantly improved our understanding of DS leukemias and the way we manage these disorders in patients. Understanding DS leukemogenesis is important given that worldwide approximately 200 000 children every year are born with DS.3 Here, I describe how observations made almost 40 years ago by Alvin Zipursky have triggered a wealth of further studies that provide insight into the prenatal origin of leukemias in DS as well as the fundamental processes governing the biological properties and gene expression of hematopoietic cells.4 Leukemia and DS Population-based studies show an increased frequency of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (Table 1).1,2 Given that the incidence of nonhematologic cancers is half that reported in individuals without DS,1,2,5 this points to the specific susceptibility of hematopoietic cells to the leukemogenic effects of T21 and also hints at effects on multiple lineages. A second distinctive feature of DS leukemias is the age at presentation, which indicates, particularly for the specific subtype of AML known as ML-DS, that there is a defined time window during which T21-driven leukemic transformation occurs. Dr Prakash Singh Shekhawat Leukemogenesis in infants with trisomy 21 | 1 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 Irene Roberts Table 1. Increased susceptibility to leukemia in young children with DS Type of malignancy Standardized incidence ratio AML Aged 0-4 years (ML-DS) 114 Aged 0-60 years 12 TAM and ML-DS: a multistep model of childhood leukemogenesis ALL Aged 0-4 years 27 Aged 0-60 years 13 0.45 Solid tumorsa megakaryocytic markers, an uncommon finding in children without DS,8 while DS-ALL, unlike non-DS ALL, is almost exclu sively B-lineage.9 Finally, DS leukemias are molecularly distinct, as described in detail later. Data reproduced with permission from Marlow et al2 and Hasle et al.5 ML-DS is now known to originate in utero and usually presents with a self-limiting, neonatal leukemic syndrome known as tran sient abnormal myelopoiesis (TAM), or transient myeloprolifera tive disorder, that is virtually unique to DS; while most cases of TAM resolve within 3 to 4 months, subsequent transformation to full-blown leukemia is limited to the first 4 years of life (Figure 1).2,4-6 By contrast, ALL is rare in infants with DS but otherwise has an age distribution similar to ALL in children without DS.5,7 DS leukemias also exhibit distinct immunophenotypic characteris tics. Most notably, blast cells in ML-DS coexpress erythroid and Transient leukemia (TAM) Fetal hematopoiec stem/progenitor Cells 1. Trisomy 21 perturbs fetal hematopoiesis ~25% Leukemia (ML-DS) 80-90% Birth +21 Spontaneous remission +21 GATA1s 2. Somac N-terminal truncang GATA1 mutaons (GATA1s) in T21 cells cause neonatal leukemia (TAM) unique to DS 10-20% +21 GATA1s + other Age 4 yrs 3. Acquision of addional mutaons (eg in cohesin) causes myeloid leukemia (ML-DS) in persisng GATA1 mutant cells. Figure 1. TAM and ML-DS, a multistep model of myeloid leukemogenesis in DS. Trisomy 21 causes an increase in the frequency of fetal MK-erythroid stem and progenitor cells in tandem with a severe reduction of B-cell progenitors. On this cellular background, somatic N-terminal truncating mutations in the GATA1 transcription factor gene that encode a shorter than normal GATA1 protein (GATA1s) are acquired at a high frequency during fetal life. The expression of GATA1s causes a fetal/neonatal leukemic syndrome known as TAM that is virtually unique to DS. Although TAM may be a severe disease, in 80% to 90% of cases it resolves spontaneously over the first 4 months of life. Where GATA1s-producing blast cells persist, the acquisition of mutations in additional genes, particularly those encoding cohesin complex proteins, leads to the development of ML-DS within the first 4 years of life. 2 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 a Aged 0-60 years; the only solid tumor with an increased incidence in DS is testicular cancer, which presents in young adults, and the only solid tumor in a child under the age of 5 years was a case of retinoblastoma.5 In the 2022 World Health Organization classification, TAM and ML-DS are collectively known as “myeloid proliferations associ ated with DS typically associated with exon 2 or 3 GATA1 muta tions.”10 The key discovery of the link between somatic N-terminal mutations in the transcription factor gene GATA1 and TAM was made by John Crispino’s lab, which had also first demonstrated the same type of GATA1 muta tion in chil dren with DS acute megakaryoblastic leukemia (now termed ML-DS).11,12 Further, his lab showed that such mutations result in the exclusive produc tion of a short GATA1 protein (GATA1s) that lacks the N-terminal activation domain. As the GATA1 gene is on the X chromosome, cells bearing GATA1s-causing mutations also lose expression of the full-length GATA1 protein, thus disrupting its normal activ ity in regulating hematopoietic cell development, particularly of erythroid and megakaryocyte (MK) lineages, by altering its ability to bind to and regulate downstream targets.13-16 Although almost allcases of TAM occur in neonates with DS, an identi cal clinical presentation can also affect neonates with mosaic DS.17,18 These babies lack the characteristic phenotypic features of DS, but peripheral blood karyotyping shows that some or all of the hematopoietic cells harbor T21, suggesting that both T21 and GATA1s-producing mutations are essential for the develop ment of TAM/ML-DS. Indeed, in the single reported case of TAM were also common.27,28 While these secondary events can occur in other leukemias, their coacquisition with GATA1s is virtually unique to ML-DS. Thus, together these findings support a mul tistep model of leukemogenesis in ML-DS in the vast majority of cases (Figure 1) and provide a framework both for under standing the natural history of ML-DS and for investigating the precise mechanisms responsible. Clinical features and natural history of TAM and ML-DS TAM is a fetal and neonatal condition. Most cases present at or just after birth and always by 3 months of age.25 Although TAM is usually a short-lived disease that resolves without specific ther apy, in fact it encompasses a wide spectrum of severity, from clinically silent disease to rapidly fatal multiorgan failure due to the leukemic infiltration of multiple tissues, particularly the liver and lungs.17,18,29 While clinical studies report that approximately 10% of neonates with DS have TAM,4,6,17,18 prospective studies, including our own, documenting the frequency of GATA1s muta tions in DS neonates using sensitive next generation sequencing indicate an even higher frequency of TAM, of approximately 30%, although many of these cases are clinically and hematologically silent.6 As the estimated incidence of DS worldwide is 1 per 500 to 1 per 2000 live births,30 TAM is by far the most common leuke mia in neonates. The characteristic hematologic features of TAM are key to the diagnosis and to our understanding of the path ogenesis of the disease. While neonates with silent TAM due to small GATA1s clones have no reliable hematologic markers of dis ease, most cases of clinical TAM have leukocytosis and increased peripheral blood blasts (>10%) (Table 2). While our group uses a blast per cent age of greater than 10%, oth ers rec om mend Table 2. Clinical and hematologic features of TAM and silent TAM in neonates with DS Condition Clinical features Hematologic features TAM GATA1s mutation; VAF variable but usually <80% • Hepatosplenomegaly 40% • Skin rash 20% • Pleural/pericardial effusion ± ascites 10% • Jaundice 70%-80% • Blasts >10%a • Hct usually normal • Platelet count increased, normal, or reduced • Leucocytosis usual and may exceed 100 000×109/l • MK fragments usually present on blood smear Silent TAM GATA1s mutation; VAF usually low, <10% • No increase in frequency of hepatosplenomegaly, jaundice, skin rash, pleural/pericardial effusions, or ascites compared to neonates with DS who lack GATA1s mutations • Blasts ≤10%* • Hct increased or normal • Platelets normal or reduced • WBC normal Neonate with DS and no GATA1s mutations • Jaundice is common in neonates with DS (~60%) and is not a useful indicator of TAM unless other clinical signs are present. • As up to 10% of neonates with DS have hepatosplenomegaly, rash, pleural/pericardial effusion, or ascites secondary to medical complications in the absence of GATA1 mutations, care must be taken to confirm the diagnosis by blood smear review and/or GATA1 mutation analysis. Compared with neonates without DS: • 98% have blasts on the blood smear (<20%). • Hct is often increased (24% are polycythemic, Hct >0.65). • Platelet counts are lower (40% are thrombocytopenic, <150 000×109/l). • Leukocyte, neutrophil, monocyte, and basophil counts are increased. Others have recommended defining TAM as “the presence of at least 5% blasts defined by immunophenotyping or morphology and/or the presence of a GATA1 mutation in a neonate with DS.”31 a VAF, variant allele frequency; WBC, white blood cell count. Data reproduced with permission from Roberts et al6 and Tunstall et al.29 Dr Prakash Singh Shekhawat Leukemogenesis in infants with trisomy 21 | 3 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 with a GATA1 mutation and no evidence of T21, the GATA1 muta tion was a large deletion rather than a producer of GATA1s.19 In addition, inherited GATA1s-producing mutations are not leuke mogenic in disomic individuals,20 although rare families with germline GATA1s mutations are reported where the development of ML-DS–like acute leukemia was accompanied by acquired tri somy or tetrasomy 21,21,22 further supporting the importance of GATA1s/T21 as a potent oncogenic driver. Studies on prenatal and cord blood samples from babies with DS have shown that somatic GATA1 mutations are acquired before birth, most likely during the second trimester.23-25 Furthermore, multiple GATA1 mutations occur in approximately 25% of cases, suggesting a strong selec tive advan tage for GATA1s in fetal cells.6,24 We previously showed that T21 itself causes a strik ing increase in fetal liver mega kar yo cytic-ery throid stem and progenitor cells in DS in the absence of GATA1s mutations, suggesting that it is the T21-mediated perturbation of fetal hematopoiesis that underlies the leukemogenic effects of GATA1s in neonates with DS.26 Paired ML-DS and TAM samples show the same GATA1 mutations, confirming that ML-DS and TAM are clonally linked conditions and suggesting that other factors, in addition to GATA1s, are needed to effect a full leu kemic transformation from a transient neonatal syndrome to ML-DS, a condition that is fatal in the absence of chemother apy.24,27,28 Whole-genome and targeted sequenc ing in more than 300 patient samples not only confirmed the presence of GATA1s mutations in ML-DS but also identified loss-of-function mutations in cohesin genes as the principal secondary genetic events in ML-DS; mutations in genes encoding epigenetic reg ulators and components of tyrosine kinase signaling pathways defining TAM on the basis of blasts greater than or equal to 5%.31 Importantly, however, the blast percentage can only serve as a guide for the presence of GATA1s mutations because more than one-third of DS neonates without GATA1s mutations have blasts greater than or equal to 5%, automated counts are unreliable, and manual counts require considerable experience. Typical blast cells in TAM resemble immature or partially differentiated megakaryoblasts (Figure 2), but they are often pleomorphic and may display features of erythroid, basophil, or eosinophil differ entiation,32-34 reflecting the roles of GATA1 in multiple lineages.35 Consistent with this, in addition to CD117 (c-kit) TAM blasts coexpress variable proportions of CD34, CD7, CD36, CD41/42b, and CD235a (glycophorin A).8 Morphologic evidence of the pertur bation of megakaryopoiesis by GATA1s is almost always seen on blood smears in TAM (Figure 2), although thrombocytopenia is not a reliable diagnostic indicator of TAM because the platelet count may be normal, reduced, or even increased.29 While ane mia is uncommon, the median hematocrit (Hct) is lower in neo nates with TAM compared with DS neonates without TAM (Table 2). This is consistent with some impairment of erythropoiesis by GATA1s, although less than seen in disomic models of GATA1s function, perhaps due to compensation by the underlying T21associated increase in erythropoiesis in neonates with DS.6,13,14,29 For neo na tes with severe TAM (5%-20%), fac tors for early death include pre term deliv ery, asci tes, a leu ko cyte count greater than 100×109/l, hepatomegaly, and coagulopathy.17,18,29 For such cases the main stay of treat ment is sup port ive care and the judicious use of cytarabine as recommended in recent guidelines.29,36 While cytarabine reduces the risk of early death in severe TAM, there is no evidence yet that treatment prevents MLDS.37,38 In most neonates, TAM resolves within 3 to 4 months, even when the disease is severe. As 10% to 20% of cases of clinical TAM subsequently develop ML-DS, even after apparent complete remission, it seems sensible for children with TAM to be offered regular review until the age of 4 years, the time window during 4 | Hematology 2022 | ASH Education Program DS acute lymphoblastic leukemia In contrast to ML-DS, children with DS-ALL have an inferior out come to those without DS, both because of treatment-related toxicity and intrinsic differences in ALL biology.9,40 A recent large international study matched for cytogenetic subgroup showed that compared to children without DS, those with DS had worse 5-year event-free survival (75% vs 88%), overall survival (77% vs 94%), and post-induction treatment-related mortality (12.2% vs 2.7%).40 Survival after relapse is also inferior, even after transplan tation, although chimeric antigen receptor T-cell therapy has shown promise.41 While the clinical features are similar to non-DS ALL, the pattern of cytogenetic abnormalities in DS-ALL is distinct, consistent with the leukemogenic role of T21 in this disease. The frequency of “good prognosis” cytogenetic abnormalities, such as ETV6-RUNX1 and high hyperdiploidy, is lower in DS-ALL, and intriguingly, BCR-ABL translocations and KMT2A gene rearrangements are rare or absent in most series.9,40 Instead, the most common genomic alteration in DS-ALL is the rearrangement of CRLF2 (CRLF2r), which is found in approximately 60% of cases, half of which also have activating mutations in JAK2.42-44 A higher proportion of RAS/MAPK pathway mutations is also reported in DS-ALL, usually in JAK2 wild-type cases or subclones.45,46 In addi tion, increasing evidence suggests the potential importance of heritable risk loci (such as PAX5 or IKZF1 deletion) both in the risk of developing ALL in children with DS and also in treatment outcome, particularly for IKZF1 dele tion in which a poten tial mechanism involving decreased enhancer activity, differential protein binding, and effects on the cell growth of IKZF1 variants has been reported.40,47 Finally, it is notable that in children with out DS, T21 is one of the most frequent chromosomal abnormal ities in B-ALL, particularly in high hyperdiploid ALL, where up to 100% of cases have 1 to 3 additional copies of Hsa21,48 supporting a key role for increased Hsa21 dosage in lymphoid leukemo genesis. Although immunophenotypic and transcriptome analysis sug gests that DS-ALL blast transcriptomes are mainly shaped by their molecular cytogenetic subgroup,45,49 clinical and mechanistic studies also support an important role for T21 at several stages in leukemogenesis. Indeed, the acquisition of a supernumerary Hsa21 is always a nonrandom event restricted to certain cyto Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 Figure 2. Peripheral blood smear from a neonate with TAM. Blood smear of a 3-day-old neonate with DS and an exon 2 mutation in the GATA1 gene showing pleomorphic blast cells with features of immature and partially differentiated megakaryoblasts and a giant dysplastic platelet. May Grunwald Giemsa stain, ×100 magnification. which these children are at risk of ML-DS.2,5,17,18,29 At present, no clinical, hematologic, or molecular features at birth reliably pre dict which children develop ML-DS, although molecular or flow cytometric evidence of residual disease at 3 months confers an increased risk of subsequent ML-DS.37,38 In our experience, the persistence of residual GATA1s clones beyond 4 months makes subsequent ML-DS almost inevitable, although such clones are usually too small to produce detectable molecular, hematologic, or clinical evidence of their presence. Although the median age at diagnosis of ML-DS is 12 to 18 months, the evolution of TAM to ML-DS usually manifests as slow, progressive pancytopenia over several months with occasional circulating blasts. However, in some cases leukemic transformation is much more acute, with rapidly increasing blasts, so any change in the blood count dur ing follow-up should prompt a careful review of the blood smear. Adaptations to ML-DS chemotherapy regimens to minimize the increased toxicity of many drugs for patients with DS have improved disease-free survival to 90%, although the outcome for children who relapse remains poor.39 genetic subtypes of DS-ALL (eg, ETV-RUNX1 and P2RY8-CRLF2); abnormalities of Hsa21 have also been estimated to affect up to 65% of allchildhood B-ALL cases.50,51 The existence of a prena tal stage of DS-ALL, although postulated, has never been dem onstrated and might seem unlikely given the rarity of DS-ALL in children under 12 months of age.9 On the other hand, there is increasing evidence that T21 severely impairs fetal B-progenitor devel op ment in humans, due both to intrin sic hema to poi etic stem/progenitor defects and extrinsic regulation through an altered hematopoietic microenvironment,26,52 and that quantita tive and qualitative defects in B cells are common in children with DS. This suggests that prenatal perturbation of B lymphopoiesis by T21 could shape the cellular context for leukemia initiation. While clinical studies provide irrefutable evidence that T21 itself is an important factor in DS leukemogenesis, model systems have been used to more precisely decipher how the supernumerary copy of Hsa21 in hematopoietic cells provides such a strong leukemogenic stimulus. Hsa21 has approximately 230 proteincoding genes, many of which have key roles in normal and malig nant hematopoiesis (such as DYRK1A, ERG, ETS2, and RUNX1), as well as almost twice as many non–pro tein-cod ing genes, including 5 microRNAs (miRNAs).3 Most studies of DS leukemo genesis have focused on the dosage of genes on Hsa21, either using mouse models based on segmental trisomies or induced pluripotent stem cells (iPSC) (Table 3).14,53,54 While some Hsa21 genes and tissues exhibit the expected 1.5-fold higher expres sion levels than the corresponding disomic tissues, differences in gene expression are highly tissue-specific and cell populationspecific. This may explain why, even though the increased dos age of several Hsa21 genes has been shown to be leukemogenic in model systems, direct links between altered gene expression and hematopoietic phenotypes in DS have been so difficult to establish and why none of these phenotypes has so far been explained by a single Hsa21 gene acting alone. Furthermore, many studies now show that the perturbation of gene expres sion by T21 is genome-wide.3,52,55 This suggests that epigenetic mechanisms of cellular adaptation to aneuploidy are likely to be involved, particularly during fetal life when the exquisite control of gene expression is crucial for normal development. In line with this, recent work has found genome-wide effects on the DNA Table 3. Principal genes on Hsa21 implicated in DS leukemogenesis Gene Role in DS leukemia DYRK1A Promotes AMKL and B-ALL in murine models.62,63 ERG Cooperates with GATA1s to promote a TAM-like disease in a mouse model58; cooperates with ETS2, RUNX1, and GATA1s to promote expansion of fetal MK progenitors in a T21 iPSC model.54 ETS2 Cooperates with ERG, RUNX1, and GATA1s to promote expansion of fetal MK progenitors in a T21 iPSC model.54 HMGN1 Overexpression of HMGN1 promotes B-cell self-renewal, increased histone H3K27 acetylation, and downstream B-lineage gene expression; cooperates with BRC-ABL or CRLF2, JAK2, PAX5, and IKZF to promote DS-ALL.64,65 RUNX1 Cooperates with ERG, ETS2, and GATA1s to promote expansion of fetal MK progenitors in a T21 iPSC model.54 MicroRNAs Overexpression of miR-125b-2 cooperates with GATA1s to promote murine fetal MK progenitor proliferation and self renewal in vitro59; miR-99a, miR-125b-2, and miR-155 cooperate with GATA1s and cohesin insufficiency (STAG2 KO) to promote AMKL in human fetal cells in xenograft models.61 AMKL, acute megakaryoblastic leukemia. Dr Prakash Singh Shekhawat Leukemogenesis in infants with trisomy 21 | 5 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 Role of trisomy 21 in leukemogenesis in DS methylation of hematopoietic cells in cord blood from neona tes with DS as well as significant changes in the methylation of regions close to genes key to normal hematopoietic develop ment, such as RUNX1 and FLI1.56 Clinical studies indicate that leukemia initiation in DS, at least for ML-DS, critically depends upon a fetal cell context as well as the presence of T21. Euploid mouse models highlight devel opmental-specific differences in GATA1s function, but these do not develop leukemia.15 Similarly, several mouse models based on segmental trisomies, iPSC, or the overexpression of a sin gle Hsa21 ortholog have not so far fully recapitulated human TAM/ML-DS despite identifying several potent oncogenic can didates (eg, DYRK1A, ERG, ETS2) that can trigger myeloprolifer ative or leukemia disorders,57,58 perhaps because they lack key features of human fetal cells. In line with this, models based on inser tional muta gen e sis in murine fetal liver cells more closely mimic the immunophenotypic and molecular charac teristics of TAM/ML-DS.27,59,60 Furthermore, Wagenblast and col leagues used clustered regularly interspaced short palindromic repeats-Cas9 editing targeting GATA1 and STAG2 in human DS fetal liver cells to investigate the mechanism of cooperation between T21, GATA1s, and cohesin loss in DS leukemogene sis and successfully created a model that recapitulated many aspects of TAM and ML-DS.61 Using a combination of genomewide analysis of GATA1-binding sites, miRNA profiling, and overexpression and knockout (KO) experiments, they went on to show that a subset of Hsa21 miRNAs (miR-99a, miR-125b-2, and miR-155) was responsible, at least in part, for a GATA1s-induced TAM-like leukemia in fetal cells,61 supporting earlier data show ing a role for Hsa21 miRNAs.59,60 Intriguingly, in their model, T21 appeared unnecessary for the development of ML-DS as both DS and normal fetal liver cells could be transformed to a simi lar degree when dual edited to produce GATA1s in the setting of STAG2 KO. If T21 is mostly important for leukemia initiation in fetal cells in DS myeloid leukemias, as these data suggest, this may explain why TAM and the acquisition of GATA1s muta tions are confined to fetal and neonatal life and why ML-DS is restricted to a time window during which fetal cells can still survive postnatally. The role of T21 in leukemia initiation and progression in DSALL has mainly been studied using mouse models in tandem with the molec u lar anal y sis of pri mary patient sam ples.7,62-65 with BCR-ABL or a combination of 4 DS-ALL cytogenetic events (CRLF2 overexpression, an activating JAK2 mutation (JAK2R683G), Pax5 haploinsufficiency, and expression of a dominant negative Ikzf isoform).64,65 Conclusion Clinical and experimental studies, only some of which are sum marized here, have together yielded fascinating insights into DS leukemogenesis and underscore the complexity of the role of T21 in hematopoietic cells. The effects of T21 are developmental stage–specific and are most evident before birth, when striking abnormalities of erythro-megakaryopoiesis and B lymphopoiesis are present in the liver and bone marrow (Figure 3). The pres ence of T21 and somatic GATA1s mutations in fetal hematopoietic cells is essential for leukemia initiation in TAM/ML-DS. Whether rewiring of gene expression programs in fetal early lymphoid or B progenitors persists into early childhood and cooperates with aberrant CRLF2/JAK2 and/or RAS function to initiate DS-ALL is not known. Many questions remain to be answered, not least the reasons for the high frequency of somatic GATA1 mutations in DS fetal blood cells and of CRLF2/JAK2 aberrations in DS-ALL. Figure 3. Impact of T21 on hematopoiesis and leukemia in DS. Cartoon summarizing the putative mechanisms that link T21 with altered hematopoiesis and leukemia in early childhood in children with DS. A trisomy 21–mediated genome-wide perturbation of gene expression from early in embryonic/fetal development causes the expansion of a rapidly proliferating hematopoietic stem and myeloid progenitor pool with erythroid/MK bias in fetal liver and BM (bone marrow). These effects are hematopoietic cell-intrinsic but supported by T21-driven alterations in the microenvironment. The acquisition of somatic GATA1 mutations that encode a short GATA1 protein (GATA1s), possibly as a mutagenic effect of T21, cause further expansion of erythro-megakaryocytic cells and selective expansion of GATA1s clones, leading to the fetal/neonatal leukemia TAM. In 10% to 20% of cases of clinical TAM, ML-DS develops when GATA1s clones persist and acquire secondary mutations, most often in cohesin genes. The expansion of fetal megakaryopoiesis in DS occurs at the expense of B-progenitor development due to a T21-driven failure to properly activate the B-lineage molecular programs. This may lead to postnatal expansion of a depleted abnormally programmed B-progenitor pool, perhaps secondary to infections in early childhood, susceptible to transformation by aberrations in CRLF2, JAK2, and or RAS pathway signaling. HSC, hematopoietic stem cell; HSPC, hematopoietic stem/progenitor cell. 6 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 Several genes on Hsa21 have been shown to be important for normal B-cell growth and differentiation, including DYRK1A, ERG, ETS2, HMGN1, and RUNX1; overexpression of DYRK1A and also of HMGN1 have been directly linked to the pathogenesis of B-ALL.6265 Increased expression of DYRK1A has been reported in a num ber of leukemias, including B-ALL, and has recently been shown to be necessary for the growth of B-ALL cells.63 Although not spe cifically investigated in DS-ALL, this work is particularly interest ing because it showed that small-molecule inhibitors targeting DYRK1A were effective in animal models of B-ALL.63 Also taking differential gene expression in primary patient cells as a starting point, Lane et al. performed experiments in the Ts65Dn mouse, one of the most well-characterized segmental T21 mouse mod els, and not only narrowed down the putative region of Hsa21 (21q22) to a region of 31 genes sufficient to alter B-cell self-renewal and differentiation but also attributed it to overexpression of just 1 of these, HMGN1, which encodes a nucleosome-binding protein.64 They went on to demonstrate that overexpression of HMGN1 caused both a global increase in histone H3K27 acet y la tion and down stream B-lin e age gene expres sion and also pro moted the devel op ment of B-ALL, albeit in coop er a tion Evidence for a mutagenic phenotype or a generalized defect in DNA repair in DS tissues is sparse66 and precisely how hemato poietic cells to adapt to the potentially devastating effects of aneuploidy, and the extent to which this varies in different cell types, remains to be determined. Conflict-of-interest disclosure Irene Roberts: no competing financial interests to declare. Off-label drug use Irene Roberts: nothing to disclose. Correspondence References 1. Hasle H, Clemmensen IH, Mikkelsen M. Risks of leukaemia and solid tumours in individuals with Down’s syndrome. Lancet. 2000;355(9199):165-169. 2. Marlow EC, Ducore J, Kwan ML, et al. Leukemia risk in a cohort of 3.9 million children with and without Down syndrome. J Pediatr. 2021;234(July):172180.e3180e3. 3. Antonarakis SE, Skotko BG, Rafii MS, et al. Down syndrome. Nat Rev Dis Primers. 2020;6(1):9. 4. Zipursky A. Transient leukaemia—a benign form of leukaemia in newborn infants with trisomy 21. Br J Haematol. 2003;120(6):930-938. 5. Hasle H, Friedman JM, Olsen JH, Rasmussen SA. Low risk of solid tumors in persons with Down syndrome. Genet Med. 2016;18(11):1151-1157. 6. Roberts I, Alford K, Hall G, et al; Oxford-Imperial Down Syndrome Cohort Study Group. 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Mundschau G, Gurbuxani S, Gamis AS, Greene ME, Arceci RJ, Crispino JD. Mutagenesis of GATA1 is an initiating event in Down syndrome leukemo genesis. Blood. 2003;101(11):4298-4300. 12. Wechsler J, Greene M, McDevitt MA, et al. Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet. 2002; 32(1):148-152. 13. Byrska-Bishop M, VanDorn D, Campbell AE, et al. Pluripotent stem cells reveal erythroid-specific activities of the GATA1N-terminus. J Clin Invest. 2015;125(3):993-1005. 14. Ling T, Birger Y, Stankiewicz MJ, et al. Chromatin occupancy and epige netic analysis reveal new insights into the function of the GATA1N terminus in erythropoiesis. Blood. 2019;134(19):1619-1631. 15. Li Z, Godinho FJ, Klusmann JH, Garriga-Canut M, Yu C, Orkin SH. Developmental stage-selective effect of somatically mutated leukemogenic tran scription factor GATA1. Nat Genet. 2005;37(6):613-619. 16. Kadri Z, Shimizu R, Ohneda O, et al. Direct binding of pRb/E2F-2 to GATA-1 regulates maturation and terminal cell division during erythropoiesis. PLoS Biol. 2009;7(6):e1000123. 17. Klusmann JH, Creutzig U, Zimmermann M, et al. Treatment and prognos tic impact of transient leukemia in neonates with Down syndrome. Blood. 2008;111(6):2991-2998. Dr Prakash Singh Shekhawat Leukemogenesis in infants with trisomy 21 | 7 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 Irene Roberts, Department of Paediatrics, MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford OX3 9DS, United Kingdom; e-mail: irene.roberts@imm.ox.ac.uk, irene.roberts@paediatrics .ox.ac.uk. 18. Gamis AS, Alonzo TA, Gerbing RB, et al. Natural history of transient mye loproliferative disorder clinically diagnosed in Down syndrome neona tes: a report from the Children’s Oncology Group Study A2971. Blood. 2011;118(26):6752-6759, quiz 6996. 19. Lukes J Jr, Danek P, Alejo-Valle O, et al. Chromosome 21 gain is dispensable for transient myeloproliferative disorder driven by a novel GATA1 mutation. Leukemia. 2020;34(9):2503-2508. 20. Hollanda LM, Lima CS, Cunha AF, et al. An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet. 2006;38(7):807-812. 21. Hasle H, Kline RM, Kjeldsen E, et al. Germline GATA1s generating mutations predispose to leukemia with acquired trisomy 21 and Down syndrome-like phenotype. Blood. 2021;139(21):3159-3165. 22. Parrella S, Aspesi A, Quarello P, et al. Loss of GATA-1 full length as a cause of Diamond-Blackfan ane mia phe no type. Pediatr Blood Cancer. 2014;61(7):1319-1321. 23. Rainis L, Bercovich D, Strehl S, et al. Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21. Blood. 2003;102(3):981-986. 24. Ahmed M, Sternberg A, Hall G, et al. Natural history of GATA1 mutations in Down syndrome. Blood. 2004;103(7):2480-2489. 25. Tamblyn JA, Norton A, Spurgeon L, et al. Prenatal therapy in transient abnormal myelopoiesis: a systematic review. Arch Dis Child Fetal Neonatal Ed. 2016;101(1):F67-F71. 26. Roy A, Cowan G, Mead AJ, et al. Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21. Proc Natl Acad Sci USA. 2012;109(43):17579-17584. 27. Yoshida K, Toki T, Okuno Y, et al. The landscape of somatic mutations in Down syndrome-related myeloid disorders. Nat Genet. 2013;45(11):12931299. 28. Labuhn M, Perkins K, Matzk S, et al. Mechanisms of progression of myeloid preleukemia to transformed myeloid leukemia in children with Down syn drome. Cancer Cell. 2019;36(3):340. 29. Tunstall O, Bhatnagar N, James B, et al. Guidelines for the investigation and management of transient leukaemia of Down syndrome. Br J Haematol. 2018;182(2):200-211. 30. de Graaf G, Buckley F, Skotko BG. Estimation of the number of people with Down syndrome in Europe. Eur J Hum Genet. 2021;29(3):402-410. 31. Goemans BF, Noort S, Blink M, et al. Sensitive GATA1 mutation screening reliably identifies neonates with Down syndrome at risk for myeloid leuke mia. Leukemia. 2021;35(8):2403-2406. 32. Bozner P. Transient myeloproliferative disorder with erythroid differentia tion in Down syndrome. Arch Pathol Lab Med. 2002;126(4):474-477. 33. Maroz A, Stachorski L, Emmrich S, et al. GATA1s induces hyperproliferation of eosinophil precursors in Down syndrome transient leukemia. Leukemia. 2014;28(6):1259-1270. 34. Bain BJ. Basophilic differentiation in transient abnormal myelopoiesis. Am J Hematol. 2016;91(8):847. 35. Katsumura KR, Bresnick EH; GATA Factor Mechanisms Group. The GATA fac tor revolution in hematology. Blood. 2017;129(15):2092-2102. 36. Al-Kershi S, Golnik R, Flasinski M, et al. Recommendations for diagnosis and treatment of children with transient abnormal myelopoiesis (TAM) and myeloid leukemia in Down syndrome (ML-DS). Klin Padiatr. 2021;233(6):267277. 37. Flasinski M, Scheibke K, Zimmermann M, et al. Low-dose cytarabine to pre vent myeloid leukemia in children with Down syndrome: TMD Prevention 2007 study. Blood Adv. 2018;2(13):1532-1540. 38. Yamato G, Deguchi T, Terui K, et al. Predictive factors for the development of leukemia in patients with transient abnormal myelopoiesis and Down syndrome. Leukemia. 2021;35(5):1480-1484. 39. Hitzler J, Alonzo T, Gerbing R, et al. High-dose AraC is essential for the treatment of ML-DS independent of post-induction MRD: results of the COG AAML1531 trial. Blood. 2021;138(23):2337-2346. 40. Michels N, Boer JM, Enshaei A, et al. Minimal residual disease, long-term outcome, and IKZF1 deletions in children and adolescents with Down syn drome and acute lymphocytic leukaemia: a matched cohort study. Lancet Haematol. 2021;8(10):e700-e710. 41. Laetsch TW, Maude SL, Balduzzi A, et al. Tisagenlecleucel in pediatric and young adult patients with Down syndrome-associated relapsed/refractory acute lymphoblastic leukemia. Leukemia. 2022;36(6):1508-1515. 42. Mullighan CG, Collins-Underwood JR, Phillips LA, et al. Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute lympho blastic leukemia. Nat Genet. 2009;41(11):1243-1246. 8 | Hematology 2022 | ASH Education Program 55. Liu B, Filippi S, Roy A, Roberts I. Stem and progenitor cell dysfunction in human trisomies. EMBO Rep. 2015;16(1):44-62. 56. Muskens IS, Li S, Jackson T, et al. The genome-wide impact of trisomy 21 on DNA methylation and its implications for hematopoiesis. Nat Commun. 2021;12(1):821. 57. Grimm J, Heckl D, Klusmann JH. Molecular mechanisms of the genetic pre disposition to acute megakaryoblastic leukemia in infants with Down syn drome. Front Oncol. 2021;11(June):636633. 58. Birger Y, Goldberg L, Chlon TM, et al. Perturbation of fetal hematopoiesis in a mouse model of Down syndrome’s transient myeloproliferative disorder. Blood. 2013;122(6):988-998. 59. Klusmann JH, Li Z, Böhmer K, et al. miR-125b-2 is a potential oncomiR on human chromosome 21 in megakaryoblastic leukemia. Genes Dev. 2010; 24(5):478-490. 60. Alejo-Valle O, Weigert K, Bhayadia R, et al. The mega kar yo cytic tran scription factor ARID3A suppresses leukemia pathogenesis. Blood. 2022; 139(5):651-665. 61. Wagenblast E, Araújo J, Gan OI, et al. Mapping the cellular origin and early evolution of leukemia in Down syndrome. Science. 2021;373(6551). 62. Laurent AP, Kotecha RS, Malinge S. Gain of chromosome 21 in hematolog ical malignancies: lessons from studying leukemia in children with Down syndrome. Leukemia. 2020;34(8):1984-1999. 63. Bhansali RS, Rammohan M, Lee P, et al. DYRK1A regulates B cell acute lym phoblastic leukemia through phosphorylation of FOXO1 and STAT3. J Clin Invest. 2021;131(1):e135937. 64. Lane AA, Chapuy B, Lin CY, et al. Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 Lys27 trimethylation. Nat Genet. 2014;46(6):618-623. 65. Mowery CT, Reyes JM, Cabal-Hierro L, et al. Trisomy of a Down syndrome critical region globally amplifies transcription via HMGN1 overexpression. Cell Rep. 2018;25(7):1898-1911.e51911e5. 66. Hasaart KAL, Bertrums EJM, Manders F, Goemans BF, van Boxtel R. Increased risk of leukaemia in children with Down syndrome: a somatic evolutionary view. Expert Rev Mol Med. 2021;23(April):e5. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000395 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/1/2021737/1roberts.pdf by guest on 09 December 2022 43. Russell LJ, Capasso M, Vater I, et al. Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell pre cursor acute lymphoblastic leukemia. Blood. 2009;114(13):2688-2698. 44. Hertzberg L, Vendramini E, Ganmore I, et al. Down syndrome acute lym phoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group. Blood. 2010;115(5):1006-1017. 45. Schwartzman O, Savino AM, Gombert M, et al. Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leu kemia in Down syndrome. Proc Natl Acad Sci USA. 2017;114(20):E4030E4039. 46. Nikolaev SI, Garieri M, Santoni F, et al. Frequent cases of RAS-mutated Down syndrome acute lymphoblastic leukaemia lack JAK2 mutations. Nat Commun. 2014;5(August):4654. 47. Brown AL, de Smith AJ, Gant VU, et al. Inherited genetic susceptibility to acute lymphoblastic leukemia in Down syndrome. Blood. 2019;134(15):12271237. 48. Paulsson K, Forestier E, Lilljebjörn H, et al. Genetic land scape of high hyperdiploid childhood acute lymphoblastic leukemia. Proc Natl Acad Sci USA. 2010;107(50):21719-21724. 49. Kubota Y, Uryu K, Ito T, et al. Integrated genetic and epigenetic analy sis revealed heterogeneity of acute lymphoblastic leukemia in Down syn drome. Cancer Sci. 2019;110(10):3358-3367. 50. Schwab C, Harrison CJ. Advances in B-cell precursor acute lymphoblastic leukemia genomics. Hemasphere. 2018;2(4):e53. 51. Abbasi MR, Nebral K, Haslinger S, et al. Copy number changes and allele distribution patterns of chromosome 21 in B cell precursor acute lympho blastic leukemia. Cancers (Basel). 2021;13(18):4597. 52. Jardine L, Webb S, Goh I, et al. Blood and immune development in human fetal bone mar row and Down syn drome. Nature. 2021;598(7880):327331. 53. MacLean GA, McEldoon J, Huang J et al. Downregulation of endothelin receptor B contributes to defective B cell lymphopoiesis in trisomy 21 plu ripotent stem cells. Sci Rep. 2018;8(1):8001. 54. Banno K, Omori S, Hirata K, et al. Systematic cellular disease models reveal synergistic interaction of trisomy 21 and GATA1 mutations in hematopoietic abnormalities. Cell Rep. 2016;15(6):1228-1241. ACUTE MYELOID LEUKEMIA: IMPROVING OUTCOMES IN CHALLENGING SUBSETS Achieving MRD negativity in AML: how important is this and how do we get there? Laboratory of Myeloid Malignancies, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD Multiple studies have demonstrated that patients with acute myeloid leukemia (AML) who have measurable residual disease (MRD) detected during or after treatment have higher relapse rates and worse survival than similar patients testing negative. Updated response criteria for AML reflect the understanding that achievement of complete remission (CR) with no detectable MRD using high-sensitivity tests represents a superior response over conventional cytomorphological CR alone. Potential use cases for AML MRD testing are diverse and include patient selection for clinical trials and therapeutic assignment, early relapse detection and intervention during sequential monitoring, and drug development, including deep quantification for antileukemia efficacy and as a surrogate endpoint for overall survival in regulatory approvals. Testing for AML MRD has not, however, been harmonized, and many technical and clinical questions remain. The implications of MRD test results for specific therapeutic combinations, molecular subsets, test types, treatment time points, sample types, and patient characteristics remain incompletely defined. No perfect AML MRD test or testing strategy currently exists, and the evidence basis for clinical recommendations in this rare disease is sparse but growing. It is unproven whether conversion of an MRD test result from positive to negative by additional therapeutic intervention improves relapse risk and survival. Several national- and international-level consortia have recently been initiated to advance the generation and collection of evidence to support the use of AML MRD testing in clinical practice, drug development, and regulatory approvals. LEARNING OBJECTIVES • Explain why the updated AML response criteria now include a best possible response of CRMRD− • Describe how tests validated for diagnostic profiling purposes only are likely insufficient for use in MRD • Understand the objectives of ongoing national and international collaborative efforts to validate AML MRD testing for multiple purposes Introduction CLINICAL CASE A man in his 70s with significant medical comorbidities presents to your clinic with AML. Flow cytometry dem onstrates abnormal CD34+ blasts in both blood (1%) and marrow (~5%). Metaphase cytogenetics are reported as 47XY, +8, inv(16)(p13.1q22), del(20)(q11.2q13.3), and next generation sequencing (NGS) by a “myeloid panel” reported 2 mutations in DNMT3A and 1 in TET2. You are delighted when he achieves a complete remission (CR) by cytomorphology after 1 cycle of treatment until your med ical student asks, “But what about measurable residual disease [MRD]?” Your institution has the tests described in the above diagnostic workup available. How, and why, should you measure MRD? Detectable disease after treatment is, by definition, refrac tory therapyresistant disease. Patients and their doctors generally strongly prefer no evidence of residual cancer. With a blood cancer like AML, which is typically widely disseminated throughout the body at initial diagnosis, the key issue in assessing posttreatment response is how accurately the evaluation of a small sample of the patient reflects the total remaining leukemic burden in the body with the capacity to lead to a subsequent clinically evident “relapse.” An insufficient sample and/or suboptimal assess ment of a sample from a patient after treatment may lead to false reassurance that a patient in “complete remission” has achieved a state of disease clearance, a conceit quickly shattered by the “relapse” that follows. While it has been stated that the most pressing problem in AML is relapse, Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 9 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 Christopher S. Hourigan When, where, and with what? Given the strong evidence that testing for MRD in patients with AML in remission can stratify them into groups with higher and lower risks of relapse and survival, there is great appeal in devel oping a universal “best” single test by which to monitor AML MRD. This potentially unrealistic aspiration may be motivated by the examples of great success in MRD monitoring for other blood cancers, such as chronic myeloid leukemia, acute promyelocytic leukemia (both entities with a single pathognomonic structural variant expressed at the transcript level for tracking by quan titative polymerase chain reaction), and the lymphoid malig nancies and multiple myeloma (in which a distinct cell surface immunophenotype and a clonally rearranged immune receptor provide 2 ideal options for MRD monitoring). In contrast, AML is a name given to a genetically heterogeneous collection of mye Table 1. Potential use cases for AML MRD • Deep quantification of antileukemia efficacy (eg, log reduction after 2 cycles of therapy) • Early relapse detection and intervention during sequential monitoring • Therapeutic assignment (eg, selection of transplant intensity where otherwise equipoise) • Patient selection for clinical trials (eg, high-risk group of unmet need) • As a surrogate endpoint for overall survival for regulatory approval 10 | Hematology 2022 | ASH Education Program Figure 1. Some validated molecular targets for AML MRD testing. The frequency and co-occurrence of those molecular tar gets with evidence of utility for AML MRD testing, based on 200 adult AML patients from the Cancer Genome Atlas database. loid malignancies, which can have changing clonal proportions even within 1 patient over time. While multiple individual targets for the molecular monitoring of AML MRD have been described (Figure 1), there remains great inter est in a “one-size-fits-all ” approach to MRD monitoring in this diverse set of blood can cers using, for example, multiparameter flow cytometry or NGS. Currently, there is no “one best test” for allcases of AML MRD. Flow cytometry is widely available, is used routinely in the initial diagnostic workup, and has a potentially rapid turnaround time. Limitations include the need for highly specialized expert interpretation for best results, difficulties in test harmonization when not performed and, in par tic u lar, ana lyzed centrally,3,11 and suboptimal relapse risk prediction even in the best centers (a recent study of 743 consecutive adults undergoing their first alloHCT for AML in remission at a single center showed that pre transplant flow cytometry identified just 96 of 230 subsequent relapses).10,16 The ELN guidelines currently state that flow cytom etry for AML MRD should be used when a validated molecular test is not available.2,3 NGS of DNA is also widely available and used routinely in the initial diagnostic workup. A longer turnaround time than other methods is balanced by objective output that allows for a decen tralized interpretation.17 NGS is not currently ELN recommended for detecting AML MRD as a stand-alone test, however, due to insufficient data on appropriate targets, performance character istics of optimal testing, and clinical utility.18 It is already clear that the full spectrum of somatic mutations detected at initial AML diagnosis are not alluseful as AML MRD targets.2,12,19,20 The concordance of AML MRD testing using flow cytometry and NGS has been observed to be incomplete,19,21 with many potential explanations (Table 2). The remarkable opportunities presented Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 a very reasonable counterargument is that the most pressing problem is instead that AML therapy is suboptimal and appears adequate only due to insufficiently stringent response criteria. In recognition of this, the European LeukemiaNet (ELN) in 2017 updated the response criteria in AML with the addition of a best possible response category of complete remission without MRD (CRMRD−).1 The case for MRD assess ments in AML response cri te ria is clear, even if the logistics associated with widespread harmo nized clinical implementation remain to be defined.2,3 For 40 years the concept that MRD in AML exists and may be detectable and treatable has been well understood.4-6 In the past 5 years, sys tematic reviews with meta-analyses have demonstrated that, regardless of the MRD methodology used, patients testing pos itive, either at a specific time point prior to allogeneic hemato poietic cell transplantation (alloHCT) or more generally at any time during treatment,7,8 have worse survival than those who test MRD negative. This stratification at the cohort level can assign patients with AML in CR after treatment into groups with higher and lower risks of subsequent relapse. However, when currently measured at a single time point, MRD testing is suboptimal at an individual patient level for relapse prediction—particularly, per haps, when baseline patient and disease characteristics are con sidered.9-12 Nevertheless, given the additional insight provided there is increasing interest in using AML MRD testing results for a variety of potential clinical use cases (Table 1).13-15 Achieving MRD negativity in AML is an important signifier of response to initial treatment, but additional evidence is required to determine if this is the most appropriate goal and, if so, the best ways to get there both in an individual patient and as an international community. Table 2. Potential reasons for discrepancy between AML MRD testing methods AML has no appropriate mutation for tracking (rare) NGS test does not include mutation of interest (common) • Core binding factor leukemia without KIT or other RTK mutation • Complex karyotype AML without TP53 mutation • Chromosomal aneuploidy or structural variants NGS test is insufficient sensitivity/not validated for MRD target (common) NGS true positive, flow cytometry false negative Lack of target (No LAIP/insufficient DfN) (rare) Limit of detection technique mismatch (eg, NPM1-mutated AML) (common) Heterogeneous or unstable immunophenotype, differentiated cells after therapy (common) NGS false positive, flow cytometry true negative Technical error (NGS test not validated for MRD use) Inappropriate NGS target selection (eg, isolated DNMT3A mutation) Mutation germ line or present only in cells incapable of causing relapse (eg, lymphocytes) Flow cytometry false positive, NGS true negative Regeneration after chemotherapy or allogeneic transplant DfN, difference from normal; LAIP, leukemia-associated immunophenotype; RTK, receptor tyrosine kinase. Figure 2. Comparison of performance of tests used for AML diagnosis vs MRD. Cartoon approximation of wide differences in target coverage (ie, number of features tracked, “breadth”) vs detection limit (ie, analytical sensitivity, “depth”) between tests validated for use at initial diagnosis (blue) vs MRD tests in use or development. dPCR, digital PCR; UMI, unique molecular identifier. by NGS mean it will almost certainly play a large role in AML MRD as appropriate targets are validated and the performance char acteristics of different approaches are better understood and optimized (Figure 2). Other potential forms of NGS, including cell-free DNA, transcript expression, and methylation signatures, do not yet have sufficient evidence for AML MRD monitoring. RNA-based NGS for the ELN-recommended AML MRD molecu lar targets has been described.22 Single-cell sequencing has the potential to elucidate clonal structure at diagnosis to determine features associated specifically with the malignant clone, includ ing linking genotype with immunophenotype (the differentiation state of the cell with a detected mutation may have implications for both therapy resistance and the ability to lead to relapse).23-25 Beyond the specific details of the current and future technol ogy used for this purpose, an optimal AML MRD testing strategy for relapse prediction requires the optimization of multiple factors, including intervals between testing time points, the type (mar row vs blood) and amount of sample tested, and an accounting for AML disease biology and kinetics, as well as patient charac teristics that include age, antecedent disorders, time points of treatment, and the intensity and nature of therapy.26-29 Is MRD clinically actionable, or does it just portend fate? AML patients in a conventional, cytomorphological CR have a higher risk of relapse and worse survival if evidence of residual disease is detected compared to those who test negative—that is, AML MRD test positivity is prognostic. Such patients are high risk, are underserved by the current standard of care, and should be offered a clinical trial where possible. It is, however, important to also say that MRD test negativity does not equal patient MRD negativity. Patients testing MRD negative by flow cytometry prior to alloHCT still have a 20% to 30% relapse rate,30 and dein tensification of standard treatment based on an MRD test result should only be attempted cautiously as part of a clinical trial. Patients testing MRD-positive may relapse, may die of a com peting risk before relapse, be false positive for technical (assay Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 11 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 Flow cytometry true positive, NGS false negative autologous transplant if MRD negative, and saw no difference between groups, suggesting a benefit to treatment intensifi cation.34 Understanding if an MRD test result is just fate (ie, a prognostic biomarker) or if treatment modification can change outcomes (ie, also a predictive biomarker), and if so in which patients and AML types, is currently one of the most important questions in AML. Observational and registry studies are poorly suited to answer this question, highlighting the need to gener ate higher-quality evidence for MRD in AML. Recent initiatives generating AML MRD evidence AML is a rare disease, further divided into subsets with diverse genetic etiologies and prognoses and, increasingly, with multi ple treatment alternatives. In this context, single investigators or institutions can make only limited generalizable contributions to the evidence base supporting AML MRD test uses, motivating the formation of several large national or multinational coopera tive efforts (Table 3). Alongside updating the assessment criteria after AML treat ment in 2017 by includ ing a new, best pos si ble response of CRMRD−,1 the ELN in 2016 also established a panel of international experts (initially 24 from 20 countries, including laboratory sci entists, pathologists, and leukemia physicians) to recommend laboratory and clinical guidelines for the use of AML MRD test ing. The first edition of these consensus standard of care guide lines was published in 2018 and provided technical guidance for performing molecular and flow cytometry–based MRD testing along with clinical recommendations, including that AML with mutated NPM1,35,36 core-binding factor AMLs, acute promyelo cytic leukemia, and the rare cases of AML with BCR-ABL1 should be monitored with a validated molecular test, while all others should be monitored by flow cytometry.3 Updated guidelines were published in 2021, with a focus on the available evidence base underlying these expert recommendations and optimized consensus generation using a two-stage Delphi poll approach.2 This group, recently renamed ELN-DAVID, will likely continue to release updated consensus recommendations every 2 to 4 years as new high-quality evidence becomes available. Table 3. Some recent initiatives generating evidence for MRD testing in AML Initiative Goal Membership ELN AML MRD guidelines: European LeukemiaNet Evidence-based clinical standard of care consensus guidelines for AML MRD testing International committee of physicians and scientists with expertise in AML MRD MPAACT: Measurable Residual Disease Partnership and Alliance in Acute Myeloid Leukemia Clinical Treatment Industry-led research alliance advancing efforts to establish MRD as a surrogate endpoint for overall survival in the treatment of AML Founded in 2018 by Janssen, Genentech, Novartis, and Celgene (now Bristol Myers Squibb), with recent additions of Amgen, AbbVie, and Kronos Bio.40 FNIH: Foundation of the NIH Biomarkers Consortium for AML MRD Establish and validate new methods for detecting MRD in AML, including a library of reference standards and evidence of clinical utility. FDA, NIH, 2 academic partners, and 15-25 private sector industry partners Pre-MEASURE NIH-led retrospective project on >1000 patients to determine the impact of pre-alloHCT MRD testing in CR1 blood using ultradeep NGS In collaboration with the CIBMTR MEASURE: Molecular Evaluation of AML Patients After Stem Cell Transplant to Understand Relapse Events Prospective multicenter protocol to determine clinical utility of MRD testing in up to 1000 AML patients undergoing alloHCT (NCT05224661) National Marrow Donor Program, CIBMTR, NIH, with initially up to 16 US-based high-volume alloHCT centers NCI MyeloMATCH Upcoming national precision medicine master protocol for AML. Rapid drug efficacy screening using genetic assignments and MRD testing. National Cancer Institute, ECOG-ACRIN, SWOG, The Alliance, Canadian Cancer Trials Group Children’s Oncology Group 12 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 analytical failure) or clinical (analyte detected but not associated with relapse risk—eg, wrong target or right target but in the wrong cellular context) reasons, or may have their relapse risk reduced by subsequent antileukemic factors (additional therapy, allogeneic or autologous immune responses). Careful system atic study to understand the nature of false-negative and falsepos i tive MRD tests are nec es sary to allow iter a tive improve ments to MRD tests and MRD testing strategies. In addition to MRD testing in AML being prognostic, it has been shown to be possible, in some circumstances, to convert a patient testing positive to a negative test status by additional treatment (for example, alloHCT).31,32 The conversion of MRD test result status from positive to negative, however, does not neces sarily imply a clinical benefit in terms of increased overall survival (from decreased relapse) or improved quality of life. It is possi ble to imagine a worst-case scenario in which additional therapy made the biomarker test turn negative but with increased tox icity and no survival benefit. Some patients with MRD in remis sion are incompletely treated and have chemo-sensitive disease left to treat. Alternatively, MRD may reflect the residual chemoresistant clone, which may or may not be resis tant to novel agents. Both these possibilities are testable. A large retrospective European Society for Blood and Mar row Transplantation reg is try suggested that myeloablative (MAC) rather than reduced-intensity conditioning (RIC) in youn ger patients mitigated some of the risk associated with pre transplant MRD positivity.30 The phase 3 randomized controlled trial (RCT) BMT-CTN 0901 study (NCT01339910) demonstrated reduced relapse and improved overall survival for younger adults in CR randomized to MAC rather than RIC; subsequent analy sis showed the greatest benefit for those who were NGS pos itive before conditioning.20 The poor outcomes of those who were MRD-positive prior to RIC alloHCT were confirmed by the FIGARO trial, an RCT that demonstrated that additional cytore ductive che mo ther apy prior to RIC (in patients inel i gi ble for MAC due to age or comorbidity) did not improve outcomes.33 The GIMEMA AML1310 trial (NCT01452646) assigned youn ger, intermediate-risk patients who were MRD positive to alloHCT, or CLINICAL CASE (Cont inu ed) Clinical NGS DNA sequenc ing “mye loid pan els” used for AML diagnostic profiling are 10 to 500 times less sensitive than AML MRD tests and is a poor choice here. Additionally, which somatic mutations detected in remission are most associated with relapse risk remain to be fully described. There is evidence that DNMT3A and TET2 mutations should not be used for MRD testing; this was reinforced by research showing that these mutations were only Figure 3. The upcoming NCI Precision Medicine Initiative MyeloMATCH. Schematic showing potential journey of a patient enrolled in MyeloMATCH through the protocol tiers. found in subclones unrelated to the AML of this patient.24 Flow cytometry performed to MRD standards is a reasonable choice but is not optimal given the presence of a defining molecular feature in this inversion-16 AML (CBFB-MYH11), quantifiable using a well-validated test.2,3 The patient achieved MRD negativity by poly mer ase chain reac tion test ing and enjoyed a prolonged remission despite being ineligible for consolidative alloHCT. Conclusion Testing AML MRD negative is preferable to testing positive, all other factors being equal. The phenomena of higher-sensitivity tools allowing refined, but imper fect, prog nos ti ca tion for patients with AML in remission have been well described in the literature but incompletely translated to the clinic. Because the AML MRD test status reflects only the sample that was tested, not the entire patient, false-positive and false-negative results are expected and have multifactorial causes. On an individual patient level, AML MRD test status can help risk-stratify but is not a guar antee of fate; serial MRD measurement kinetics are likely supe rior to single landmark assessments. The coming years will see the generation of high-quality evidence for the many potentialuse cases for AML MRD testing and collaboration on the first logistical steps toward a harmonized national-level approach for measurable patient benefit. Acknowledgments This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute of the National Institutes of Health. The Visual Abstract, Figure 1, and Figure 2 are courtesy of Alan Hoofring and Dr. Laura Dillon. Figure 3 is courtesy of Dr. Rich Little. Conflict-of-interest disclosure Christopher S. Hourigan: funding: Sellas, Foundation for the Na tional Institutes of Health Acute Myeloid Leukemia Measurable Residual Disease Biomarkers Consortium. Off-label drug use Christopher S. Hourigan: nothing to disclose. Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 13 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 Two important initiatives in AML MRD from, or in partnership with, the biopharmaceutical industry are worthy of comment. First, fol low ing the US Food and Drug Administration (FDA)convened Duke Margolis Center Public Meeting on Minimal Resid ual Disease as a Surrogate Endpoint in Hematologic Cancer Trials in 2016, a partnership of 4 pharmaceutical companies was formed (led initially by Sharon McBain, then of Janssen, the partnership was formalized and expanded in 2022 and is now known as MPAACT) to advance efforts in this area, including the planned performance of a meta-analysis of clinical trial data to evaluate the association of MRD with overall survival. Complementary to this, focusing on the generation of new standards, tests, and data, rather than the analysis of existing data sets, in early 2022 the Foundation of the National Institutes of Health (NIH) Bio markers AML MRD Consortium was launched as a collaboration between public-sector (NIH, FDA), private-sector (~20 pharma ceutical, biotechnology, research, or diagnostic testing compa nies), and academic (Fred Hutch and Dana Farber) partners. AlloHCT is a key therapeutic intervention to reduce subse quent relapse risk for many patients with AML in CR, with good evi dence that MRD before trans plant is prog nos tic.7,9,16,20,30,33,37 The NIH-funded Pre-MEASURE study eval u ated pretransplant blood samples from 1075 patients transplanted in first remission at one of 111 Center for International Blood and Marrow Trans plant Research (CIBMTR) sites between 2013 and 2019 to estab lish the clinical utility of NGS-based AML MRD testing for FLT3-ITD and NPM1 mutations.38 Following this retrospective study, the National Marrow Donor Program and the CIBMTR sponsored a prospective protocol, MEASURE, at 16 major US transplant cen ters to establish a national framework for introducing MRD test ing into the clinical care of AML patients undergoing alloHCT (https://clinicaltrials.gov/ct2/show/NCT05224661). The National Cancer Institute precision medicine initiative for patients with AML, MyeloMATCH, is also launching officially in early 2023. This national umbrella trial will test treatments for AML, typically in randomized phase 2 designs comparing against the current best standard of care therapy, evaluating early endpoint efficacy signals in specific molecular and clinical risk groups. The novel design will assign a unique single patient identifier upon enrollment for initial therapy, allowing subjects to be followed throughout their treatment journey while partici pating in up to 4 different RCTs based on scheduled reassess ments (Figure 3). The intent is to use MRD testing as an efficacy endpoint, as an inclusion criterion for subsequent “tiers” of ther apy, while also facilitating the validation of novel, highly sensitive MRD assays such as duplex sequencing. Finally, regulatory guidance for the use of MRD, including in AML, for drug development has been published and presumably updated based on evidence resulting from the initiatives above.39 Correspondence Christopher S. Hourigan, Laboratory of Myeloid Malignancies, Hematology Branch, National Heart, Lung, and Blood Institute, NIH Clinical Ctr, Room 10CRC 6-5142, 10 Center Dr, Bethesda, MD 20892; e-mail: hourigan@nih.gov. References 1. 14 | Hematology 2022 | ASH Education Program DOI 10.1182/hematology.2022000323 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/9/2021982/9hourigan.pdf by guest on 09 December 2022 Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 2. Heuser M, Freeman SD, Ossenkoppele GJ, et al. 2021 update measurable residual disease in acute myeloid leukemia: European LeukemiaNet Work ing Party consensus document. Blood. 2021;138(26):2753-2767. 3. Schuurhuis GJ, Heuser M, Freeman S, et al. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2018;131(12):1275-1291. 4. Löwenberg B, Hagenbeek A. Minimal Residual Disease in Acute Leukemia. 1st ed. Dordrecht: Springer; 1984. 5. Martens AC, Hagenbeek A. Detection of minimal disease in acute leukemia using flow cytometry: studies in a rat model for human acute leukemia. Cytometry. 1985;6(4):342-347. 6. Hagenbeek A, Martens AC. BCG treatment of residual disease in acute leu kemia: studies in a rat model for human acute myelocytic leukemia (BNML). Leuk Res. 1983;7(4):547-555. 7. Buckley SA, Wood BL, Othus M, et al. Minimal residual disease prior to allogeneic hematopoietic cell transplantation in acute myeloid leukemia: a meta-analysis. Haematologica. 2017;102(5):865-873. 8. Short NJ, Zhou S, Fu C, et al. Association of measurable residual disease with survival outcomes in patients with acute myeloid leukemia: a system atic review and meta-analysis. JAMA Oncol. 2020;6(12):1890-1899. 9. Othus M, Gale RP, Hourigan CS, Walter RB. Statistics and measurable resid ual disease (MRD) testing: uses and abuses in hematopoietic cell transplan tation. Bone Marrow Transplant. 2020;55(5):843-850. 10. Othus M, Wood BL, Stirewalt DL, et al. Effect of measurable (“minimal”) residual disease (MRD) information on prediction of relapse and survival in adult acute myeloid leukemia. Leukemia. 2016;30(10):2080-2083. 11. Paiva B, Vidriales MB, Sempere A, et al; PETHEMA (Programa para el Estu dio de la Terapéutica en Hemopatías Malignas) Cooperative Study Group. Impact of measurable residual disease by decentralized flow cytometry: a PETHEMA real-world study in 1076 patients with acute myeloid leukemia. Leukemia. 2021;35(8):2358-2370. 12. Murdock HM, Kim HT, Denlinger N, et al. Impact of diagnostic genetics on remission MRD and transplantation outcomes in older patients with AML. Blood. 2022;139(24):3546-3557. 13. Gui G, Hourigan CS. Measurable residual disease assessment as a surrogate marker in new drug development in acute myeloid leukemia. Cancer J. 2022;28(1):73-77. 14. Spyridonidis A. How I treat measurable (minimal) residual disease in acute leukemia after allogeneic hematopoietic cell transplantation. Blood. 2020;135(19):1639-1649. 15. Hourigan CS, Gale RP, Gormley NJ, Ossenkoppele GJ, Walter RB. Mea surable residual disease testing in acute myeloid leukaemia. Leukemia. 2017;31(7):1482-1490. 16. Morsink LM, Sandmaier BM, Othus M, et al. Conditioning intensity, pretransplant flow cytometric measurable residual disease, and outcome in adults with acute myeloid leukemia undergoing allogeneic hematopoietic cell transplantation. Cancers (Basel). 2020;12(9):2339. 17. Heuser M. Standardizing NGS-MRD in AML: an international study by the ELN-MRD Working Party. Paper presented at: European Hematology Asso ciation Annual Congress; 2021, virtual meeting. 18. Ghannam J, Dillon LW, Hourigan CS. Next-generation sequencing for mea surable residual disease detection in acute myeloid leukaemia. Br J Hae matol. 2020;188(1):77-85. 19. Jongen-Lavrencic M, Grob T, Hanekamp D, et al. Molecular minimal residual disease in acute myeloid leukemia. N Engl J Med. 2018;378(13):1189-1199. 20. Hourigan CS, Dillon LW, Gui G, et al. Impact of conditioning intensity of allogeneic transplantation for acute myeloid leukemia with genomic evi dence of residual disease. J Clin Oncol. 2020;38(12):1273-1283. 21. Patkar N, Kakirde C, Shaikh AF, et al. Clinical impact of panel-based error-corrected next generation sequencing versus flow cytometry to detect mea sur able resid ual dis ease (MRD) in acute mye loid leu ke mia (AML). Leukemia. 2021;35(5):1392-1404. 22. Dillon LW, Hayati S, Roloff GW, et al. Targeted RNA-sequencing for the quantification of measurable residual disease in acute myeloid leukemia. Haematologica. 2019;104(2):297-304. 23. Bottomly D, Long N, Schultz AR, et al. Integrative anal y sis of drug response and clinical outcome in acute myeloid leukemia. Cancer Cell. 2022;40(8):850-864.e9864e9. 24. Dillon LW, Ghannam J, Nosiri C, et al. Personalized single-cell proteog enomics to distinguish acute myeloid leukemia from non-malignant clonal hematopoiesis. Blood Cancer Discov. 2021;2(4):319-325. 25. Miles LA, Bowman RL, Merlinsky TR, et al. Single-cell mutation analysis of clonal evolution in myeloid malignancies. Nature. 2020;587(7834):477-482. 26. Pratz KW, Jonas BA, Pullarkat V, et al. Measurable residual disease response and prognosis in treatment-naïve acute myeloid leukemia with venetoclax and azacitidine. J Clin Oncol. 2022;40(8):855-865. 27. Roboz GJ, Ravandi F, Wei AH, et al. Oral azacitidine prolongs survival of patients with AML in remission independently of measurable residual dis ease status. Blood. 2022;139(14):2145-2155. 28. Maiti A, DiNardo CD, Wang SA, et al. Prognostic value of measurable resid ual disease after venetoclax and decitabine in acute myeloid leukemia. Blood Adv. 2021;5(7):1876-1883. 29. Platzbecker U, Middeke JM, Sockel K, et al. Measurable residual diseaseguided treat ment with azacitidine to pre vent haematological relapse in patients with myelodysplastic syn drome and acute mye loid leu kae mia (RELAZA2): an open-label, multicentre, phase 2 trial. Lancet Oncol. 2018;19(12):1668-1679. 30. Gilleece MH, Labopin M, Yakoub-Agha I, et al. Measurable residual dis ease, conditioning regimen intensity, and age predict outcome of allo geneic hematopoietic cell transplantation for acute myeloid leukemia in first remission: a registry analysis of 2292 patients by the Acute Leukemia Working Party European Society of Blood and Marrow Transplantation. Am J Hematol. 2018;93(9):1142-1152. 31. Heuser M, Heida B, Büttner K, et al. Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations. Blood Adv. 2021;5(9):2294-2304. 32. Paras G, Morsink LM, Othus M, et al. Conditioning intensity and peritrans plant flow cytometric MRD dynamics in adult AML. Blood. 2022;139(11):16941706. 33. Craddock C, Jackson A, Loke J, et al. Augmented reduced-intensity regi men does not improve postallogeneic transplant outcomes in acute mye loid leukemia. J Clin Oncol. 2021;39(7):768-778. 34. Venditti A, Piciocchi A, Candoni A, et al. GIMEMA AML1310 trial of riskadapted, MRD-directed therapy for young adults with newly diagnosed acute myeloid leukemia. Blood. 2019;134(12):935-945. 35. Dillon R, Hills R, Freeman S, et al. Molecular MRD status and outcome after transplantation in NPM1-mutated AML. Blood. 2020;135(9):680-688. 36. Kapp-Schwoerer S, Weber D, Corbacioglu A, et al. Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial. Blood. 2020;136(26):3041-3050. 37. Thol F, Gabdoulline R, Liebich A, et al. Measurable residual disease mon itoring by NGS before allogeneic hematopoietic cell transplantation in AML. Blood. 2018;132(16):1703-1713. 38. Hourigan CS, Dillon LW, Gui G, et al. Pre-MEASURE: multicenter evaluation of the prognostic significance of measurable residual disease testing prior to allogeneic transplantation for adult patients with AML in first remission. J Clin Oncol. 2022;40(suppl 16):7006. 39. US Food and Drug Administration. Hematologic malignancies: regulatory considerations for use of minimal residual disease in development of drug and biological products for treatment. https://www.fda.gov/regulatory -information/search-fda-guidance-documents/hematologic-malignancies -regulatory-considerations-use-minimal-residual-disease-development -drug-and. 40. Johnson and Johnson. MPAACT consortium unites industry and academia to establish measurable residual disease as a surrogate endpoint in acute myeloid leukemia drug development. https://www.jnj.com/mpaact -consortium-unites-industry-and-academia-to-establish-measurable -residual-disease-as-a-surrogate-endpoint-in-acute-myeloid-leukemia -drug-development. ACUTE MYELOID LEUKEMIA: IMPROVING OUTCOMES IN CHALLENGING SUBSETS Kieran D. Sahasrabudhe and Alice S. Mims The James Cancer Center, The Ohio State University, Columbus, OH The treatment landscape in acute myeloid leukemia (AML) is rapidly evolving, with multiple new therapies approved in recent years. However, the prognosis for patients with high-risk genetic subsets of AML remains poor, and the development of more effective treatment options for these patients is ongoing. Three of these high-risk AML patient subsets include TP53-mutated AML, FLT3-internal tandem duplication (ITD)-mutated AML, and AML harboring rearrangements affecting the KMT2A locus (KMT2A-r AML). The prognosis for TP53-mutated AML remains poor with both intensive and targeted regimens, including those incorporating the BCL-2 inhibitor, venetoclax. Allogeneic hematopoietic stem cell transplantation is the only potentially curative therapy for these patients, but posttransplant relapse rates remain high. Patients with FLT3-ITD-mutated AML continue to have suboptimal outcomes with standard therapies and experience high rates of relapse following transplant. KMT2A-r AML is also associated with poor outcomes with current treatment approaches, and effective standards of care are lacking for patients with relapsed/refractory disease. This article discusses current treatment approaches, along with the investigational agents being explored for the treatment of these 3 AML subsets, focusing primarily on agents that are further along in development. LEARNING OBJECTIVES • Recognize the highrisk subsets of TP53mutated, FLT3ITD mutated, and KMT2Arearranged AML, in which better treatment options are needed • Understand that patients with TP53mutated AML have poor outcomes with current standard therapies and should be treated on clinical trials when possible • Review the novel FLT3 inhibitors being studied in combination with many other agents in patients with FLT3 mutated AML • Learn about the novel inhibitors of the meninKMT2A interaction that have demonstrated promise in the treatment of KMT2Arearranged AML Current approaches CLINICAL CASE 1 A 60yearold man with a history of stage III colon can cer treated with surgery and adjuvant chemotherapy 8 years ago develops pancytopenia with circulating blasts. A bone marrow biopsy reveals acute myeloid leukemia (AML) with 30% myeloid blasts. Cytogenetic analysis shows a complex karyotype including monosomy 7. A next generation sequencing panel reveals a pathogenic TP53 mutation with a variant allele frequency of 30%. He is oth erwise feeling well without any functional limitations and has no other notable comorbidities. The TP53 protein is a transcription factor and tumor sup pressor that regulates many target genes with diverse functions. The mechanism of action of TP53 along with the therapeutic agents being investigated in TP53mutated AML is shown in Figure 1. TP53 mutations occur in 5% to 10% of de novo AML and have a higher incidence in therapyrelated AML (tAML), at 20% to 35%. TP53mutated AML is also often associated with a complex karyotype.1,2 Most AML cases har boring TP53 derangements involve biallelic inactivation of the gene. TP53 derangements caused by mutations, dele tions, and other disruptions of the 17p13 locus are associated with a very poor prognosis in AML with current approaches. Highrisk AML novel approaches: TP53, KMT2A, FLT3 | 15 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 Novel investigational approaches for high-risk genetic subsets of AML: TP53, KMT2A, FLT3 For patients treated with intensive chemotherapy, complete remission (CR) rates are less than 30%, and median overall sur vival (OS) is less than 1 year.1,3 For patients who respond to induc tion therapy, subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only potentially curative treatment. However, posttransplant outcomes also remain poor, with a median posttransplant OS of less than 1 year and a 2-year OS rate of less than 30%.4,5 There has been interest in less intensive treatments, includ ing hypomethylating agents (HMAs), in this patient population given the poor prognosis associated with intensive chemother apy. One single-arm study found that 10-day decitabine therapy was associated with favorable response rates in TP53-mutated AML.6 However, this study included only 21 patients with TP53 mutations. Other studies have found poor OS of less than 1 year in patients with TP53 mutations treated with azacitidine, 5-day decitabine, and 10-day decitabine.7,8 One of these was a random ized study involving 71 patients, 30% of whom had TP53 muta tions, that compared 5-day vs 10-day decitabine. Among patients with TP53 mutations, there was no difference in response rates with 5-day vs 10-day decitabine. Median OS also did not differ 16 | Hematology 2022 | ASH Education Program significantly and was less than 6 months in both groups, indicat ing no advantage of 10-day over 5-day decitabine. The addition of venetoclax, a BCL-2 inhibitor, to HMA ther apy has, unfortunately, not shown the improvement in patient outcomes to the level seen in other genetic subtypes of AML. Among patients with TP53-mutated disease treated in the phase 3 VIALE-A study, the com bi na tion of azacitidine-venetoclax yielded an encour ag ing CR with an incom plete hema to logic recov ery (CRi) rate of 55% (95% CI, 38.3-71.4) com pared to 0% in patients treated with azacitidine plus placebo (P<.001).9 However, a retrospective review of patients with TP53-mutated AML and poor-risk cytogenetics treated both in VIALE-A and the phase 1b study preceding VIALE-A found that the combination of venetoclax plus an HMA did not translate into improved duration of response or OS compared to an HMA alone.10 Another study of 10-day decitabine plus venetoclax in patients with newly diagnosed AML found that OS was significantly worse in TP53mutated vs wild-type patients, with a median OS of less than 6 months in patients with TP53 mutations.11 These results further indicate the lack of improvement with 10-day decitabine with or without venetoclax in this patient population. The overall poor Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 Figure 1. Mechanism of action of TP53 and agents under investigation for TP53-mutated AML. (A) Mechanism of action of TP53: cel lular stress and DNA damage activate the TP53 protein, which leads to downstream cell cycle arrest, DNA repair, and apoptosis. Cells containing mutations affecting the TP53 protein are not able to respond to stress and DNA damage appropriately, which increases the risk of neoplastic transformation. (B) Mechanism of action of magrolimab: the binding of CD47 on tumor cells to SIRPα on macro phages inhibits phagocytosis of tumor cells. Magrolimab is a monoclonal antibody that binds to CD47 and prevents it from binding to SIRPα, which thereby facilitates tumor cell phagocytosis. (C) Mechanism of action of APR-246: APR-246 is converted to MQ under physiological conditions. MQ binds to mutated TP53 and facilitates thermodynamic stabilization of the protein, thereby shifting equi librium to the active form of TP53. APR-246 also exhibits TP53-independent activity through glutathione depletion, which increases lipid peroxidases and other reactive oxygen species, thereby promoting cell death through ferroptosis (not depicted). Created with BioRender.com. outcomes with the current available standards of care highlight the need to develop additional novel therapies beyond veneto clax to improve survival and long-term outcomes in patients with TP53-mutated AML. Targeting CD47 in TP53-mutated AML TP53 stabilization in TP53-mutated AML APR-246 (epranetapopt) acts as a TP53 stabilizer and is a prodrug that converts to methylene quinuclidinone (MQ ) under physio logical conditions. MQ binds to cysteine residues in mutant TP53, which leads to thermodynamic stabilization of the protein and shifts equilibrium toward the functional conformation (Figure 1C).15 APR-246 has also been shown to deplete glutathione. Glutathione depletion increases lipid peroxidases and other reactive oxygen species, which increases susceptibility to ferroptosis.16 Ferropto sis induction has been shown to be an important mechanism of the early leukemic cell death induced by APR-246 regardless of TP53 mutation status.17 A phase 1b/2 study of APR-246–azacitidine in patients with TP53-mutated myelodysplastic syndromes (MDS) or oligoblastic AML (20%-30% blasts) found an overall response rate (ORR) of 71%, with 44% of patients achieving CR.18 Despite the promising results, a subsequent phase 3 trial (NCT03745716) failed to meet its primary end point of a superior CR rate with APR-246–azacitidine compared to azacitidine alone in patients with TP53-mutated MDS. However, a subsequent phase 2 study of APR-246–azacitidine as maintenance therapy following allo-HSCT in patients with TP53-mutated AML or MDS showed promising results compared to historical data, with a median relapse-free survival of 368 days and a median OS of 586 days.19 Another phase 1 study of the triplet combination of APR-246–venetoclax– azacitidine for the pri mary treat ment of TP53-mutated AML showed a CR rate of 37% and a CR/CRi rate of 53%, meeting the Simon’s 2-stage efficacy criteria.20 The patient presented here is a fit 60-year-old man with TP53mutated, therapy-related AML and minimal comorbidities. An up-front discussion regarding the limited treatment options and poor prognosis is very important for shared decision-making that aligns with the patient’s treatment goals. If possible, he should be referred for a clinical trial focusing on TP53-mutated AML. A summary of our approach for the treatment of newly diagnosed TP53-mutated AML is presented in Figure 2. CLINICAL CASE 2 A 50-year-old man with no major comorbidities and good per formance status presents to discuss treatment options for AML diagnosed in the context of fatigue and circulating blasts. A bone mar row biopsy dem on strates 80% blasts. Polymerase chain reaction with capillary electrophoresis for FLT3 mutations detects the presence of an FLT3 internal tandem duplication (ITD) mutation. The karyotype is normal, and no other muta tions are detected on next generation sequencing. Background Mutations in the FMS-like tyrosine kinase 3 (FLT3) protein are present in approximately 30% of newly diagnosed AML cases. These mutations are classified as ITD mutations affecting the juxtamembrane domain or point muta tions in the tyro sine kinase domain (TKD mutations). FLT3-ITD mutations have his torically been established as a poor prognostic factor.21 FLT3TKD mutations have not demonstrated a consistent prognostic impact.22 The 2017 European LeukemiaNet prognostic classifi cation system separated FLT3-ITD-mutated AML into different categories based on the ITD allelic ratio and the presence of a concurrent NPM1 mutation.23 However, the updated 2022 ELN classification includes all FLT3-ITD-mutated AML in the intermediate risk category.24 The current standard of care for most patients with FLT3-ITD mutations who are candidates is to undergo allo-HSCT in first CR. However, patients with FLT3ITD mutations are also at increased risk of relapse following transplant.25 Recent efforts in FLT3-mutated AML have focused on developing novel FLT3 inhibitors, combining FLT3 inhibi tors with other therapies to overcome therapeutic resistance, expanding treatment options for patients with relapsed/refrac tory (R/R) disease, and studying posttransplant maintenance therapies to reduce the risk of posttransplant relapse. The path ogenesis of FLT3-mutated AML and the mechanism of action of FLT3 inhibitors is shown in Figure 3A. Current approaches The current standard frontline therapy for patients with newly diagnosed FLT3-mutated AML who are eligible for intensive che motherapy is induction therapy with midostaurin combined with traditional “7+3” followed by consolidation with midostaurincytarabine. Midostaurin is a first-generation type 1 FLT3 inhib i tor with activ ity against both ITD- and TKD-mutated recep tors. This approach was informed by the phase 3 RATIFY trial in which midostaurin outperformed pla cebo when com bined High-risk AML novel approaches: TP53, KMT2A, FLT3 | 17 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 The CD47 pro tein is overexpressed in mye loid malig nan cies, including in AML leukemic stem cells. The CD47 protein acts as a “do not eat me” signal that inhibits phagocytosis of tumor cells by binding to the signal regulatory protein alpha on mac rophages. In preclinical studies, CD47 blockade overcomes this inhibition and stimulates tumor cell killing by macrophages (Figure 1B).12 A phase 1b study combining the CD47 antibody mag rolimab with azacitidine for the treatment of patients with newly diagnosed AML ineligible for intensive chemotherapy showed promising efficacy in both TP53-mutated and TP53-wild-type patients. The objective response rate was 65% for allcomers and 71% for TP53-mutated patients. Median OS was 18.9 months for TP53-wild-type patients and 12.9 months for TP53-mutated patients.13 A triplet combination of magrolimab-azacitidinevenetoclax was subsequently studied in a phase 1b/2 clinical trial. In patients with newly diagnosed AML, the CR/CRi rate was 94% for allpatients and 100% for patients with TP53-mutated dis ease.14 The most common grade 3/4 adverse events were pneu monia, febrile neutropenia, hyperbilirubinemia, elevated alanine aminotransferase, and skin infection. CD47 is expressed on red blood cells, and it is common to see a drop in hemoglobin, par ticularly early in the course of treatment. The ongoing phase 3 trials Enhance-2 (NCT04778397) and Enhance-3 (NCT05079230) are further addressing the role of magrolimab in TP53-mutated and TP53-wild-type disease, respectively. Other CD47 antibod ies are currently in development in earlier-phase clinical trials. CLINICAL CASE 1 (Continued) with chemotherapy in patients aged 18 to 59 (hazard ratio for death, 0.78; one-sided P=.009).26 Though the RATIFY study only included patients aged 18 to 59, there is no age limitation on the US Food and Drug Administration’s approval. For patients who are ineligible for intensive chemotherapy, the combination of venetoclax plus HMA therapy can be given front line. The com pos ite CR rate was 72.4% in patients with FLT3-mutated AML treated with frontline venetoclax-azacitidine on the VIALE-A trial.9 The cur rent stan dard of care for patients with R/R FLT3mutated AML is monotherapy with the second-generation type 1 FLT3 inhibitor gilteritinib. This practice was based on results of the phase 3 Admiral trial, in which gilteritinib was found to be supe rior to standard of care chemotherapy in this setting (median OS, 9.3 months vs 5.6 months; hazard ratio, 0.64; 95% CI, 0.49-0.83; P<.001).27 For patients with FLT3-ITD-mutated AML who undergo allo-HSCT, sorafenib (a type 2 FLT3 inhibitor active against ITDmutated but not TKD-mutated AML) is sometimes given off-label 18 | Hematology 2022 | ASH Education Program as posttransplant maintenance therapy based on the results of 2 clinical trials, one of which was placebo controlled.28,29 Investigational approaches Intensive chemotherapy candidates Several clin i cal tri als are ongo ing involv ing gilteritinib in patients with FLT3-mutated AML, many of which combine gil teritinib with intensive chemotherapy. A phase 1 study com bining gilteritinib with 7 + 3 was well tolerated and associated with a FLT3 mutation clearance rate of 70%.30 Additional FLT3 inhibitors are also under investigation. Quizartinib is a secondgeneration type 2 inhibitor that has been compared to placebo in combination with 7 + 3 in patients with newly diagnosed, FLT3ITD-mutated AML in the Quantum First trial (NCT02668653). At a median follow-up of 39.2 months, OS was significantly lon ger in the quizartinib arm (31.9 months) vs the pla cebo arm (15.1 months; haz ard ratio, 0.776; 95% CI, 0.615-0.979; 2-sided P = .0324). CR/CRi rates were 71.6% in the quizartinib Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 Figure 2. Treatment approach for newly diagnosed TP53-mutated AML. # Many clinicians prefer less intensive regimens to reduce toxicity, even in patients who are eligible, given the poor outcomes associ ated with intensive chemotherapy. Robust data supporting this practice are lacking. * Adding venetoclax to the treatment regimen may improve the likelihood of response, but mounting data suggest that this is unlikely to improve survival. ^ Even though allo-HSCT is the only potentially curative therapy, posttransplant outcomes remain poor. Consequently, the risk/ benefit assessment ends up not favoring allo-HSCT for many patients. Created with BioRender.com. arm vs 64.9% in the placebo arm.31 It is notable that this trial included patients aged 18 to 75 compared to the RATIFY trial, which had an upper age cutoff of 59. Crenolanib is a secondgeneration, type 1 FLT3 inhibitor that has also demonstrated prom is ing results in early-phase tri als.32,33 FLT3 inhibitors are also being inves ti gated as main te nance ther apy post trans plant for patients under go ing allo-HSCT. Posttrans plant midostaurin was found to be asso ci ated with supe rior event-free sur vival com pared to a his toric con trol in 1 study.34 Another study found that posttransplant midostau rin improved relapse-free sur vival com pared to stan dard of care only when FLT3 phos phor y la tion was inhibited to less than 70% of base line.35 Gilteritinib is also being com pared to placebo as maintenance therapy following allo-HSCT through the Blood and Marrow Transplant Clinical Trials Network Pro tocol 1506 (NCT02997202) and fol low ing che mo ther apy in patients who are not undergoing allo-HSCT (NCT02927262). Nonintensive chemotherapy candidates For those patients who are not candidates for intensive chemo therapy, gilteritinib is also being studied in combination with HMA therapy and other targeted therapies. The recently com pleted phase 3 LACEWING trial compared the combination of gilteritinib-azacitidine to azacitidine alone in patients with newly diagnosed FLT3-mutated AML who were ineligible for intensive chemotherapy. The combination arm yielded superior response rates but not superior OS, and the study therefore did not meet its primary end point.36 There is evidence that BCL-2 upregulation may play a role in resistance to FLT3 inhibition and that adding venetoclax may help to overcome this.37 A phase 1b study of gilteritinib-venetoclax in patients with R/R disease found a favorable ORR of 90% in patients with FLT3-mutated disease.38 An ongo ing phase 1/2 study (NCT04140487) of gilteritinib-venetoclax-azacitidine found an ORR of 67% with a median OS of 10.5 months at a median follow-up of 9.9 months in patients with R/R disease. The ORR was 100% in newly diagnosed patients ineligible for chemother apy, with median OS not reached at a median follow-up of 3.8 months.39 Another study of venetoclax-decitabine-FLT3 inhibitor of investigator’s choice found an ORR of 62% with 2-year OS of 29% in patients with R/R disease and an ORR of 92% with 2-year OS of 80% in patients over 60 years of age with newly diagnosed disease.40 CLINICAL CASE 2 (Continued) The patient presented in this case has AML harboring an FLT3ITD mutation and is an intensive induction candidate. Again, it is impor tant to deter mine the patient’s under ly ing treat ment goals through shared deci sion-mak ing. A sum mary of our approach and investigational strategies for the treatment of FLT3-mutated AML, both newly diagnosed and R/R, is pre sented in Figure 4. High-risk AML novel approaches: TP53, KMT2A, FLT3 | 19 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 Figure 3. Pathogenesis of FLT3-mutated AML and KMT2A-r AML. (A) FLT3-ITD and FLT3-TKD mutations lead to increased downstream signaling from the FLT3 receptor, which thereby promotes increased cell growth, proliferation, and survival. FLT3 inhibitors block this downstream signaling. (B) Binding of the KMT2A protein to menin allows the KMT2A/fusion partner protein to facilitate down stream transcription of HOX and other developmental proteins that contribute to leukemic pathogenesis. Inhibitors of the meninKMT2A interaction inhibit this transcription. AKT, Akt serine/threonine protein kinases; MAPK, mitogen-activated protein kinases; PI3K, phosphoinositide-3 kinases; STAT5, signal transducer and activator of transcription 5. Created with BioRender.com. CLINICAL CASE 3 A 65-year-old woman without major comorbidities and normal performance status is diagnosed with AML harboring a cytoge netic translocation involving 11q23. She is treated with frontline venetoclax-azacitidine and achieves CR. However, 4 months later she is noted to have relapsed AML still harboring the 11q23 translocation. Background The KMT2A gene (previously known as MLL1) codes for the histone-lysine N-methyltransferase 2A protein and is located on chromosome 11q23. Translocations involving the KMT2A locus occur in approximately 3% of AML cases and have a negative impact on prognosis.23,41 Translocations involving the KMT2A locus result in the expression of fusion proteins that enable an aberrant transcription program characterized by the overexpression of HOX and other developmental genes.42,43 The binding of these fusion proteins to menin, a scaffold protein, leads to the nuclear localization of the fusion 20 | Hematology 2022 | ASH Education Program proteins, thereby facilitating the aberrant transcription that is central to the pathogenesis of KMT2A-rearranged (KMT2A-r) AML.44,45 The interaction of menin with the wild-type KMT2A protein has also been shown to play a role in the pathogenesis of NPM1-mutated AML.46 Targeting menin inhibition in KMT2A-rearranged AML SNDX-5613 is an oral small-molecule inhibitor that binds with high affinity to the KMT2A-binding pocket of menin. This inhib its the interaction between menin and KMT2A, which thereby inhibits oncogenic expression and cellular proliferation. SNDX5613 has dem on strated activ ity against KMT2A-r and NPM1mutated leukemia in preclinical and xenograft models. Augment-1 was the first-in-human phase 1 study involving SNDX-5613 and included patients with R/R KMT2A-r and NPM1-mutated acute leukemias (both acute lymphocytic leukemia and AML). This study demonstrated promising response rates, with a composite CR of 44% and a median duration of response of 5.2 months. The only dose-limiting toxicity was grade 3 QTc prolongation, which occurred in 8% of patients, allof whom were clinically asymp Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 Figure 4. Treatment recommendations for FLT3-Mutated AML. Color code: Blue background with white text: standard of care; green background with red text: investigational approaches. Aza, azacitidine; ELN, European Leukemia Network; Gilt, gilteritinib; IC, inten sive chemotherapy; LDAC, low dose cytarabine; ND, newly diagnosed; R/R, relapsed/refractory; Ven, venetoclax. *Data exist supporting allo-HSCT for all patients with FLT3-ITD-mutated AML, even if they are classified as favorable risk by the 2017 ELN guidelines based on a low ITD allelic ratio and concurrent NPM1 mutation. The updated 2022 ELN guidelines classify all FLT3-ITD-mutated AML as intermediate risk. **Sorafenib maintenance after allo-HSCT is approved per National Comprehensive Cancer Network guidelines but not commonly used in clinical practice. Sorafenib is only effective against FLT3-ITD mutations, not FLT3-TKD mutations. Created with BioRender.com. CLINICAL CASE 3 (Cont inu ed) Patients with newly diagnosed KMT2A-r AML who are candidates should undergo allo-HSCT if they achieve remission with front line therapy. There is no current standard of care for R/R disease, but investigational inhibitors of the menin-KMT2A interaction represent promise in this setting. Conclusions TP53 mutations, FLT3-ITD mutations, and KMT2A rearrangements represent high-risk genetic features in AML, and improved outcomes are needed in these AML patient subsets. Multiple novel agents and therapeutic combinations are being studied in these AML subsets that have the potential to foster these needed improvements. This is an exciting time in AML research, with hope for better outcomes in patients with high-risk disease moving forward. Conflict-of-interest disclosure Kieran D. Sahasrabudhe: no com pet ing finan cial inter ests to declare. Alice S. Mims: scientific advisory committee: Syndax Pharmaceu ticals, AbbVie, Genentech, BMS, Astellas, Servier Pharmaceuti cals, Ryvu Therapeutics; data and safety monitoring committee: Jazz Pharmaceuticals, Daiichi Saynko; senior medical director: Leukemia and Lymphoma Society Beat AML study. Off-label drug use Kieran D. Sahasrabudhe: the off-label use of sorafenib is discussed. Alice S. Mims: the off-label use of sorafenib is discussed. Correspondence Alice S. Mims, The Ohio State University, 1800 Cannon Dr, 1120G Lincoln Tower, Columbus, OH 43210; e-mail: alice.mims@osumc .edu. References 1. Bowen D, Groves MJ, Burnett AK, et al. TP53 gene mutation is frequent in patients with acute myeloid leukemia and complex karyotype, and is asso ciated with very poor prognosis. Leukemia. 2009;23(1):203-206. 2. Ok CY, Patel KP, Garcia-Manero G, et al. Mutational profiling of therapyrelated myelodysplastic syndromes and acute myeloid leukemia by next generation sequencing, a comparison with de novo diseases. Leuk Res. 2015;39(3):348-354. 3. Grossmann V, Schnittger S, Kohlmann A, et al. A novel hierarchical prog nos tic model of AML solely based on molec u lar muta tions. Blood. 2012;120(15):2963-2972. 4. Poiré X, Labopin M, Maertens J, et al. Allogeneic stem cell transplantation in adult patients with acute myeloid leukaemia and 17p abnormalities in first complete remission: a study from the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT). J Hematol Oncol. 2017;10(1):20. 5. Ciurea SO, Chilkulwar A, Saliba RM, et al. Prognostic factors influencing survival after allogeneic transplantation for AML/MDS patients with TP53 mutations. Blood. 2018;131(26):2989-2992. 6. Welch JS, Petti AA, Miller CA, et al. TP53 and decitabine in acute myeloid leukemia and myelodysplastic syndromes. N Engl J Med. 2016;375(21):20232036. 7. Bories P, Prade N, Lagarde S, et al. Impact of TP53 muta tions in acute mye loid leu ke mia patients treated with azacitidine. PLoS One. 2020;15(10):e0238795. 8. Short NJ, Kantarjian HM, Loghavi S, et al. Treatment with a 5-day versus a 10-day schedule of decitabine in older patients with newly diagnosed acute myeloid leukaemia: a randomised phase 2 trial. Lancet Haematol. 2019;6(1):e29-e37. 9. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in pre viously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617629. 10. Pollyea DA, Pratz KW, Wei AH, et al. Outcomes in patients with poor-risk cytogenetics with or without TP53 mutations treated with venetoclax combined with hypomethylating agents. Blood. 2021;138(suppl 1):224. 11. Kim K, Maiti A, Loghavi S, et al. Outcomes of TP53-mutant acute mye loid leukemia with decitabine and venetoclax. Cancer. 2021;127(20):37723781. 12. Chao MP, Takimoto CH, Feng DD, et al. Therapeutic targeting of the mac rophage immune checkpoint CD47 in myeloid malignancies. Front Oncol. 2019;9(22 January):1380. 13. Sallman D, Asch A, Kambhampati S, et al. AML-196: the first-in-class antiCD47 antibody magrolimab in combination with azacitidine is well tol erated and effective in AML patients: phase 1b results. Clin Lymphoma Myeloma Leuk. 2021;21(suppl 1):s290. 14. Daver N, Konopleva M. Phase I/II study of azacitidine (AZA) with venetoclax (VEN) and magrolimab (Magro) in patients (Pts) with newly diagnosed old er/unfit or high-risk acute myeloid leukemia (AML) and relapsed/refractory (R/R) AML. Blood. 2021;138(suppl 1):371. 15. Lambert JM, Gorzov P, Veprintsev DB, et al. PRIMA-1 reactivates mutant p53 by covalent binding to the core domain. Cancer Cell. 2009;15(5):376388. 16. Liu DS, Duong CP, Haupt S, et al. Inhibiting the system xC-/glutathione axis selectively targets cancers with mutant-p53 accumulation. Nat Commun. 2017;8(27 March):14844. 17. Birsen R, Larrue C, Decroocq J, et al. APR-246 induces early cell death by ferroptosis in acute myeloid leukemia. Haematologica. 2022;107(2):403416. 18. Sallman DA, DeZern AE, Garcia-Manero G, et al. Eprenetapopt (APR-246) and azacitidine in TP53-mutant myelodysplastic syndromes. J Clin Oncol. 2021;39(14):1584-1594. 19. Mishra A, Tamari R, Byrne M, et al. Phase II trial of eprenetapopt (APR-246) in combination with azacitidine (AZA) as maintenance therapy for TP53 mutated acute mye loid leu ke mia (AML) or myelodysplastic syn dromes (MDS) following allogeneic hematopoietic cell transplantation (HCT). Paper presented at: BMT Tandem Meetings; 23-26 April 2022; Salt Lake City, UT; Abstract 39. High-risk AML novel approaches: TP53, KMT2A, FLT3 | 21 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 tomatic. Other common treatment-related adverse events were nausea (22%), vomiting (17%), differentiation syndrome (DS; 15%), and diarrhea (11%).47 A follow-up phase 2 study is ongo ing. SNDX-5613 is also being studied in combination with inten sive chemotherapy in a phase 1 trial in patients with R/R AML or acute lymphocytic leukemia harboring a KMT2A rearrangement or NPM1 mutation (NCT05326516). An all-oral triplet regimen of SNDX-5613–ASTX727–venetoclax is being studied in a phase 1/2 clinical trial in patients with R/R AML or mixed phenotype acute leukemia (NCT05360160). The Beat AML Master Clinical trial is also studying the combination of SNDX-5613–azacitidine– venetoclax in patients older than 60 with newly diag nosed KMT2A-r or NPM1-mutated AML (NCT03013998). KO-539 and JNJ-75276617 are 2 other novel inhibitors of the menin-KMT2A interaction that are being investigated in earlyphase clin i cal tri als in patients with R/R AML (NCT04067336, NCT04811560). DS is an important adverse event for clinicians to be aware of and, if unrecognized, can be fatal. DS was typically low grade in patients treated on Augment-1 but did lead to the US Food and Drug Administration placing a temporary hold on KO-539, which has since been lifted. Signs and symptoms of DS include unexplained fever, weight gain, edema, pleural or peri cardial effusions, radiographic opacities, dyspnea, hypotension, renal dysfunction, rash, and/or a rapidly increasing white blood cell count. Treatment involves the prompt initiation of steroids and following the investigator brochure guidelines regarding continuation of the investigational agent. The pathogenesis of KMT2A-r AML is shown in Figure 3B. 22 | Hematology 2022 | ASH Education Program maintenance therapy in acute myeloid leukemia with FLT3-ITD. Blood. 2019;133(8):840-851. 35. Maziarz RT, Levis M, Patnaik MM, et al. Midostaurin after allogeneic stem cell transplant in patients with FLT3-internal tandem duplication-positive acute myeloid leukemia. Bone Marrow Transplant. 2021;56(5):1180-1189. 36. Wang ES, Montesinos P. Phase 3, open-label, randomized study of gilter itinib and azacitidine vs azacitidine for newly diag nosed FLT3-mutated acute myeloid leukemia in patients ineligible for intensive induction che motherapy. Blood. 2021;138(suppl 1):700. 37. Yamatani K, Tabe Y, Saito K, et al. Upregulation of Bcl-2 confers resistance to FLT3 inhibition in FLT3-ITD AML with secondary acquired mutations. Blood. 2018;132(suppl 1):3944. 38. Perl AE, Daver NG, Pratz KW, et al. Venetoclax in combination with gilteri tinib in patients with relapsed/refractory acute myeloid leukemia: a phase 1b study. Blood. 2019;134(suppl 1):3910. 39. Short NJ, DiNardo CD, Daver N, et al. A triplet combination of azacitidine, venetoclax and gilteritinib for patients with FLT3-mutated acute myeloid leukemia: results from a phase I/II study. Blood. 2021;138(suppl 1):696. 40. Maiti A, DiNardo CD, Daver NG, et al. Triplet therapy with venetoclax, FLT3 inhibitor and decitabine for FLT3-mutated acute myeloid leukemia. Blood Cancer J. 2021;11(2):25. 41. Schoch C, Schnittger S, Klaus M, Kern W, Hiddemann W, Haferlach T. AML with 11q23/MLL abnormalities as defined by the WHO classification: inci dence, partner chromosomes, FAB subtype, age distribution, and prog nostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood. 2003;102(7):2395-2402. 42. Meyer C, Burmeister T, Gröger D, et al. The MLL recombinome of acute leu kemias in 2017. Leukemia. 2018;32(2):273-284. 43. Del Rizzo PA, Trievel RC. Substrate and product specificities of SET domain methyltransferases. Epigenetics. 2011;6(9):1059-1067. 44. Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O, Meyerson M, Cleary ML. The menin tumor suppressor protein is an essential onco genic cofactor for MLL-associated leukemogenesis. Cell. 2005;123(2):207218. 45. Caslini C, Yang Z, El-Osta M, Milne TA, Slany RK, Hess JL. Interaction of MLL amino terminal sequences with menin is required for transformation. Cancer Res. 2007;67(15):7275-7283. 46. Kühn MW, Song E, Feng Z, et al. Targeting chromatin regulators inhibits leukemogenic gene expression in NPM1 mutant leukemia. Cancer Discov. 2016;6(10):1166-1181. 47. Stein EM, Aldoss I, DiPersio JF, et al. Safety and efficacy of menin inhibition in patients (Pts) with MLL-rearranged and NPM1 mutant acute leukemia: a phase (Ph) 1, first-in-human study of SNDX-5613 (AUGMENT 101). Blood. 2021;138(suppl 1):699. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000325 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/15/2021947/15sahasrabudhe.pdf by guest on 09 December 2022 20. Garcia-Manero G, Goldberg AD, Winer ES, et al. Phase I and expansion study of eprenetapopt (APR-246) in combination with venetoclax (VEN) and azacitidine (AZA) in TP53-mutant acute mye loid leu ke mia (AML). Blood. 2021;138(suppl 1):3409. 21. Fröhling S, Schlenk RF, Breitruck J, et al; AML Study Group Ulm. Acute myeloid leukemia. Prognostic significance of activating FLT3 muta tions in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood. 2002;100(13):4372-4380. 22. Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a signifi cantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood. 2007;110(4):1262-1270. 23. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 24. Döhner H, Wei AH, Appelbaum FR, et al. Diagnosis and management of AML in adults: 2022 ELN recommendations from an international expert panel. Blood. 2022;140(12):1345-1377. 25. Brunet S, Labopin M, Esteve J, et al. Impact of FLT3 internal tandem dupli cation on the outcome of related and unrelated hematopoietic transplan tation for adult acute myeloid leukemia in first remission: a retrospective analysis. J Clin Oncol. 2012;30(7):735-741. 26. Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus chemother apy for acute mye loid leu ke mia with a FLT3 muta tion. N Engl J Med. 2017;377(5):454-464. 27. Perl AE, Martinelli G, Cortes JE, et al. Gilteritinib or chemotherapy for relapsed or refractory FLT3-mutated AML. N Engl J Med. 2019;381(18):1728-1740. 28. Burchert A, Bug G, Fritz LV, et al. Sorafenib main te nance after allo ge neic hematopoietic stem cell transplantation for acute myeloid leukemia with FLT3–internal tandem duplication mutation (SORMAIN). J Clin Oncol. 2020;38(26):2993-3002. 29. Xuan L, Wang Y, Huang F, et al. Sorafenib maintenance in patients with FLT3-ITD acute myeloid leukaemia undergoing allogeneic haematopoietic stem-cell transplantation: an open-label, multicentre, randomised phase 3 trial. Lancet Oncol. 2020;21(9):1201-1212. 30. Pratz KW, Cherry M, Altman JK, et al. A phase 1 study of gilteritinib in com bination with induction and consolidation chemotherapy in patients with newly diagnosed AML: final results. Blood. 2020;136(suppl 1):16-17. 31. Abstract Book for the 27th Congress of the European Hematology Associ ation. HemaSphere. 2022;6:1-4130. 32. Cortes JE, Kantarjian HM, Kadia TM, et al. Crenolanib besylate, a type I panFLT3 inhibitor, to demonstrate clinical activity in multiply relapsed FLT3-ITD and D835 AML. J Clin Oncol. 2016;34(suppl 15):7008. 33. Goldberg AD, Geyer MB, Kell J, et al. Clinical benefit of crenolanib, with or without salvage chemotherapy, in multiply relapsed, FLT3 mutant AML patients after prior treatment with gilteritinib. Blood. 2020;136(suppl 1):8-9. 34. Schlenk RF, Weber D, Fiedler W, et al; Ger man-Austrian AML Study Group. Midostaurin added to chemotherapy and continued single-agent ACUTE MYELOID LEUKEMIA: IMPROVING OUTCOMES IN CHALLENGING SUBSETS Optimizing outcomes in secondary AML Abramson Cancer Center, Hospital of University of Pennsylvania, Philadelphia, PA Acute myeloid leukemia (AML) secondary to antecedent hematologic disorder or prior therapeutics for cancer represent a diverse group of leukemias often associated with inferior outcomes. Conventional therapy with cytarabine-based chemotherapy has been the mainstay of care for the past 30 years with disappointing overall outcomes. Novel therapies, including liposomal cytarabine/daunorubicin, and venetoclax-based therapies have emerged as options in recent years based on studies showing improvement in outcomes over standard-of-care therapies. Despite these advances, mutations in TP53 are associated with inferior response to both therapies and represent an area of unmet clinical need. Novel strategies with immune-targeted therapies such as CD47 monoclonal antibodies appear active in early-phase studies, but randomized studies have yet to report outcomes leading to approval. Allogeneic transplant remains the only known curative therapy for many of these cases. Nonetheless, pretransplant high-risk molecular features of secondary AML are associated with inferior outcome despite transplantation. An optimal approach to secondary AML is yet to be determined. LEARNING OBJECTIVES • Secondary acute myeloid leukemia (AML) encompasses both therapy-related myeloid neoplasms and antecedent myelodysplastic syndrome and/or myeloproliferative neoplasm • Favorable risk secondary AML is rare but can be managed like de novo favorable risk disease • Allogeneic stem cell transplant remains the preferred option in first complete remission for intermediate- and unfavorable-risk disease • Venetoclax and hypomethylating agent combination may be used as a bridge to transplant or destination therapy • Emerging therapies may further expand treatment options for this historically older, frailer population with complex genetic changes and TP53 mutations Introduction Secondary acute myeloid leukemia (AML) has been historically divided into 2 categories based on known risks for development of disease from either (1) antecedent myeloid neoplasm such as myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) or (2) exposure to ionizing radiation and/or cytotoxic chemotherapy (Table 1). In population-based cohort series, secondary AML accounts for 20% to 30% of all AML cases.1,2 The previous World Health Organization (WHO) 2016 classification included the separate categories of AML with myelodysplastic changes (AML-MRC) and therapy-related myeloid neoplasms (tMNs), which were the formal categories of secondary AML.3 The WHO classification of tMN was subdivided into therapy-related acute myeloid leukemia and therapy-related myelodysplastic syndrome. The fact that both MDS and AML are included in the single category of “therapy-related myeloid neoplasm” emphasizes that regardless of precise blast count above or below 20%, similar biology, pathogenesis, and risk of rapid progression with poor prognosis guide the need for treatment and transplant evaluation for most patients. The latest editions of the WHO classification4 and the International Consensus Classification5 further emphasize disease biology and genetic features while softening the boundary between MDS and AML. The WHO classification now specifically eliminates blast cutoffs for most AML types with defining genetic alterations but retains a 20% blast cutoff to delineate MDS from AML in cases lacking such genetic alterations. AML-MRC has been updated to AML, myelodysplasia related (AML-MR). AML-MR harbors specific mutational or cytogenetic abnormalities and can no longer be defined by morphology alone. Either the presence of 1 or more molecular and cytogenetic abnormalities or a history of MDS/MPN is required for a diagnosis of AML-MR. Further complicating these distinctions, patients with aplastic anemia or inherited bone marrow failure syndromes may also develop AML, particularly if dominant clones emerge. Far more commonly, patients have had prior cytotoxic Dr Prakash Singh Shekhawat Optimizing outcomes in secondary AML | 23 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 Andrew Matthews and Keith W. Pratz Table 1. Summary of World Health Organization AML Classifications3 Category Key features De novo AML • AML with defining genetic abnormalities Defined balanced translocation/inversions and gene mutations (eg, mutated NPM1, biallelic CEBPA, RUNX1::RUNX1T1). Only AML with BCR::ABL1 fusion and AML with CEBPA mutation require ≥20% blast count in PB or BM. • AML defined by differentiation* ≥20% PB or BM blasts with further subcategories based on morphology, cytochemistry, and immunophenotype Secondary myeloid neoplasms ≥20% PB or BM blasts and prior therapy has been excluded with either specific cytogenetic abnor malities or somatic mutations.† This includes patients with known history of MDS or MDS/MPN. • AML, post–cytotoxic therapy (previously therapy-related myeloid neoplasms) ≥20% PB or BM blasts and patient-developed myeloid neoplasms following cytotoxic therapy • Myeloid neoplasms with germline predisposition Includes myeloid neoplasms with germline pathologic/likely pathologic mutations in CEPBA, DDX41, TP53, RUNX1, ANKRD26, ETV6, GATA2, SAMD9, SAMD9L, and BLM along with telomere biology disor ders, RASopathies, Down syndrome, and bone marrow failure syndromes. Phenotype of AML vs MDS with germline predisposition based on presence or absence of >20% PB or BM blasts, respectively. *AML not otherwise specified is no longer recognized as a category. †Cytogenetic abnormalities include 5q deletion or loss of 5q due to unbalanced translocation; monosomy 7, 7q deletion, or loss of 7q due to unbal anced translocation; 11q deletion; 12p deletion or loss of 12p due to unbalanced translocation; monosomy 13 or 13q deletion; 17p deletion or loss of 17p due to unbalanced translocation; isochromosome 17q; and idic(X)(q13). Molecular somatic mutations include SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2. BM, bone marrow; PB, peripheral blood. therapy or known MDS/MPN, or they can be classified into AMLMR based solely on the pres ence of myelodysplasia-related chromosomal or mutational findings. Meanwhile, tMN has been replaced by mye loid neo plasm post–cyto toxic ther apy, still requiring a documented history of chemotherapy treatment or large-field radiation therapy for an unrelated neoplasm. Within both categories of AML-MR and AML, post–cytotoxic therapy (AML-pCT), similar chromosomal and molecular abnor malities are found, suggesting a common final pathway. Secondary leukemia from antecedent MDS/MPN appears to share a typical evolutionary pathway, with several genes distinguishing secondary AML from de novo AML with high specificity.6 Secondary leukemias after cytotoxic therapy may evolve through a sim ilar MDS evolutionary pathway or acquire TP53 or other driver mutations directly from chemotherapy, radiation, or immu nosuppression. Common cytogenetic and molecular features reflect this variability, with 50% of patients with AML-pCT having poor-risk cytogenetics. The most frequent molecular aberration is TP53 (33%), and only 15% of patients with AML-pCT present with favorable-risk fusion genes (RUNX1::RUNX1T1, CBFB::MYH11, PML::RARA). Age-related increase in clonal hematopoiesis7 may predispose patients to develop secondary leukemia as an accre tion of mutations leads to myeloid neoplasms, especially under the selective pressure of chemotherapy.8 Therapy-related myeloid neoplasms may arise after a vari ety of treatments, with classic examples being alkylating agents leading to AML-pCT with antecedent MDS, with abnormalities of chromosome 5 and/or 7 or topoisomerase II inhibitors leading to KMT2A gene rearrangements at 11q23. Beyond recurring gene or chromosomal rearrangements, other therapies can lead to leuke mia by selecting preexisting clones resistant to chemotherapy, with recent novel therapy examples including poly (ADP-ribose) polymerase inhibitors and lenalidomide (see Figure 1).1,9 Outcomes in both AML-MR and AML-pCT appear sim i lar across genetic subsets. Rare subsets of favorable-risk treat 24 | Hematology 2022 | ASH Education Program ment-related disease such as AML with inv16 or t(8;21) and acute promyelocytic leukemia t(15;17) appear to have dimin ished overall survival compared with de novo AML. However, the impact of confounding from the antecedent malignancy or advanced age is often not examined in retrospective studies comparing outcomes in secondary AML to de novo AML. Clinically, diagnosis requires integration of clinical history, morpho logic changes, cytogenetics, and molecular analyses. Each of these components may change treatment strategies and ulti mate outcome. Herein we describe the genetic and molecular features associated with the secondary AMLs and approaches and outcomes of conventional and emerging therapies. CLINICAL CASE 1 A 55-year-old woman with a remote his tory of Bur kitt lym phoma presented for routine annual examination and was found on complete blood count to have pancytopenia. White blood cell count was 900/µL, hemoglobin was 7.4 g/dL, and plate let count was 101 000/µL. Bone marrow examination revealed 28% blasts by morphology, and karyotype was complex 43,XX, del(5)(q11.2q33),-13,add(14) (p11.2),add(17)(p11.2),dic(18;19)(p11;2; q13.1),psu dic(22;1)(q13;p13)[16]/45,XX,del(5),der(13;14) (q10;q10), add(17),add(20)(q13.1)[2],46,XX[2]. Molecular stud ies revealed TP53 mutation at R248Q with 62% variant allele frequency (VAF). Performance sta tus was excel lent, and she had nor mal endorgan function. Her lymphoma history was notable for 30 years of ongoing remission after multiagent chemotherapy in her early 20 s. She had an unknown quantity of anthracycline exposure, and records of the therapy were unavailable. Echocardiogram at time of diagnosis revealed normal ejection fraction without regional wall motion abnormalities. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 • AML, myelodysplasia related Dr Prakash Singh Shekhawat Optimizing outcomes in secondary AML | 25 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 Figure 1. Descriptive data by AML type. (A) Incidence of de novo AML, secondary AML (sAML), and therapy-related AML (tAML) in Denmark by year of diagnosis. Distribution of previous disease in (B) 203 patients with tAML and (C) 603 patients with sAML. Originally published in Østgård LSG, Medeiros BC, Sengeløv H, et al. Epidemiology and clinical significance of secondary and therapy-related acute myeloid leukemia: a national population-based cohort study. J Clin Oncol. 2015;33(31):3641-3649.1 This patient has myeloid neoplasm, post–cytotoxic therapy, and her blasts >20% put her in the acute myeloid leukemia cat egory. Cohorts of AML-pCT tend to contain more women than men as the most common primary malignancy is breast can cer followed by non-Hodgkin lym phoma.10 Despite the long latency from treatment to AML-pCT presentation, her complex karyotype and TP53 mutation are common features of AML-pCT. Multiple treatments are options in this case. Standard-of-care therapy with “7+3” (7 days of cytarabine and 3 days of anthracycline) would be the most common approach in this case, given a 33% to 55% overall response rate in secondary AML with intensive chemotherapy.1,11 However, the full molecular and kar yotypic pattern would predict for a low overall response rate. Based on karyotypic changes alone, she also meets criteria for AML, myelodysplasia related,3,5 and as such, therapy with lipo somal cytarabine and daunorubicin (CPX-351) could be consid ered.12 The overall response rates associated with secondary AML and TP53-mutant leu ke mia for both 7+3 and lipo so mal cytarabine/daunorubicin are summarized in Table 2. The longterm survival with either therapy is very limited and only found in patients who can proceed with allogeneic transplantation.13,14 The patient’s his tory of high-grade lym phoma and exten sive remote chemotherapy exposure would also complicate her tol erance of high-intensity chemotherapy. With limited knowledge about her anthracycline exposure, the choice of a regimen with anthracyclines becomes problematic even with normal ejection fraction and use of cardioprotection with dexrazoxane. There were lower-intensity options offering reasonable remis sion rates as a bridge to curative intent transplant. Azacitidine and venetoclax (aza/ven) has been associated with complete response (CR) and complete remission with incomplete blood count recov ery rate in AML with poor-risk cytogenetics and TP53 mutations of 41% in a recent review of older adults who were unfit for induction chemotherapy in phase 1b and phase 3 studies of aza/ven.15 Given no prospective comparative studies and a growing body of retro spective studies that show similar overall survival with venetoclax and hypomethylating agents com pared with more tra di tional intensive chemotherapy approaches, a patient-centered discus sion of putative risks and benefits is appropriate.16,17 This patient had a strong preference for outpatient-based therapy, and as such, we proceeded with this therapy. She achieved CR after 1 course and completed 5 cycles of aza/ven before proceeding to myeloablative allogeneic stem cell transplant 26 months ago with under 5% VAF TP53 mutation and normal karyotype at the time of transplant. She has ongoing remission and normal blood counts without active graft-vs-host disease. Data on transplant in this secondary AML subset reflect out comes predicted by pretransplant molecular and cytogenetic risks in de novo AML. Several stud ies sup port myeloablative conditioning over reduced intensity conditioning in improved relapse-free sur vival and equal or supe rior over all sur vival, although historically for TP53 mutation, the benefit has been less clear, especially in MDS.18 In addition, the presence of measurable residual disease (MRD), defined as detection of any of a subset of 13 commonly mutated genes on ultra-deep error-corrected sequenc ing of preconditioning blood, appears to con fer the highest risk of relapse on patients who receive a nonmyeloablative transplant.19 It is unknown if MRD-negative patients may forgo Table 2. Representative efficacy outcomes in AML subsets Regimen Response rates Overall survival All groups TP53 All groups TP53 7+3 (cytarabine and anthracycline)2,11,12,37 CR: 35%-71% CR/CRi: 40%-71% sAML subset: CR: 26%-52% CR/CRi: 33%-55% CR: 30%-34% CR/CRi: 40% sAML subset: 5-10 mo 5-6 mo CPX-351 (liposomal cytarabine and daunorubicin)12,16,37 sAML subset: CR: 7%-12% CR/CRi: 45%-48% CR: 29% CR/CRi: 29% sAML subset: 10-13 mo 4-6 mo Azacitidine and venetoclax16,17,21 CR: 40% CR/CRi: 65% sAML subset: CR/CRi: 60% CR: NA CR/CRi: 50%-55% 11-16 mo sAML subset: 11-16 mo 5-7 mo Azacitidine or decitabine monotherapy21,38 CR: 13%-24% CR/CRi: 18%-27% sAML subset: CR/CRi: 25% CR: 24%-40% CR/CRi: 0%-40% 6-11 mo sAML subset: 7-8 mo 2-7 mo Low-dose cytarabine (LoDAC)38-40 CR: 3%-24% CR/CRi: 5%-34% sAML subset: CR: 0% CR: 0%-11% CR/CRi: 0%-22% 4-5 mo sAML subset: 4 mo 2-3 mo Glasdegib and LoDAC41,42 CR: 18% CR/CRi: 24% sAML subset: CR: 24% CR: 24%* CR/CRi: NA 8-9 mo sAML subset: 9 mo 5-6 mo* CD47/SIRPα inhibitors with HMA (eg, magrolimab)35 CR: 44% CR/CRi: 56% CR: 48% CR/CRi: 67% 18.9 mo 12.9 mo TIM-3 inhibitors with HMA (eg, sabatolimab)43 NA CR: 25% CR/CRi: 30% NA NA Bispecific antibody therapies (eg, flotetuzumab) in R/R AML44 NA CR: 27% CR/CRi: 40% NA 4.5 mo Intensive regimens Experimental regimens Representative efficacy outcomes in subsets of acute myeloid leukemia with myelodysplasia-related changes, therapy-related myeloid neoplasm, and TP53. Early clinical trial data are presented for representative experimental regimens with listed agents. *Poor cytogenic risk group reported, no separate TP53 mutated subset data available. CRi, complete remission with incomplete count recovery; HMA, hypomethylating agent; NA, not available; R/R, relapsed refractory disease; sAML, secondary AML. myeloablative regimens.20 In this case, the achievement of remis sion allowed for transplantation to proceed. Despite the mod est response rate published with aza/ven, the durability of these responses is often limited to less than 6 months, highlighting the need for more effective and durable therapy in this subset.15,21 Our current approach favors stem cell transplant when possible, but for intermediate- or adverse-risk patients who are ineligible or borderline candidates for transplant, novel therapies are needed. CLINICAL CASE 2 A 52-year-old woman with a history of triple-negative breast cancer following adjuvant radiation therapy along with doxo rubicin, cyclophosphamide, and docetaxel was found to have 40% blasts on routine blood work 12 months after completion of chemotherapy. Bone marrow biopsy specimen confirmed 26 | Hematology 2022 | ASH Education Program AML with 73% blasts, and chromosome analysis revealed 48,XX, +8,+13,t(16;16) (p13.1;q22)[20]. Molecular stud ies revealed a NRAS G13D mutation with a VAF of 38%. Her lifetime anthracycline dose was 240 mg/m2. She has no evidence of breast cancer at the time of diagnosis of AML. Acute leukemia after exposure to ionizing radiation or chemo therapy rarely presents with otherwise favorable genetic markers such as fusions of RUNX1-RUNXT1, CBFB-MYH11, and PML-RARA in ~15% of cases.10 In the case of acute promyelocytic leukemia after chemotherapy, the outcomes appear as favorable as the out comes in patients who had never been exposed to chemother apy.22 On occasion, core binding factor AML is found in patients with prior chemotherapy exposure, and review of the outcomes of these patients suggests standard-of-care therapies are asso ciated with shorter overall survival than de novo cases. That said, Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 Nonintensive regimens CLINICAL CASE 3 A 69-year-old woman presented for shortness of breath and was found to have pancytopenia. She had no prior diagnosis of an antecedent MDS or MPN and no history of cytotoxic thera pies. Bone marrow biopsy specimen revealed AML, myelodysplasia related. Blasts were enumerated at 68%, and dysplasia was greater than 50% in 2 cell lines. Karyotype was normal. Molecular testing revealed NPM1 (46% VAF), IDH1 R132H (48% VAF), SRSF2 (45% VAF), and FLT3-ITD (4.5% VAF). She is fit for induction chemotherapy. AML-MR falls under a category of AML included in the sec ondary subset. This case highlights a common scenario where a patient presents without a known antecedent disorder and is still man aged as hav ing sec ond ary AML. In this case, the founder mutation in the IDH1 gene at R132H likely predisposed the patient to the dysplastic features seen in this presentation. Therapeutic options include conventional therapy with 7+3 or CPX-351, which has been approved for patients with AML-MR.12 In this case, before knowing the results of the NPM1 and FLT3 testing, the patient initiated therapy with liposomal cytarabine/ daunorubicin. Upon determination of the low-level FLT3-ITD muta tion, midostaurin was added on days 8 to 21 based on improved overall survival with standard 7+3 induction therapy plus midostaurin in the RATIFY trial.26 She was found to have MRD-negative remission at the completion of induction via assessment of NPM1 with quantitative RT-PCR and proceeded to nonmyeloablative stem cell transplant given her age over 65 years. This case highlights some complexity in the diagnostics and characterizations of AML with the evolution of novel therapies and evolved risk categorizations to include molecular changes. By the WHO 2016 classification system, the presence of dyspla sia alone would have been sufficient for a diagnosis of AML-MRC, but by the 2022 classification system criteria, the presence of SRSF2 or other molecular or cytogenetic changes is necessary for AML-MR. By the European LeukemiaNet 2017 criteria, she would fall into favorable-risk disease.27 In younger patients, over all survival (OS) is similar in those who receive an allogenic stem cell transplant in first CR and those who do not. It is unclear if this finding can be extrapolated to older patients.28 While favorable via this method, the median OS in the phase 3 study of CPX351 was only 9.7 months.12 As such, our standard practice is to consolidate remissions achieved in patients given CPX-351 who are transplant eligible with an allogeneic stem cell transplant. In the registrational study, the outcomes in patients who went to transplant after CPX-351 appear to be improved over those who received 7+3, although MRD assessments were not available.14 An interesting subanalysis of these OS data suggested lower transplant-related (nonrelapse) mortality in the CPX-351 arm.29 Specific to the NPM1 subset, quantitative RT-PCR monitoring of blood or marrow after induction can identify patients likely to relapse and those with intermediate-risk disease who would benefit from transplant, monitoring of blood once in remission can identify favorable-risk patients likely to relapse, and mon itoring of blood or bone marrow before and after transplant can help prognosticate risk of relapse.30 In older adults in whom improvements from favorable-risk disease appear less,31 stem cell transplant still appears to be advantageous.32 This patient was fit for intensive therapy, but venetoclax and azacitidine would be any option for many other 69-year-old patients. We would have favored that approach over, say, ivosidenib and azacitidine given the higher response rate and faster time to response. CLINICAL CASE 4 A 77-year-old man presented for evaluation of dizziness and shortness of breath and was found to have a white blood cell count of 7480/µL with 17% peripheral blood blasts, hemoglo bin of 6.1 g/dL, and platelet count of 70 000/µL. Bone marrow examination revealed 48% blasts and chromosome analysis revealed a complex karyotype with evidence of monosomy 5, 7, and 17. Molecular studies revealed mutation in TP53 at R273 with a VAF of 80%. His performance status prior to this presen tation was excellent for his age. AML with complex karyotype and TP53 mutations appears to have the worst outcomes of the secondary AMLs. We estimate the likelihood of achieving remission with standard 7+3 and CPX351 to be about 30% in these cases (Table 2). This patient may be considered ineligible for standard induction by age alone, but the biologic features of his disease make a stronger case for alternative therapies. In the phase 3 study of azacitidine and Dr Prakash Singh Shekhawat Optimizing outcomes in secondary AML | 27 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 over all out comes appear deter mined by addi tional adverse genetic features more so than by prior exposure to cytotoxic therapies.23 A retrospective collection of 69 patients with treat ment-related core bind ing fac tor leu ke mia revealed patients with secondary cytogenetic abnormalities had longer overall survival than those without abnormalities. Presence of trisomy 8 and trisomy 22 and loss of X or Y chromosome were associated with improved survival in both the post–cytotoxic therapy and de novo subsets.23 In these situations, we advocate standardof-care therapies with cytarabine-containing induction regimens plus gemtuzumab ozogamicin.24 This highlights a principle that the key feature of secondary AML is the specific genetic changes present rather than prior therapy or antecedent MDS or MPN. In these cases, monitoring MRD via high-sensitivity polymer ase chain reaction (PCR)–based testing of the blood should be performed to document an MRD-negative remission for therapy to continue with curative intent consolidation. RUNX1::RUNX1T1 or CBFB::MYH11 fusion genes cor re spond ing to t(8;21) or inv(16/t(16;16) changes can be monitored by blood-level realtime quantitative PCR with high sensitivity (10–4 to 10–6), allowing potential early intervention.25 For these assays to be most reliable, assessment via molecular assays at the time of diagno sis is critical to establish the ability to detect fusions that occa sionally can be missed if noncanonical gene fusions are present. Even in the sec ond ary leu ke mia set ting, patients with oth er wise favorable leukemias can have reasonable outcomes with non-transplant-based therapy. Measurement of RUNX1-RUNX1T1 MRD via reverse transcription (RT)–PCR after course 1 of 7+3 with gemtuzumab and course 2 of high-dose cytarabine revealed no detectable evidence of disease, and the patient has completed 4 courses of consolidation without transplant in first CR. Conclusions Secondary AML represents a heterogenous group of patients with features often overlapping de novo cases with similar genet ics. Management and risks are often comparable to de novo dis ease with similar genetic features. Secondary AML encompasses a wide spectrum of molecular features. The historically poor out comes in secondary AML reflect the higher proportion of neg ative features like poor risk cytogenetics and TP53 mutation. Furthermore, patients are frequently older with complex med ical histories, limiting treatment options. The complete profiling of histopathology and genetic drivers is critical with the evolu tion of alternatives to standard 7+3 therapy. Patients with more favorable underlying cytogenetic and molecular features may do well with therapy, especially those who are candidates for allo geneic stem cell transplantation. Overwhelmingly, patients have dis ease with poor-risk fea tures. Only rarely do patients have AML-pCT that is truly low risk. Maintenance therapy or transplan tation is appropriate in most cases. Venetoclax-based regimens have expanded treatment options for frailer, older patients with sta ble comorbidities. Emerging treat ments seek to open the therapeutic window by targeting specific genetic vulnerabilities or altering the microenvironment. Current treatment decisions 28 | Hematology 2022 | ASH Education Program require a careful integration of clinical history, genetic features of disease, and evaluation for allogeneic transplant. Conflict-of-interest disclosure Andrew Matthews: no competing financial interests to declare. Keith W. Pratz: research funding from AbbVie, Agios, Daiichi Sankyo, and Millennium; advisory board member for AbbVie, Astellas, Boston BioMedical, BMS, Celgene, Novartis, Jazz Pharmaceuticals, and Servier. Off-label drug use Midostaurin is approved in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation. Case discussion gives an example of a patient starting CPX-351 with addition of midostaurin after FLT3 mutation resulted. Correspondence Keith W. Pratz, University of Pennsylvania, PCAM South 12155, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA; e-mail: keith.pratz@pennmedicine.upenn.edu. References 1. Granfeldt Østgård LS, Medeiros BC, Sengeløv H, et al. Epidemiology and clin i cal sig nifi cance of sec ond ary and ther apy-related acute mye loid leu ke mia: a national pop u la tion-based cohort study. J Clin Oncol. 2015;33(31):3641-3649. 2. Martínez-Cuadrón D, Megías-Vericat JE, Serrano J, et al. Treatment patterns and outcomes of 2310 patients with secondary acute myeloid leukemia: a PETHEMA registry study. Blood Adv. 2022;6(4):1278-1295. 3. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391-2405. 4. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours: myeloid and histio cytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 5. Arber DA, Orazi A, Hasserjian RP, et al. International Consensus Classification of myeloid neoplasms and acute leukemia: integrating morphological, clinical, and genomic data. Blood. 2022;140(11):1200-1228. 6. Lindsley RC, Mar BG, Mazzola E, et al. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations. Blood. 2015;125(9):1367-1376. 7. Zink F, Stacey SN, Norddahl GL, et al. Clonal hematopoiesis, with and with out can di date driver muta tions, is com mon in the elderly. Blood. 2017;130(6):742-752. 8. Bolton KL, Ptashkin RN, Gao T, et al. Cancer therapy shapes the fitness landscape of clonal hematopoiesis. Nat Genet. 2020;52(11):1219-1226. 9. McNerney ME, Godley LA, Le Beau MM. Therapy-related mye loid neo plasms: when genetics and environment collide. Nat Rev Cancer. 2017;17(9):513-527. 10. Kayser S, Döhner K, Krauter J, et al; German-Austrian AMLSG. The impact of therapy-related acute myeloid leukemia (AML) on outcome in 2853 adult patients with newly diagnosed AML. Blood. 2011;117(7):2137-2145. 11. Löwenberg B, Ossenkoppele GJ, van Putten W, et al; Dutch-Belgian Cooperative Trial Group for Hemato-Oncology (HOVON); German AML Study Group (AMLSG); Swiss Group for Clinical Cancer Research (SAKK) Collaborative Group. High-dose daunorubicin in older patients with acute myeloid leukemia. N Engl J Med. 2009;361(13):1235-1248. 12. Lancet JE, Uy GL, Cortes JE, et al. CPX-351 (cytarabine and daunorubicin) liposome for injection versus conventional cytarabine plus daunorubicin in older patients with newly diagnosed secondary acute myeloid leukemia. J Clin Oncol. 2018;36(26):2684-2692. 13. Nilsson C, Hulegårdh E, Garelius H, et al. Secondary acute myeloid leuke mia and the role of allogeneic stem cell transplantation in a populationbased setting. Biol Blood Marrow Transplant. 2019;25(9):1770-1778. 14. Lancet JE, Uy GL, Newell LF, et al. CPX-351 versus 7+3 cytarabine and daunorubicin chemotherapy in older adults with newly diagnosed high-risk or secondary acute myeloid leukaemia: 5-year results of a randomised, open-label, multicentre, phase 3 trial. Lancet Haematol. 2021;8(7):e481-e491. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 venetoclax, the remission rates were modest (55%), but the OS was short (6.5 months).21 There are several novel strategies under development specifically in the TP53 subset, including TP53 acti vating therapy in the form of eprenetapopt (APR-246). In early-phase studies, the reported response rates were high33 but, in subsequent randomized studies compared with azacitidine alone, failed to reach a primary end point of improvement in CR rates (Aprea press release 12/2020). The more recent strategies involve the use of immune target activation. There are several emerging immunotherapy strategies, including immune check point blockade (such as Tim-3), bispecific antibody therapies (such as flotetuzumab, an inves ti ga tional bispecific anti bodybased molecule to CD3ε and CD123), and the most advanced candidate: monoclonal antibodies to CD47 (such as magrolimab). The expression of CD47 on myeloid cells acts to block immune response to malignant cells directly at the site of phagocyto sis. Interestingly, CD47 messenger RNA expression varies across cytogenic and molecular subgroups, with lower expression in cases with t(8;21), a favor able-risk trans lo ca tion, and higher expression in cases harboring FLT3-ITD mutations. High CD47 expression has been shown to be an independent prognostic factor for poor OS in AML patient cohorts.34 The monoclonal anti body magrolimab has been developed to block the CD47 sig naling and in early-phase development shows promising activity against TP53 AML, with an overall response rate of 71% (48% CR and 19% CR with incomplete count recovery, 5% morphologic leukemia-free state) in 21 patients.35 The definitive randomized phase 3 studies in AML and MDS are enrolling. In addition, triplet therapy with venetoclax and a hypomethylating agent has been reported in abstract form, with a 64% CR rate in TP53-mutant disease. Fifty-five percent of these CRs were MRD negative by flow cytometry, suggesting deep remissions in a subset of the responders.36 In this patient, we elected to enroll him on a clin ical trial examining the efficacy of a CD47 monoclonal antibody with the knowledge that transplant in this age group is generally not an option. molecular alterations in adults with acute myeloid leukemia. J Clin Oncol. 2012;30(36):4515-4523. 32. Koreth J, Schlenk R, Kopecky KJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta-analysis of prospective clinical trials. JAMA. 2009;301(22):23492361. 33. Sallman DA, DeZern AE, Garcia-Manero G, et al. Eprenetapopt (APR-246) and azacitidine in TP53-mutant myelodysplastic syndromes. J Clin Oncol. 2021;39(14):1584-1594. 34. Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286-299. 35. Sallman D, Asch A, Kambhampati S, et al. AML-196: the first-in-class antiCD47 antibody magrolimab in combination with azacitidine is well tol erated and effective in AML patients: phase 1b results. Clin Lymphoma Myeloma Leuk. 2021;21:S290. 36. Daver N, Konopleva M, Maiti A, et al. Phase I/II study of azacitidine (AZA) with venetoclax (VEN) and magrolimab (magro) in patients (pts) with newly diag nosed older/unfit or high-risk acute mye loid leu ke mia (AML) and relapsed/refractory (R/R) AML [Abstract #371]. Paper presented at: 2021 American Society of Hematology Annual Meeting; 12 December 2021; Atlanta, GA. 37. Lindsley RC, Gibson CJ, Murdock HM, et al. Genetic characteristics and outcomes by mutation status in a phase 3 study of CPX-351 versus 7+3 in older adults with newly diagnosed, high-risk/secondary acute myeloid leukemia (AML). Blood. 2019;134(suppl 1):15. 38. Boddu P, Kantarjian H, Ravandi F, et al. Outcomes with lower inten sity therapy in TP53-mutated acute myeloid leukemia. Leuk Lymphoma. 2018;59(9):2238-2241. 39. Wei AH, Montesinos P, Ivanov V, et al. Venetoclax plus LDAC for newly diag nosed AML ineligible for intensive chemotherapy: a phase 3 randomized placebo-controlled trial. Blood. 2020;135(24):2137-2145. 40. Stone A, Zukerman T, Flaishon L, Yakar RB, Rowe JM. Efficacy outcomes in the treatment of older or medically unfit patients with acute myeloid leu kaemia: a systematic review and meta-analysis. Leuk Res. 2019;82:36-42. 41. Heuser M, Smith BD, Fiedler W, et al. Clinical benefit of glasdegib plus lowdose cytarabine in patients with de novo and secondary acute myeloid leukemia: long-term analysis of a phase II randomized trial. Ann Hematol. 2021;100(5):1181-1194. 42. Cortes JE, Heidel FH, Hellmann A, et al. Randomized comparison of low dose cytarabine with or without glasdegib in patients with newly diag nosed acute mye loid leu ke mia or high-risk myelodysplastic syn drome. Leukemia. 2019;33(2):379-389. 43. Brunner AM, Esteve J, Porkka K, et al. Efficacy and safety of sabatolimab (MBG453) in combination with hypomethylating agents (HMAs) in patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS): updated results from a phase 1b study. Blood. 2020;136(suppl 1):1-2. 44. Vadakekolathu J, Lai C, Reeder S, et al. TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immuno therapy in AML. Blood Adv. 2020;4(20):5011-5024. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000324 Dr Prakash Singh Shekhawat Optimizing outcomes in secondary AML | 29 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/23/2021835/23matthews.pdf by guest on 09 December 2022 15. Pollyea DA, Pratz KW, Wei AH, et al. Outcomes in patients with poor-risk cytogenetics with or without TP53 mutations treated with venetoclax and azacitidine. Clin Cancer Res. 2022;25;CCR-22-1183. 16. Matthews AH, Perl AE, Luger SM, et al. Real-world effectiveness of CPX351 vs venetoclax and azacitidine in acute myeloid leukemia. Blood Adv. 2022;6(13):3997-4005. 17. Cherry EM, Abbott D, Amaya M, et al. Venetoclax and azacitidine compared with induction chemotherapy for newly diagnosed patients with acute myeloid leukemia. Blood Adv. 2021;5(24):5565-5573. 18. Sengsayadeth S, Gatwood KS, Boumendil A, et al. Conditioning intensity in secondary AML with prior myelodysplastic syndrome/myeloproliferative disorders: an EBMT ALWP study. Blood Adv. 2018;2(16):2127-2135. 19. Hourigan CS, Dillon LW, Gui G, et al. Impact of conditioning intensity of allogeneic transplantation for acute myeloid leukemia with genomic evi dence of residual disease. J Clin Oncol. 2020;38(12):1273-1283. 20. Gilleece MH, Labopin M, Yakoub-Agha I, et al. Measurable residual dis ease, conditioning regimen intensity, and age predict outcome of allo geneic hematopoietic cell transplantation for acute myeloid leukemia in first remission: a registry analysis of 2292 patients by the Acute Leukemia Working Party European Society of Blood and Marrow Transplantation. Am J Hematol. 2018;93(9):1142-1152. 21. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in pre viously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617629. 22. Duffield AS, Aoki J, Levis M, et al. Clinical and pathologic features of sec ondary acute promyelocytic leukemia. Am J Clin Pathol. 2012;137(3):395402. 23. Rogers HJ, Wang X, Xie Y, et al. Comparison of therapy-related and de novo core binding factor acute myeloid leukemia: a bone marrow pathol ogy group study. Am J Hematol. 2020;95(7):799-808. 24. Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a meta-analysis of individual patient data from randomised con trolled trials. Lancet Oncol. 2014;15(9):986-996. 25. Willekens C, Blanchet O, Renneville A, et al; French AML Intergroup. Prospective long-term minimal residual disease monitoring using RQ-PCR in RUNX1-RUNX1T1-pos i tive acute mye loid leu ke mia: results of the French CBF-2006 trial. Haematologica. 2016;101(3):328-335. 26. Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus chemother apy for acute mye loid leu ke mia with a FLT3 muta tion. N Engl J Med. 2017;377(5):454-464. 27. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 28. Schlenk RF, Döhner K, Krauter J, et al; German-Austrian Acute Myeloid Leukemia Study Group. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358(18):1909-1918. 29. Uy GL, Newell LF, Lin TL, et al. Transplant outcomes after CPX-351 vs 7+3 in older adults with newly diagnosed high-risk and/or secondary AML. Blood Adv. 2022;6(17):4989-4993. 30. Falini B, Sciabolacci S, Falini L, Brunetti L, Martelli MP. Diagnostic and ther a peu tic pit falls in NPM1-mutated AML: notes from the field. Leukemia. 2021;35(11):3113-3126. 31. Mrózek K, Marcucci G, Nicolet D, et al. Prognostic significance of the Euro pean LeukemiaNet stan dard ized sys tem for reporting cyto ge netic and ACUTE MYELOID LEUKEMIA: IMPROVING OUTCOMES IN CHALLENGING SUBSETS EVIDENCE-BASED MINIREVIEW Brandon J. Aubrey and Andrew Brunner Harvard Medical School and Massachusetts General Hospital, Boston, MA LEARNING OBJECTIVES • Understand the role of panelbased molecular testing at the time of diagnosis in AML • Consider the characteristics and limitations of serial panelbased molecular testing in AML CLINICAL CASE A 52yearold woman is diagnosed with acute myeloid leukemia (AML). At diagnosis, pathology demonstrated myelomonocytic morphology with a normal karyotype. The patient underwent standard induction chemotherapy with cytarabine and daunorubicin, achieving a complete remission, and is currently having consolidation therapy with highdose cytarabine without an initial plan for allo geneic hematopoietic stem cell transplantation. What is the utility of broad panelbased molecular testing at dif ferent time points during the management of this patient? Introduction One of the great advances in understanding the pathobi ology of AML has been through the discovery and charac terization of recurrent somatic gene mutations. This is a rapidly evolving area where clinical decisionmaking may now capitalize on knowledge of a patient’s mutational pro file for diagnostic subtyping, prognostication, and choice of therapy, carried out in conjunction with standard histo pathologic and cytogenetic studies at the time of diag nosis. Somatic mutations can be identified using several techniques, including targeted sequencing of a single gene, RNA or DNAbased quantitative polymerase chain reaction (qPCR) assays, panelbased nextgeneration sequencing (NGS), and wholeexome or wholegenome sequencing, each of which exhibits varying sensitivity and specificity for the detection of gene mutations. Application of these technologies has revealed the genomic complexity of AML, with molecular heterogeneity between patients that may evolve over time and in response to treatment. A strong evidence base supports the testing of a small number of 30 | Hematology 2022 | ASH Education Program specific gene mutations in routine clinical practice, includ ing NPM1, FLT3, and CEBPA, as they may influence diagnostic subtyping and provide clear prognostic information in patients with a normal karyotype on cytogenetic testing.1 Improved technology and declining cost for gene sequenc ing has provided more widely available panelbased gene testing for patients with AML, providing accessible testing for a larger number of genes. NGSbased gene panels used in AML have grown to incorporate an everexpanding set of genes that typically include FLT3, NPM1, CEBPA, RUNX1, isocitrate dehydrogenase 1 and 2 (IDH1/2), TET2, TP53, KIT, WT1, and ASXL1, among other recurring gene mutations found in myeloid malignancy (Table 1). While NGSbased gene panels are able to detect a larger number of gene mutations and comutations in a single patient, it can be challenging to interpret and clinically use all of these data points. In this evidencebased minireview, we assess the decision to test a broad NGS panel, rather than individual genes, in AML and how this decision may affect the diag nosis, prognosis, and/or treatment selection for a patient. What role does molecular testing play in establishing a diagnosis of AML? AML diagnosis is primarily based on morphology and cyto genetics but increasingly takes molecular features into consideration. Some AML subtypes now incorporate spe cific gene mutations into the diagnostic criteria, including NPM1 and CEBPA.13 This is based on the observation that these mutations are associated with distinct clinicopatho logic features that are reflected in patient outcomes. Nota bly, AML may now be diagnosed by the presence of NPM1 mutation irrespective of bone marrow myeloblast per centage, analogous to the finding of specific cytogenetic abnormalities, such as t(8;21) and inv(16). An emerging Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/30/2021776/30aubrey.pdf by guest on 09 December 2022 Clinical utilization of panel-based molecular testing for patients with AML Table 1. Clinical utility of somatic gene mutation testing in AML and molecular features that may guide treatment strategy and can result in a benefit to patient survival Gene Clinical utility NPM1 Diagnostic, good prognosis in isolation, MRD monitoring ITD confers poorer prognosis, therapeutic target for ITD and TKD Role in diagnosis, familial risk, good prognosis with in-frame bZIP mutation IDH1/2 Therapeutic targets, proposed role in diagnosis, clonal hematopoiesis TP53 Very poor prognosis, chemotherapy refractory, may favor hypomethylating agent-based treatment (azacitidine/decitabine) or clinical trial RUNX1 Poorer prognosis, familial risk KIT Poorer prognosis in CBF AML, potential therapeutic target TET2 Associated with clonal hematopoiesis EZH2 Associated with secondary AML STAG2 Associated with secondary AML SRSF2 Associated with secondary AML BCOR Associated with secondary AML SF3B1 Associated with secondary AML U2AF1 Associated with secondary AML ASXL1 Poor prognosis, secondary AML, clonal hematopoiesis DNMT3A Associated with clonal hematopoiesis PTPN11 Associated with clonal hematopoiesis, poorer prognosis Feature Therapeutic conferring survival advantage FLT3 (ITD and TKD) midostaurin with induction therapy, gilteritinib at relapse IDH1⁺ Ivosidinib + azacitidine sAML* Cytarabine/daunorubicin liposomal (Vyxeos; CPX-351) bZIP, basic leucine zipper region; CBF, core-binding factor; ITD, internal tandem duplication; MRD, measurable residual disease; TKD, tyrosine kinase domain mutation. ⁺In the phase 3 study comparing azacytidine and venetoclax to azacytidine monotherapy, patients with IDH1 and IDH2 mutations appeared to have improved responses in subgroup analysis. *sAML may be suggested by the finding of high mutation burden and/or EZH2, STAG2, SRSF2, BCOR, RUNX1, SF3B1, U2AF1, and ASXL1, but diagnosis requires correlation with clinical history, morphology, and cytogenetic findings. Vyxeos has shown benefit to patients with sAML. area is the understanding that comprehensive molecular pro files may better characterize the origin of a given AML subtype; for instance, the detection of gene mutations in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 are associated with >95% specificity for secondary AML (sAML).4 However, to date, the formal diagnosis of sAML uses the correlation of such molec ular features with the clinical history, morphology, and cytoge netic findings. In the investigational setting, detailed molecular profiling of patients with AML has been shown to effec tively subclassify patients into diverse, clinically relevant subgroups and predicts the existence of leukemia subtypes not currently known within the existing classification system,5 suggesting the strong potential for future clinical use of in-depth molecular dis ease classification. At this time, it is important to recognize the expanding number of mutations necessary for a complete AML classification; although single-gene mutation testing can be done for a minimal set of genes, panel sequencing increasingly has an advantage in this domain. What role does molecular testing play in predicting a patient’s prognosis? FLT3, NPM1, and CEBPA were identified early as having a role in risk strat i fi ca tion of patients with AML who have nor mal cytogenetics,6 effectively identifying patient risk of relapse and survival after chemotherapy.1,2 AML with an isolated NPM1 muta tion predicts for more favorable-risk disease with good response to standard chemotherapy.7 Similarly, in-frame mutations within the basic leu cine zip per region of the CEBPA gene iden tify patients with favorable-risk disease.8,9 In contrast, the presence of FLT3 gene internal tandem duplication predicts a higher risk of relapse. Recent data suggest that broader mutation profiles— often facilitated through panel-based gene testing approaches— can improve upon risk stratification and disease prognosis (Table 1). For example, TP53 mutations confer a distinctly poor prognosis, have a high risk for failure with standard chemother apy, and may prompt alternative treatment approaches such as those with a hypomethylating agent backbone.10,11 RUNX1,12 Dr Prakash Singh Shekhawat Molecular testing for patients with AML | 31 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/30/2021776/30aubrey.pdf by guest on 09 December 2022 FLT3 CEBPA ASXL1,13 and other secondary-like mutations may also indicate higher risk of patient relapse or therapy resistance. In addition, high mutational burden or the detection of certain mutations (Table 1) may suggest a diagnosis of sAML, which has clinical importance, in that these gene mutations identify a group of patients with very poor prognosis, and increasingly guides the choice of induction chemotherapy and consolidation strategy. Can gene panel testing affect treatment selection for AML differently than testing for single mutations? Where is molecular testing likely to play an impact after initial AML diagnosis? Molecular testing has a growing role following a patient’s ini tial diagnosis during treatment. There is a strong interest and active research to optimize molecular monitoring of measurable residual disease to guide postremission treatment strategies for AML.18 In the setting of NPM1-mutant AML, specific detection of the mutant NPM1 transcript by qPCR is a powerful indicator of relapse risk that can be used to risk-adapt patient treatment.19 A limitation to the universal application of such techniques is the genetic heterogeneity of AML; NGS-based gene panel assays are being evaluated as a way to monitor disease over time and are better able to track clonal heterogeneity compared with genespecific qPCR assays. This is relevant as most patients with AML will not have a mutation as easy to track as NPM1. If such track ing is validated, NGS panels have an advantage compared with qPCR assays because they assess a larger selection of genes, and most patients will have at least 1 genetic alteration detected by the panel assay that can be monitored over time.5 Furthermore, broad NGS-based gene panel testing enables detection of the emergence of additional molecular abnormalities over time that may not have been present at the time of diagnosis. NGS-based measurable residual disease monitoring faces several limitations, including relatively lower sensitivity compared with dedicated qPCR assays, including a lack of standardization, and significant chal lenges related to distinguishing what rep re sents resid ual leukemia from what may be background clonal hematopoiesis (clonal hematopoiesis of indeterminate potential or clonal cyto penias of unknown significance).20 At this time, inter val NGS 32 | Hematology 2022 | ASH Education Program What is the role of molecular testing in patients with AML after relapse? Finally, AML relapse after chemotherapy, like at diagnosis, repre sents a time point to reassess disease in order to develop a sal vage strategy. In contrast to monitoring in remission, at relapse, there is more emphasis on immediately actionable mutations. These include muta tions in FLT3, IDH1, and IDH2. Whether a broad NGS-based panel offers significant advantage over tar geted sequencing at relapse is less clear, however. Particularly for late relapses, NGS can help to clar ify the rela tion of the relapsed leukemia to the initial diagnosis. Since most disease at relapse has a universal poor prognosis, risk assessment has less immediate utility. It is nonetheless important to assess for FLT3 mutations, which can emerge during treatment and for which several targeted therapies are available. Mutations in IDH1 or IDH2 appear more stable over time but can be subject to sub clonal evolution that may evade detection at low levels early in the disease course. An increasing number of targeted therapies in clinical development also require the presence of a mutated gene target; given the importance of clinical trials as an option for patients with relapsed or refractory AML, such testing can be informative. Conclusions Gene panel testing, now commonly performed by NGS-based technology, is an increasingly available tool for patients with AML. We recommend allpatients with AML have broad gene panel testing at diagnosis, which includes the most common recurrently mutated genes in this disease where testing may influence diagnosis, prognosis, and treatment selection. At this time, there is an evolving evidence base for serial NGS-based broad gene panel testing during treatment in routine clinical practice. We await robust evidence to support the routine use of this outside of a clinical trial, although it may be valuable in specific clinical scenarios such as prior to allogeneic transplant. Many open questions remain, including optimal timing for serial panel mutation assessment, thresholds for detection in NGSbased methods that balance sensitivity and specificity, and bet ter defining which gene mutations, and combinations thereof, are most predictive of patient outcomes. Finally, a major barrier remains regarding the accessibility, cost, and standardization of these techniques to make them more accessible in routine clinical practice. Recommendations for NGS-based broad molecular testing in AML 1. Broad NGS-based gene panel testing should be performed at the time of AML diagnosis. (Strong recommendation, moder ate certainty of the evidence) 2. At AML relapse, patients should have molecular testing repeat ed, although it is less clear whether NGS-based panel testing has an advantage over testing single actionable genes at this time. (Strong recommendation for testing in general, low cer tainty of evidence between testing modalities) Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/30/2021776/30aubrey.pdf by guest on 09 December 2022 Several mutation profiles affect the initial treatment strategy. The presence of FLT3 internal tandem duplication or FLT3 mutations in the tyrosine kinase domain, for instance, informs the use of drug therapies that target the FLT3 kinase. Incorporating FLT3 inhibi tors with standard chemotherapy provides a survival advantage for patients with this AML subtype.14 More challenging perhaps is the entity of sAML, which may be more variably defined but for which CPX-351 (Vyxeos; Jazz Pharmaceuticals) may be advanta geous to standard induction therapy.15 A growing number of avail able targeted therapies may be better suited to other genetic alterations. In patients with IDH1/2 mutations, small-molecule inhib i tors of IDH1 or IDH2, com bined with azacitidine, can improve outcomes in select patients,16 although these patients also have high response rates to other regimens such as azac itidine in combination with venetoclax.17 There is also evidence that TP53-mutated AML—and perhaps other secondary-like mutations—may be equally if not more responsive to alternative therapies to induction, such as hypomethylating agent-based therapies. With the expanding availability of targeted drug ther apies, the value of panel-based gene testing is likely to expand. test ing in remis sion remains under inves ti ga tion but may be most valuable at the time of clear treatment decisions where estimation of patient risk is clinically important, such as with allo geneic transplant.21 3. At this time, routine serial NGS-based gene panel testing for patients with AML in remis sion remains inves ti ga tional, al though may have value in specific scenarios. (Low certainty of evidence) Conflict-of-interest disclosure Off-label drug use Brandon J. Aubrey: nothing to disclose. Andrew Brunner: nothing to disclose. Correspondence Andrew Brunner, Harvard Medical School and Mas sa chu setts General Hospital, Zero Emerson Place, Suite 118, Bos ton, MA 02114, USA; e-mail: abrunner@mgh.harvard.edu References 1. 2. 3. 4. 5. 6. 7. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Orga nization Classification of Haematolymphoid Tumours: myeloid and Histio cytic/Dendritic Neoplasms. Leukemia. 2022;36(7):1703-1719. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. Arber DA, Orazi A, Hasserjian RP, et al. International consensus classifica tion of myeloid neoplasms and acute leukemia: integrating morphological, clinical, and genomic data. Blood. 2022;140(11):1200-1228. Lindsley RC, Mar BG, Mazzola E, et al. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations. Blood. 2015;125(9):1367-1376. Papaemmanuil E, Gerstung M, Bullinger L, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med. 2016;374(23):22092221. Schlenk RF, Döhner K, Krauter J, et al; German-Austrian Acute Myeloid Leu kemia Study Group. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358(18):1909-1918. Falini B, Mecucci C, Tiacci E, et al; GIMEMA Acute Leukemia Working Party. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005;352(3):254-266. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000417 Dr Prakash Singh Shekhawat Molecular testing for patients with AML | 33 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/30/2021776/30aubrey.pdf by guest on 09 December 2022 Brandon J. Aubrey is a former employee of the Walter and Eliza Hall Institute, Melbourne, Australia, and has received payments relat ing to roy al ties and mile stone pay ments for the BCL-2 antagonist venetoclax/ABT-199. Andrew Brunner is a con sul tant for Acceleron, Agios, BMS/Celgene, CTI Biopharma, Gilead, Keros Therapeutics, No vartis, Taiho, and Takeda. 8. Taube F, Georgi JA, Kramer M, et al; Study Alliance Leukemia (SAL). CEBPA mutations in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome. Blood. 2022;139(1):87-103. 9. Wakita S, Sakaguchi M, Oh I, et al. Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia. Blood Adv. 2022;6(1):238-247. 10. Welch JS, Petti AA, Miller CA, et al. TP53 and decitabine in acute myeloid leukemia and myelodysplastic syndromes. N Engl J Med. 2016;375(21):20232036. 11. Grob T, Al Hinai ASA, Sanders MA, et al. Molecular char ac ter iza tion of mutant TP53 acute myeloid leukemia and high-risk myelodysplastic syn drome. Blood. 2022;139(15):2347-2354. 12. Mendler JH, Maharry K, Radmacher MD, et al. RUNX1 mutations are associ ated with poor outcome in younger and older patients with cytogeneti cally normal acute myeloid leukemia and with distinct gene and microRNA expression signatures. J Clin Oncol. 2012;30(25):3109-3118. 13. Schnittger S, Eder C, Jeromin S, et al. ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome. Leukemia. 2013;27(1):82-91. 14. Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus chemother apy for acute mye loid leu ke mia with a FLT3 muta tion. N Engl J Med. 2017;377(5):454-464. 15. Lancet JE, Uy GL, Cortes JE, et al. CPX-351 (cytarabine and daunorubicin) liposome for injection versus conventional cytarabine plus daunorubicin in older patients with newly diagnosed secondary acute myeloid leukemia. J Clin Oncol. 2018;36(26):2684-2692. 16. Montesinos P, Recher C, Vives S, et al. Ivosidenib and azacitidine in IDH1-mutated acute myeloid leukemia. N Engl J Med. 2022;386(16):15191531. 17. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in pre viously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617629. 18. Ghannam J, Dillon LW, Hourigan CS. Next-generation sequencing for mea surable residual disease detection in acute myeloid leukaemia. Br J Haematol. 2020;188(1):77-85. 19. Ivey A, Hills RK, Simpson MA, et al; UK National Cancer Research Institute AML Working Group. Assessment of minimal residual disease in standardrisk AML. N Engl J Med. 2016;374(5):422-433. 20. Pløen GG, Nederby L, Guldberg P, et al. Persistence of DNMT3A muta tions at long-term remission in adult patients with AML. Br J Haematol. 2014;167(4):478-486. 21. Getta BM, Devlin SM, Levine RL, et al. Multicolor flow cytometry and multi gene next-generation sequencing are complementary and highly predic tive for relapse in acute myeloid leukemia after allogeneic transplantation. Biol Blood Marrow Transplant. 2017;23(7):1064-1071. ANXIETY PROVOKING CONSULTATIONS: MAST CELLS AND EOSINOPHILS Jason Gotlib Division of Hematology, Stanford Cancer Institute/Stanford University School of Medicine, Stanford, CA The historically poor prognosis of patients with advanced systemic mastocytosis (AdvSM) and primary eosinophilic neoplasms has shifted to increasingly favorable outcomes with the discovery of druggable targets. The multikinase/KIT inhibitor midostaurin and the highly selective KIT D816V inhibitor avapritinib can elicit marked improvements in measures of mast cell (MC) burden as well as reversion of MC-mediated organ damage (C-findings) and disease symptoms. With avapritinib, the achievement of molecular remission of KIT D816V and improved survival compared with historical therapy suggests a potential to affect disease natural history. BLU-263 and bezuclastinib are KIT D816V inhibitors currently being tested in trials of AdvSM. In the new World Health Organization and International Consensus Classifications, the category of “myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase (TK) gene fusions” is inclusive of rearrangements involving PDGFRA, PDGFRB, FGFR1, JAK2, FLT3, and ETV6::ABL1. While the successful outcomes with imatinib in FIP1L1::PDGFRA-positive cases and PDGFRB-rearranged neoplasms have become the “poster children” of these disorders, the responses of the other TK-driven neoplasms to small-molecule inhibitors are more variable. The selective FGFR inhibitor pemigatinib, approved in August 2022, is a promising therapy in aggressive FGFR1-driven diseases and highlights the role of such agents in bridging patients to allogeneic transplantation. This review summarizes the data for these approved and investigational agents and discusses open questions and future priorities regarding the management of these rare diseases. LEARNING OBJECTIVES • Summarize the efficacy and safety data for targeted therapy of advanced systemic mastocytosis and primary eosinophilic neoplasms • Discuss the impact of these novel agents on current treatment algorithms and how they shape future management priorities CLINICAL CASE A 61-year-old man with systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML) presented with severe fatigue and a 30-pound weight loss over the prior 6 months. He did not respond to PEG-interferon-alfa-2a. For several months, his paracenteses requirement increased to twice weekly for a volume of 10 to 15 L. Examination was notable for temporal wasting and marked atrophy of muscles in the bilateral supraclavicular regions. Imaging findings of hepatosplenomegaly were not palpable on examination due to his marked ascites. Laboratory studies revealed a white blood cell count of 17.8 × 109/L (51% monocytes), hemoglobin of 11.9 g/dL, platelet count of 34 | Hematology 2022 | ASH Education Program 106 × 109/L, albumin of 2.3 g/dL (normal, 3.5-5.0 g/dL), and serum tryptase level of 416 ng/mL (normal, <11.5 ng/mL). The bone marrow biopsy specimen revealed 30% mast cells, increased monocytes, and 8% blasts/blast equivalents; karyotype revealed del(13q). Next-generation sequencing revealed KIT D816V and TET2 Q243X mutations (variant allele frequencies of 11% and 44%, respectively). The patient was referred for management. Advanced systemic mastocytosis Systemic mastocytosis (SM), defined by established World Health Organization (WHO) criteria, is divided into nonadvanced forms (indolent SM [ISM], bone marrow Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 Available and emerging therapies for bona fide advanced systemic mastocytosis and primary eosinophilic neoplasms from both the MC and AHN compartments.14,15,27,28 The experi ence with midostaurin and avapritinib has validated this clinical strategy but has also revealed challenges of KIT inhibitor monotherapy. In particular, patients with SM-AHN and/or cases with a complex molecular landscape are vulnerable to AHN progression or transformation to secondary AML.4,5,22-24 Midostaurin Midostaurin (Novartis) is a multikinase/KIT inhibitor that tar gets not only D816V-mutated KIT but also wild-type (WT) KIT, PDGFRα/β, VEGFR2, and FLT3.29 Based on an encour ag ing partial response to midostaurin in a patient with SM-MDS/MPNunclassifiable30 and an overall response rate (ORR) of 69% in an investigator-initiated trial,31 the drug was eval u ated in a global registrational, phase 2, single-am, open-label study of 89 evaluable patients with AdvSM with ≥1 C-findings. The ORR was 60% (45% major responses) by (mod i fied) Valent and Cheson criteria.32 The European Medicines Agency (EMA) and FDA performed post hoc analyses of the trial using International Working Group–Myeloproliferative Neoplasms Research and Treatment-Euro pean Competence Network on Mastocytosis (IWG) response criteria. The EMA and FDA identified IWG ORRs of 28% and 17%, respec tively, the dif fer ence reflecting the FDA’s decision not to include the category of “clinical improve ment” in their calculation of response rate.33,34 Midostaurin elicited a median best percent reduction of serum tryptase level and BM MC burden by −58% and −59%, respectively; reduced spenomegaly; and elicited a significant reduction in symptoms (using the Memorial Symptom Assessment Scale) except for nausea and vomiting, which are common midostaurin-related adverse events.32,35 The median progression-free survival was 14.1 months, and the overall survival (OS) was 28.7 months.32 These data led to midostaurin’s approval for advanced SM in 2017 by the FDA and EMA. Additional real-world cohorts of AdvSM patients have cor rob o rated midostaurin’s activ ity, including an analysis that showed that a ≥25% reduction in KIT D816V RNA expressed allele burden was the strongest variable associated with prolonged survival.36-39 A retrospective regis try analysis also confirmed the superior survival of midostaurin compared with cladribine.40 Avapritinib Avapritinib (BLU-265; Blueprint Medicines) was designed as a highly selective, type 1 inhibitor of D816V-mutated KIT.41 It exhib its a 10-fold lower 50% inhibitory concentration (IC50) compared with midostaurin in an assay of KIT D816V kinase activity (0.27 vs 2.9nM). Avapritinib’s restricted tar get pro file also includes plate let-derived growth fac tor recep tor alpha (PDGFRα) with neg li gi ble activ ity against WT KIT; the drug received FDA approval in 2020 for adults with an unresectable or metastatic gastrointestinal stromal tumor harboring a PDGFRA exon 18 mutation, including the D842V mutation.42 Avapritinib’s FDA approval in 202143 as first-line therapy for adults with AdvSM (recommended plate let count ≥50×109/L) was based on the phase 1 EXPLORER study44 and an interim analysis of the phase 2 PATHFINDER study.45 The centrally adju dicated EXPLORER study consisted of a dose escalation phase evaluating doses of 30 to 400 mg daily in patients with AdvSM with ≥1 eligible organ damage finding by IWG criteria. The sub sequent dose expansion phase evaluated 2 dosing cohorts of 200 mg and 300 mg daily. Ultimately, 200 mg daily was cho Therapies for advanced SM & eosinophilic neoplasms | 35 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 mastocytosis, smol der ing SM) and advanced SM var i ants (aggres sive SM [ASM], SM with an asso ci ated hema to logic neoplasm [SM-AHN], and mast cell leukemia [MCL]).1-3 ASM is defined by ≥1 C-findings (neoplastic mast cell [MC]–related organ damage). Diagnostic criteria for SM-AHN include the presence of SM and an associated (myeloid) neoplasm (most commonly CMML),4 and MCL is histopathologically defined by ≥20% MCs on a bone marrow (BM) aspirate.1-4 Advanced SM (AdvSM) variants carry a poor prognosis (typically less than 4 years),5-9 and the worst outcomes are observed in patients with MCL (eg, <6 months to ~2 years).5,10-13 In both the International Consensus Classification (ICC) and WHO fifth edition,1,2 modifications to current minor diagnostic criteria include addition of CD30 as an immunohistochemical marker of SM, and an activating mutation in KIT besides D816V may also qualify. In the ICC,1 “AHN” is changed to “AMN” (associated myeloid neoplasm), and a core biopsy specimen may be used to diagnose MCL if the aspirate is a dry tap. While bone mar row mastocytosis is maintained as a subtype of ISM in the ICC,1 it is a distinct subtype from ISM in the WHO classification.2 Using high-sensitivity assays such as digital droplet polymer ase chain reaction (PCR) and allele-specific PCR, KIT D816V can be detected in ~95% of patients with AdvSM.14,15 Compared with nonadvanced SM, AdvSM variants are more frequently charac terized by multilineage involvement by KIT D816V and a com plex, multimutated genetic land scape, best exem pli fied by SM-AHN.16-18 SRSF2, ASXL1, and RUNX1 (S/A/R panel) are high-risk mutations,19,20 but other myeloid mutations are commonly iden tified (eg, TET2, DNMT3A, JAK2, NRAS, CBL, EZH2),15 some of which have been incorporated into prognostic scoring systems based on clinical, laboratory, and molecular features.6-9 Historically, therapy of AdvSM has consisted of off-label use of PEG-interferon (IFN) α±corticosteroids or cladribine. Studies evaluating these agents have often consisted of an admixture of patients with nonadvanced and AdvSM with overall responses in the range of 30% to 50% using heterogenous response criteria and variable reporting of biomarkers of response, including changes in BM MC bur den and serum tryptase level.21-24 Imatinib was approved by the US Food and Drug Administration (FDA) in 2005 for patients with ASM without KIT D816V or unknown mutation status, an exceedingly rare patient population. While exon 17 KIT D816V is an imatinib-resistant mutation, the drug can be effective in patients with SM with exon 8 to 11 mutations/juxtamembrane variants such as F522C, which is associated with a well-differenti ated SM phenotype.25 Acute myeloid leukemia (AML)–type induc tion chemotherapy has been used in patients with kinetically aggressive or refractory/relapsed disease to the aforementioned ther a pies. For patients with SM-AHN, in whom AHN-directed therapy is required, hydroxyurea has been used to control leu kocytosis and splenomegaly. Hypomethylating agents are com monly employed when the AHN is a higher-risk myelodysplastic syndrome (MDS) or MDS/myeloproliferative neoplasm (MPN). A retrospective analysis of allogeneic hematopoietic stem cell transplantation (HSCT) revealed a 3-year overall survival of 57% among 57 patients, with a diagnosis of MCL and reduced inten sity (vs myeloablative) conditioning being identified as adverse prognostic factors.26 These data were derived before the use of KIT inhibitors; therefore, the role of transplant in the modern KIT inhibitor era is not well defined. The rationale for KIT inhibition in AdvSM is based on the high frequency of the KIT D816V mutation and its presence in cells Table 1. Response rates to avapritinib in the EXPLORER and PATHFINDER studies Characteristic Phase 1 EXPLORER Phase 2 Interim PATHFINDER Response evaluable, n 53 32 ASM 3 2 SM-AHN 37 26 MCL 13 4 75 75 CR 15 0 CRh 21 19 PR 34 31 CI 6 25 SD 23 13 PD 0 3 NE 2 9 ASM 100 100 SM-AHN 76 81 MCL 69 25 Yes 69 74 No 86 78 Prior midostaurin 59 82 Midostaurin naive 83 67 Yes 74 71 No 77 80 AdvSM subtype, n ORR by modified IWG criteria, % (CR + CRh + PR + CI) Best response, % ORR by any prior therapy, % ORR by midostaurin history, % ORR by S/A/R comutation status, % CI, clinical improvement; CRh, complete remission with partial hematologic recovery; NE, not evaluable; PD, progressive disease; S/A/R, SRSF2, ASXL1, RUNX1; SD, stable disease. sen as the recommended phase 2 dose based on a composite analysis of safety/tolerability, pharmacokinetics, efficacy, and biomarkers of response, including dynamic changes in BM MC burden and serum tryptase levels. Table 1 shows the overall rates of response by modified IWG criteria, AdvSM variant, prior ther apy and midostaurin exposure, and S/A/R mutation status in the EXPLORER study (n=69 evaluable patients)44 and from an interim anal y sis of PATHFINDER (n=32 evaluable patients).45 Figure 1 highlights the marked decreases in measures of MC disease from the EXPLORER study, including BM MC burden, serum tryptase level, and KIT D816V variant allele fraction from BM using digi tal droplet PCR with a limit of detection of 0.17%. Importantly, a com plete molec u lar remis sion (CMR) was achieved in 30% of patients, which represents a new response benchmark for AdvSM. Reversion of MC skin lesions and significant improve ment in symptoms using the AdvSM Symptom Assessment Form were also observed.44-46 An analysis of pooled outcomes in 53 patients treated with an avapritinib starting dose of ≤200 mg daily revealed an ORR 36 | Hematology 2022 | ASH Education Program of 72% (complete response [CR]/CR with partial hematologic recovery [28%] + partial response [PR] [28%] + clinical improve ment [15%]).47 The ORR among the 31 patients who had received prior therapy was 71%, including a CR/CR with partial hemato logic recovery rate of 19%.48 Overall survival at 12 and 24 months was 80% and 65%, respectively, and the median OS survival was not reached in this previously treated population with a median follow-up of 17.7 months.48 In 2022, the EMA granted approval for avapritinib in patients with AdvSM exposed to at least 1 prior systemic therapy.49 Long-term outcomes The esti mated pro gres sion-free sur vival rates were 84% at 12 months and 63% at 24 months in the EXPLORER response- evaluable population (n = 53).44 During a median follow-up of 23 months, 14 (20%) exhibited disease progression, including 6 patients (9%) with transformation to secondary AML. There was no consistent pattern of baseline or on-treatment myeloid mutations, changes in the variant allele frequency of specific Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 ORR by subtype, % genes, or resistance mutations in KIT in patients with or with out clinical progression.50 In the EXPLORER over all AdvSM safety pop u la tion, the median OS was not reached with a median follow-up duration of 23 months.44 Patients with S/A/R mutations or a baseline muta tion-adjusted risk score ≥2 exhibited shorter sur vival.44 Among allpatients with AdvSM, the estimated 24-month OS rate was 76% and 100%, 67%, and 92% for ASM, SM-AHN, and MCL subtypes, respectively.44 The comparative survival rates from the global midostaurin trial were 53% for allpatients with AdvSM and 86%, 49%, and 26% for patients with ASM, SM-AHN, and MCL, respectively.32 The estimated median OS for allpatients with AdvSM was 46.9 months in a pooled analy sis of patients from EXPLORER and PATHFINDER initiated with avapritinib ≤200 mg daily.47 Compared with a historical cohort of patients with AdvSM treated with best available therapy, avapritinib-treated patients exhibited sig nifi cantly improved survival (adjusted hazard ratio, 0.48; 95% confidence interval, 0.29-0.79; P = .004), longer duration of treatment (23.8 vs 5.4 months; P < .001), and a 60% greater mean difference in the percent maximum decrease in levels of serum tryptase.51 An indirect treatment comparison found that avapritinib improved survival compared with midostaurin.52 eral/periorbital edema (81%/4%), diarrhea (34%/<1%), nausea (31%/3%), fatigue/asthe nia (28%/7%), and cog ni tive effects (25%/2%).41 Hematologic AEs consisted of neutropenia (17%/16%), anemia (44%/27%), and the grouped terms thrombo cytopenia/platelet count decreased (50%/30%). In EXPLORER, intracranial bleed ing (ICB; eg, intraparenchymal hem or rhage, subdural hematoma) emerged as an AE of special interest that occurred in 9 (13%) patients, with 7 of these cases developing in the setting of thrombocytopenia (platelet count <50×109/L). Asymptomatic cases (n=5, grade 1) were detected by prespecified protocol magnetic resonance imaging of the brain; 2 events were grade 2, and one each was grade 3 and grade 5 (associ ated with head trauma). Mitigation procedures (eg, exclusion of patients with a starting platelet count <50×109/L, dose hold and reduction for emergent thrombocytopenia reaching this thresh old, platelet transfusions, increased blood count surveillance) led to a decrease in ICB in the interim PATHFINDER analysis, with only 1 patient (1.6%) experiencing a grade 2 subdural hematoma in the setting of progressive thrombocytopenia.45 The recommended frequency of platelet count monitoring is detailed in the avapritinib prescribing insert. Avapritinib: adverse events BLU-263 (Blueprint Medicines) has comparable selectivity and potency to avapritinib but limited brain penetration potential, which may mitigate cognitive changes and ICB. It is currently being evaluated in the phase 2/3 HARBOR study (NCT04910685) In the pooled outcomes from the overall safety population of 131 avapritnib-treated patients,47 the most common nonhematologic adverse events (AEs) (allgrades %/grade ≥3%) were periph Emerging KIT inhibitors BLU-263 and bezuclastinib Therapies for advanced SM & eosinophilic neoplasms | 37 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 Figure 1. Phase 1 EXPLORER Study: Avapritinib effects on measures of mast cell burden. Waterfall plots are shown for bone marrow mast cell burden, serum tryptase level, spleen volume, and KIT D816V variant allele fraction. A, other antineoplastic therapy; M, prior midostaurin exposure; MAF, mutant allele fraction; VAF, variant allele fraction. AdvSM and KIT inhibitors: open questions and future directions Avapritinib, midostaurin, or enrollment in clinical trials should be considered the first-line treatment option for AdvSM. While avapritinib is not recommend for patients with platelet count <50 × 109/L, it may otherwise merit preferred status. The drug can generate molecular remissions of KIT D816V, and recent data indicate more favorable long-term outcomes compared with historical treatments, including midostaurin. The major challenge of these KIT inhibitors is the AdvSM variant SM-AHN, in which prognosis is usually driven by the AHN, and the pres ence of a complex mutational landscape beyond KIT D816V may promote progression and resistance. In this regard, how to sequence KIT inhibition with AHN-targeted therapy is a major focus of clinical trial development in AdvSM. Table 2 highlights some of the major challenges and open questions that have emerged in the era of KIT inhibitors. CLINICAL CASE (Continued) The patient was started on avapritinib 200 mg daily. After 4 months, he became paracentesis independent. By 6 months, the albumin had increased to 4.8 g/dL, and the patient had gained 30 pounds and no longer exhibited muscle wasting. In addition, the serum tryptase level normalized to 10.1 ng/mL and the BM biopsy specimen showed no mast cell aggregates. He exhibited a 38% improvement in total symptom score on the AdvSM Symptom Assessment Form. Due to persistence of periorbital edema and achievement of a complete remission, the avapritinib dose was decreased to 100 mg daily. After 3 years of follow-up, he maintains a complete remission; the BM shows no evidence of MC aggregates and blasts are not increased. The serum tryptase level remains normal. The del(13q) abnormality and TET2 muta tion persist, but the KIT D816V mutation is no longer detectable. Primary eosinophilic neoplasms The WHO major category of “myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2” has had its title changed to “myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions” in both the fifth edition of the WHO classification of hematolymphoid tumors and the International Consensus Classification (ICC).1,2 The modification in nomenclature reflects the common molecular theme of rearranged, constitutively activated tyrosine kinases. These TK gene fusions (and associated chromosomal breakpoints) involve PDGFRA (the most com mon fusion, FIP1L1::PDGRA, is cryptic; otherwise, breakpoint 4q12), PDGFRB (5q31~q33), FGFR1 (8p11), JAK2 (9p24), FLT3 (13q12), and ETV6::ABL1. ETV6::ABL1 presenting as Philadelphia chromosome-like acute lymphoblastic leukemia (which should be distinguished from this category of TK gene fusions) can also rarely present as de novo T-cell acute lymphoblastic leukemia or a myeloid neoplasm.55,56 Table 2. Major questions and future challenges with KIT inhibitors in AdvSM • What are the most predictive biomarkers of prolonged response and survival? • Will response criteria based on pure pathologic criteria (eg, BM MC burden, serum tryptase level, blood counts), without confounding by lingering C-findings, better predict long-term end points such as overall survival? • How do we define and harmonize molecular evaluation of MRD? • Is time-limited treatment with KIT inhibitors feasible in patients who achieve a minimum duration of complete molecular remission of KIT D816V? • What is the clinical impact of KIT inhibitors on the natural history of the AHN component? • Does the presence of an AHN or multimutated molecular profile abrogate the potential benefits of molecular remission of KIT D816V or negative MRD? • How do we sequence KIT inhibitors and AHN-directed therapy in patients with SM-AHN? • What is the optimal role and timing of allogeneic HSCT in the era of KIT inhibitors and who are most appropriate candidates for transplant? • How do we use KIT inhibitors as a cytoreduction strategy before transplant? • Should KIT inhibitors be continued after transplant only in patients with detectable KIT D816V? • Will KIT inhibitors with less CNS penetration (eg, BLU-263, bezuclastinib) permit their use with therapy for high-risk AHN (eg, HMA±venetoclax) or with intensive induction chemotherapy in KIT D816V-positive AML? • Is there a role for cladribine + selective KIT inhibitors in patients with AdvSM with refractory/relapsed or rapidly progressive disease? CNS, central nervous system; HMA, hypomethylating agent; MRD, measurable residual disease. 38 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 of patients with ISM.53 Substantial clinical interest exists in eval uating BLU-263 in patients with AdvSM as monotherapy or as sequenced therapy with AHN-directed agents given the concern about the potential for thrombocytopenia-emergent ICB in this patient population with avapritinib. Bezuclastinib (CGT9486; Cogent Biosciences) is a type I TK inhibitor with activity against mutations in KIT exons 9, 11, 17, and 18, including D816V.54 Its target profile avoids other kinases such as WT KIT, PDGFRα, PDGFRβ, wild-type KIT, VEGFR2, and CSF1R and exhibits limited blood-brain barrier penetration and no cen tral nervous system toxicities in preclinical studies. The drug is currently being evaluated in a phase 2, open-label clinical trial (APEX; NCT04996875) of AdvSM in which patients are first ran domized to 1 of 4 doses of the drug in a dose optimization stage, followed by dose expansion at the recommended phase 2 dose. A phase 2/3 study of bezuclastinib in ISM/smoldering SM has also been initiated (NCT05186753). Table 3. Tyrosine kinase fusion genes and potential targeted therapies in myeloid/lymphoid neoplasms with eosinophilia Most common partner fusion gene Other partner fusion genes and chromosome breakpoint Tyrosine kinase inhibitors with established or potential activity PDGFRA (4q12) FIP1L1 (4q12) BCR (22q11) KIF5B (10p11) TNKS2 (10q23) CDK5RAP2 (9q33) ETV6 (12p13) STRN (2p24) FOXP1 (3p14) Imatinib PDGFRB (5q31~33) ETV6 (12p13) SPTBN1 (2p16) PDE4DIP (1q22) WDR48 (3p22) GOLGB1 (3q12) DIAPH1 (5q31) KANK1 (9p24) CEP85L (6q22) GIT2 (12q24) HIP1 (7q11) NIN (14q24) ERC1 (12p13) DTD1 (20p11) MYO18A (17q11) NDE1 (16p13). CPSF6 (12q15) CCDC88C (14q32) TPM3 (1q21) SPDR (2q32) GOLGA4 (3p22) PRKG2 (4q21) TNIP1 ((5q33) SART3 ((12q23) CCDC6 (10q21) NDEL1 (17p13) GPIAP1 (11p13) SPECC1 (17p11) TRIP11 (14q32) RABEP1 (17p13) MPRIP (17p11) TP53BP1 (15q22) BIN2 (12q13) Imatinib FGFR1 (8p11) ZMYM2 (13q12) FGFR1OP (6q27) LRRFIP1 (2q37) SQSTM1 (5q35) TRIM24 (7q34) HERV-K (19q13) BCR (22q11) CPSF6 (12q15) CNTRL (9q33) RANBP2 (2q13) CUX1 (7q22) TPR1 (1q25) FGFR1OP2 (12p11) MYO18A (17q11) TFG (3q12) Pemigatinib Futibatinib Midostaurin Ponatinib JAK2 (9p24) PCM1 (8p21) ETV6 (12p13) BCR (22q11) FLT3 (13q12) ETV6 (12p13) SPTBN1 (2p16) TRIP11 (14q32) LYN (8q12) ABL1 (9q34) ETV6 (12p13) Ruxolitinib Fedratinib Pacritinib Momelotinib GOLGB1 (3q12) NTRK3 (15q25) SYK (9q22) Gilteritinib Midostaurin Sorafenib Sunitinib Dasatinib Nilotinib Imatinib Bosutinib Ponatinib Asciminib Eosinophilia is not an invariable feature of these neoplasms but can serve as a useful pre-diagnostic checkpoint to think about these disease entities. At the time of writing, there have been 8 PDGFRA partner fusion genes characterized, over 30 for PDGFRB, 16 for FGFR1, 3 for JAK2, and 7 for FLT3 (Table 3).55-57 In some cases besides the cytogenetically invisible FIP1L1:: PDGFRA, standard cytogenetics and/or fluorescence in situ hybridization (FISH) may not be a ble to identify the TK gene fusion. Integrated genomic analyses, including chromo somal microarrays, whole-genome sequencing, and/or RNA sequencing, may be necessary to uncover small deletions or inversions resulting in cryptic fusion genes.58 The clinical presentation of myeloid/lymphoid neoplasms with eosinophilia (MLN-eo) with TK gene fusions can be highly complex. One should determine whether the disease involves the BM and peripheral blood (PB) only, extramedullary disease (EMD) only, or both BM/PB and EMD. If clinically suspected, imaging (preferably positron emission tomography/computed tomography) should be undertaken to identify EMD so it can be serially followed during treatment. Second, the presence of chronic phase (CP) or blast phase (BP) disease should be characterized in the BM/PB. The presence of EMD represents a BP component. Last, the disease lineages of the BM/PB and EMD components should be characterized. The most common CP presentations of MLN include myeloid neoplasms: MPN or MDS/MPN (CMML or MDS/MPN-unclassifiable) with or without eosinophilia. The BM may exhibit an atypical (usually intersti tial) infiltrate of MCs in the absence of the KIT D816V mutation. BP disease in the BM/PB and/or EMD may present as AML, B- or T-cell leukemia/lymphoblastic lymphoma, or a mixedphenotype acute leukemia. Importantly, the disease lineage in the BM/PB can be different from the EMD; therefore, biopsy of the EMD for lineage ascertainment may be helpful but not always possible. In some cases, we have seen tandem involve ment of the BM/PB by both a chronic myeloid neoplasm and B- or T-cell acute lymphoblastic leukemia. If patients have Therapies for advanced SM & eosinophilic neoplasms | 39 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 Tyrosine kinase gene BM/PB: Chronic Phase* with EMD BM/PB: Blast Phase No EMD BM/PB: Blast Phase with EMD EMD Only Treated, no morphologic or radiologic evidence of disease; + Cytogenetic/FISH, or molecular evidence of FGFR1 rearrangement Figure 2. Clinical presentations of MLN with eosinophilia and tyrosine kinase gene fusions. *BM/PB can present with a concurrent chronic myeloid neoplasm as well as acute lymphoblastic leukemia/lymphoma (B- or T-cell lineage) or mixed-phenotype acute leukemia. received prior treatment, only cytogenetic, FISH, or molecular evidence of a TK fusion gene may be evident as a marker to follow during subsequent therapy. The heterogeneous pre sentations of MLN with TK fusion genes are shown in Figure 2. Their heterogeneity may reflect the TK fusion gene, its part ner, or the presence of additional cytogenetic or molecular abnormalities (eg, RUNX1 muta tion in patients with FGFR1 fusions). Imatinib in PDGFRA and PDGFRB TK gene fusions FIP1L1::PDGFRA is the prototypic fusion TK gene and is almost always associated with eosinophilia. An increase in the vita min B12 and/or serum tryptase level often accompanies the finding of atypical, interstitial MCs in the BM.59-61 Most patients are male who present with a chronic myeloid neoplasm with eosinophilia; however, rare cases of AML or T-cell ALL, as well as isolated or concurrent EMD, have been reported.62 FIP1L1:: PDGFRA results from a submicroscopic deletion of 800 kb on chromosome 4q12 and is not detected by standard karyotyping.59 FISH for the CHIC2 deletion63 or reverse transcription PCR is used to identify the fusion and for serial monitoring; false-neg a tive cases using FISH have been reported and therefore PCR should be employed in such cases with high clinical suspicion.64 Studies have confirmed the deep and durable responses of FIP1L1::PDGFRA-positive disease to imatinib at starting doses of 100 mg daily.65-68 In patients with known or possible car diac involvement with eosinophils, concomitant initiation of corticosteroids (eg, prednisone 1 mg/kg for 7-10 days) with imatinib is recommended to mitigate potential complications from heart failure/cardiogenic shock. Complete hematologic 40 | Hematology 2022 | ASH Education Program remissions are achievable in >95% of patients and can achieved within several weeks; complete FISH and/or PCR responses are typically observed within 3 to 6 months. In patients achiev ing FISH or PCR negativity, maintenance dosing of 100 mg 3 times a week or weekly can main tain deep responses.69 Similar to chronic myeloid leukemia, increas ing experience is accumulating with imatinib discontinuation and the con cept of treatment-free remission in FIP1L1::PDGFRA-positive cases. In case series, hematologic and/or molecular relapse has been documented in <6 months, but in some cases, molecular remissions have been maintained for more than 5 years.70-73 In a retrospective French series, the relapse-free sur vival rate of patients who discontinued imatinib was 57% after a median follow-up of 58 months (range, 25-100 months).74 Relapses occurred after a median of 10 months (range, 4-23), consisting of hematologic (n = 18, 90%) or isolated molecular relapses (n = 2, 10%). Imatinib was successfully resumed in 17 of 20 (85%) relapsed patients, with complete hematologic remission within 1 month in allcases and molecular responses within 12 months in alltested patients.74 In multivariable anal ysis, duration of prior imatinib therapy treatment was a sta tistically significant factor for relapse while time to imatinib initiation showed a trend for significance. After a mean followup of 80 months, the 1-, 5-, and 10-year overall survival rates of imatinib-treated patients in this cohort were 99%, 95%, and 84%, respectively.74 In another study, 12 patients with FIP1L1::PDGFRA who achieved a CMR were followed after imatinib discontinuation.75 Median time of treatment and median time of CMR before imatinib discontinuation were 80 months (range, 43-175 months) and 66 months (range, 37-174 months), respectively. A molecular Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 BM/PB: Chronic Phase* No EMD JAK2, FLT3, and ETV6::ABL1 TK gene fusions In several case series, patients have been treated with ruxolitinib for JAK2 gene fusions or with FLT3 inhibitors (midostaurin, sunitinib, sorafenib, gilteritinib) for gene fusions involving FLT3.84-86 Hematologic and cytogenetic remissions have been described with JAK2 or FLT3 inhibitors in both TK-driven neoplasms, but they tend not to be durable.79-85 Since JAK2- and FLT3-targeting TKIs in these diseases have generally not provided sufficient dis ease control, the intent should be to use them as a cytoreductive bridge to HSCT. This was recently demonstrated in an infant with ETV6::FLT3 MLN who achieved a morphologic, immunophenotypic, and cytogenetic remission with gilteritinib before proceeding to transplant.87 In patients with CP presentations of ETV6::ABL1-positive disease, the limited experience to date suggests that the second-generation TKIs nilotinib and dasatinib may elicit more durable complete remissions than imatinib.86 FGFR1 inhibitors in MLN with FGFR1 gene fusions Patients with FGFR1-rearranged MLN-eo exhibit an aggres sive disease course.88,89 Individuals with chronic phase disease exhibit a cumulative rate of transformation to blast phase at 12 months approaching 50%, and 1-year OS of patients presenting with blast phase disease is 30%.89 Hydroxyurea and multikinase inhib i tors with anti-FGFR1 activ ity (eg, ponatinib, midostaurin) in CP and lineage-specific induction chemotherapy ± TKI in BP can lead to partial response (PR) or short-lived complete response (CR), but cytogenetic responses (CyR) are rare.90-92 Allogeneic HSCT is the only treatment modality that has been shown to produce long-term remissions in MLN with a FGFR1 fusion gene.93 Futibatinib is a selective inhibitor of FGFR1-4 that was eval uated in a patient with a PCM1::FGFR1 fusion.94 Futibatinib at a dose of 20 mg daily (dose reduced to 16 mg daily after 3 months for a grade 2 bullous rash) produced a durable, complete hema tologic and cytogenetic remission that was ongoing after >18 months of therapy. Futibatinib is currently being evaluated in a phase 2 study of MLN as well as solid tumors (NCT04189445). The most mature data are avail able for pemigatinib (INCB054828), a selective inhibitor of FGFR1-3. It has received regulatory approval for adult patients with previously treated, unresectable, locally advanced, or met a static cholangiocarcinoma with an FGFR2 fusion or other rearrangements.95 The ongoing FIGHT-203 study evaluating pemigatinib is a phase 2, multicenter trial enrolling adults with MLN-eo with a FGFR1 fusion TK gene. Initially, patients had to have received ≥1 prior ther apy with a starting dose of pemigatinib 13.5 mg daily in 3-week cycles (2 weeks on, 1 week off). With amendments, patients without prior therapy were also eligible and the starting dose was modified to 13.5 mg daily on a continuous schedule. The pri mary end point is CR rate; secondary end points include over all response (ORR [CR + PR]), complete CyR or partial CyR, and safety.96 All primary and secondary end points were investiga tor assessed and also adjudicated retrospectively by a Central Review Committee (CRC) with CRC-defined criteria based on local lab and radiologic results and central review of histopathol ogy and standard karyotyping/FISH. Thirty-four patients were enrolled and treated (1 subject with out an FGFR1 rearrangement was excluded from the efficacy analysis). The average number of prior therapies was 1.6 (range, 0-6); 3 patients had undergone prior transplant and 5 patients were treat ment naive.96 The lon gest dura tion of pemigatinib exposure was 192 weeks with a median dosing duration of 29 weeks. Patients completed a median of 10.0 treatment cycles (range, 2-65). At data cutoff (December 31, 2020), treatment was ongoing in 18 patients (53%); reasons for treatment discontin uation (n, %) included bridging to transplant (n=6, 18%), pro gressive disease (n=5, 15%), adverse event (n=3, 9%), physician decision (n=1, 3%), and patient decision (n=1, 3%).96 The base line fea tures of the 33 patients with FGFR1-rearranged MLN have been previously reported.96 Two patients had treated MLN with FGFR1 with a persistent cytogenetic abnormal ity only (no morphologic evidence of disease), and the remaining patients had CP (n=18) or BP (n=13) disease. Among these 31 patients, CR rates per investigator and CRC assessments were 64.5% and 77.4%, respectively; among the 33 patients evaluable for CyR, complete CyR rates were 72.7% and 75.8%, respectively (Figure 3). The most com mon treat ment-emer gent adverse events (TEAEs) were hyperphosphatemia (68%), alo pe cia (59%), diarrhea (50%), stomatitis (44%), and anemia (35%). Grade Therapies for advanced SM & eosinophilic neoplasms | 41 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 relapse was observed in 4 patients between 10 and 24 months. A second CMR was achieved in 3 patients after 3, 4, and 21 months. Eight patients (62%) are in ongoing CMR (median, 17 months; range, 3-71 months). Molecular relapse-free survival was 91% at 12 months and 65% at 24 months.75 No significant differences were identified between patients with and without molecular relapse regarding dose and duration of imatinib therapy or prior dura tion of CMR. Further prospective studies are needed to better define predictive factors for treatment-free remission in these patients. For patients with fusion tyrosine kinases involving PDGFRB, starting doses of imatinib 100 to 400 mg daily are recommended.55-57 A maintenance dose of 100 mg daily can be considered in patients achieving hematologic and molecular remissions following an induction dose of 400 mg daily. Long-term treatment outcomes with imatinib are similar to the experience with FIP1L1::PDGFRA.76 Long-term follow-up (median, 10.2 years) of PDGFRB-rearranged patients treated with imatinib for a median duration of 6.6 years showed a 96% response rate and a 10-year overall survival rate of 90%.77 None of the patients achieving a complete cytogenetic (n=13) or molecular (n=8) remission lost their response or exhibited progression to blast phase disease.77 Imatinib monotherapy can be effective in de novo presenta tions of blast phase disease with PDGFRA or PDGFRB fusion TK genes. In a case series of 17 patients in blast phase and/or with EMD, 15 patients treated with imatinib only achieved durable com plete hematologic and molecular remissions.78 Two of 17 patients (12%) died after a median follow-up of 65 months. While these data are encouraging, an alternate approach may be to combine lineage-specific induction chemotherapy + imatinib with consid eration of allografting patients in first complete remission. Primary resistance is not a clinical concern. Although uncom mon, secondary resistance almost always defaults to 2 canoni cal mutations: PDGFRA T674I or PDGFRA D842V. Although these mutations have exhibited variable sensitivity to tyrosine kinase inhibitors (TKIs) in vitro, clinical responses have been underwhelming.79-83 However, avapritinib’s activity against PDGFRA D842V in patients with GIST42 augurs promise for similar benefit in the context of FIP1L1::PDGFRA. ≥3 TEAEs in ≥10% of patients were anemia (18%) and pain in extremity and stomatitis (both 12%). Dose modifications due to any TEAE included dose inter rup tion (65%), reduc tion (59%), and discontinuation (12%: due to cardiac failure, multiple-organ dysfunction, increased alkaline phosphatase, and calciphylaxis).96 While Kaplan-Meier median durations of CR and ORR had not been reached in the overall efficacy population, clinical and cytogenetic responses in BP disease were less frequent and less durable than in patients with CP disease. The most frequent reason for treatment discontinuation was bridging to transplant, which was achieved in 23% of BP patients.96 Taken together, these data indicate that pemigatinib can pro duce durable and high rates of complete clinical and cyto genetic responses, most of whom had progressed on prior therapy. Pemigatinib may be a useful option for patients inel igible for transplant or may facilitate bridging to transplant in eligible patients. Pemigatinib was approved by the FDA on August 26, 2022, for relapsed or refractory MLN with FGFR1 rearrangement. Chronic eosinophilic leukemia In both the new ICC and WHO clas si fi ca tions,1,2 diagnos tic cri te ria for chronic eosin o philic leu ke mia (CEL) require both abnormal BM morphology (eg, dysplastic megakaryo cytes with or without dysplastic features in other lineages or increased blasts ≥5% in the bone marrow and/or ≥2% in the peripheral blood) as well as demonstration of a clonal cytogenetic abnormality and/or somatic mutation(s). In the absence of a clonal cytogenetic abnormality and/or somatic muta tion(s) or increased blasts, the afore men tioned BM findings are sufficient in the presence of persistent eosino philia, for which other causes have been excluded. The focus 42 | Hematology 2022 | ASH Education Program on abnormal BM histopathology97 is intended to distinguish CEL from idiopathic hypereosinophilic syndrome and hypereosinophilia of unknown significance. The ICC also requires persistent PB hypereosinophilia (eosinophil count ≥1.5 × 109/L and eosinophils ≥10% of white blood cells)1 while the WHO specifies the hypereosinophilia should endure for at least 4 weeks from the prior requirement of 6 months.2 The WHO also removed the CEL disease qualifier “NOS.”2 Notably, CEL is still included among the BCR::ABL1-negative MPNs, not MDS/MPNs, despite dysplastic marrow findings being a core histopathologic feature of these neoplasms. CEL lacks recurrent cytogenetic and/or molecular genetic abnormalities such as BCR::ABL1 or TK gene fusions asso ci ated with MLN-eo. Case reports and series have anno tated nonspecific abnormalities, including del(13q), del(20q), tri somy 8, monosomy 7, and complex karyotypes.98 In some CEL cases, next-generation sequencing (NGS) panels have uncov ered gene muta tions that are pro mis cu ous among mye loid neoplasms (eg, DNMT3A, ASXL1, TET2, EZH2, SETBP1, CBL, SF3B1, among others).99 It may not always be possible to dis tinguish whether some variants are drivers of eosinophilia or clonal hematopoiesis of indeterminate potential muta tions. Rarely, NGS may uncover poten tial druggable tar gets or pathways, such as the JAK-STAT signaling axis. In 1 study, the STAT5B N642H mutation was identified in 27 of 1715 (1.6%) cases referred for eosinophilia.100 Somatic insertion/deletion muta tions (p.L583_A586delinsS) within exon 13 of the pseudokinase domain of JAK2 have also been described in patients with a diagnosis of both CEL and polycythemia vera,101 and a somatic activating JAK1 R629_S632delinsSA mutation was identified in another patient with long-standing CEL.102 CEL carries a poor prognosis, with a median OS in the range of 1 to 2 years in 2 case series.98,103 No consensus standard Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 Figure 3. FIGHT-203 Trial: Rates of complete clinical and cytogenetic response with pemigatinib. Responses to pemigatinib were adjudicated by local investigators and according to a central review committee. Central FISH was given priority over local cytogenetic results during CRC adjudications. aIn the 2 patients with treated MLN with no morphologic evidence of disease but persistent cytogenetic abnormality, cytogenetic responses were adjudicated, but clinical responses were not. Table 4. Open questions and future priorities with novel agents in primary eosinophilic neoplasms • Need for response criteria for MLN with eosinophilia and tyrosine kinase gene fusions • Increasing diagnostic recognition of MLN with eosinophilia and tyrosine kinase gene fusions given the availability of targeted therapies • How do we incorporate standard cytogenetic/FISH and molecular monitoring of tyrosine kinase fusion genes into clinical decision-making? • Harmonization of molecular monitoring techniques for tyrosine kinase fusion genes • What is the optimal induction and maintenance dosing of tyrosine kinase inhibitors in specific MLN with eosinophilia and tyrosine kinase gene fusions? • What are the best predictors of treatment-free remission in PDGFRA- and PDGFRB-rearranged MLN treated with imatinib? • In FGFR1-rearranged MLN, is pemigatinib monotherapy sufficient for chronic phase disease, and what is the role of lineage-specific induction chemotherapy±pemigatinib in blast phase disease? • What is the role of pemigatinib and other tyrosine kinase inhibitors posttransplant to minimize disease relapse? • Unmet therapeutic need in CEL: evaluate HMAs and other therapies (eg, venetoclax) in the context of clinical trials frontline treatment exists for CEL. Corticosteroids, hydroxyurea, PEG-IFN-α, and imatinib can improve leukocytosis and hypereosinophilia, but responses tend not to be durable. PEG-IFN-α can elicit hematologic and cytogenetic responses, as well as reversion of end-organ damage, including in patients who are relapsed/refractory to corticosteroids and hydroxyurea.55,56 In the absence of a tyrosine kinase target, hematologic improvement with empiric use of imatinib may reflect nonspecific myelosuppression, and responses tend to be short-lived. Anti–interleukin 5 and anti–interleukin 5 receptor antibodies (mepolizumab and benralizumab, respectively), which demonstrate activity in idi opathic hypereosinophilic syndrome and lymphocyte variant hypereosinophilia, are generally not active in primary eosino philic neoplasms.104,105 As BM dysplasia is a prominent feature of bona fide CEL, the role of hypomethylating agents should be further explored in these cases. Novel agents in primary eosinophilic neoplasms: open questions and future priorities Imatinib has reversed the once poor survival of patients with PDGFRA and PDGFRB fusions into “good-risk” diseases. Despite the availability of TK inhibitors in MLN with rearranged FGFR1, JAK2, FLT3, and ABL1, responses to targeted agents are usually partial and less durable. Their natural histories generally remain poor and should be distinguished from the very favorable experi ence with imatinib in PDGFRA- and PDGFRB-rearranged disease. The use of pemigatinib as a bridg ing agent to trans plan ta tion in MLN with FGFR1 fusions exemplifies the approach that will need to be considered more broadly in MLN, especially in the BP of disease, where TKI response is often short-lived. For patients with CEL, the results of NGS may provide druggable targets in rare instances, but otherwise a treatment backbone with hypomethylating agents or PEG-IFN-α or enrollment in clin ical trials of novel agents is recommended with an eye toward transplantation when feasible. Table 4 highlights outstanding ques tions and future pri or i ties for the treat ment of pri mary eosinophilic neoplasms. Acknowledgments Dr. Gotlib expresses gratitude to the Charles and Ann Johnson Foundation for its support of MPN Research at Stanford, as well the Stanford Hematology Division, US and international SM investiga tors, and patients with SM and their families. Dr. Gotlib expresses special acknowledgment of members of the Stanford mastocytosis clinical research collaborative: Dr. William Shomali, Dr. Asiri Ediriwickrema, Cheryl Langford, Justin Abuel, Cecelia Perkins, Lenn Fechter, Annie Jinsuh Jung, Denise DeVore, Leslie Hwang, Robert Jones, Parveen Abidi, and Bhuvana Ramachandran. Conflict-of-interest disclosure Research funding: Incyte, Novartis, Kartos, Blueprint Medicines, Cogent Biosciences, Abbvie, Celgene, BMS, Protagonist Therapeutics. Advisory boards/consulting/honoraria: Incyte, Novartis, Kartos, Blueprint Medicines, Cogent Biosciences, Abbvie, Protagonist Therapeutics, PharmaEssentia, Imago Biosciences. Off-label drug use Bezuclastinib and BLU-263 are under investigation for indolent and advanced SM. Futibatinib is under clinical investigation for FGFR1-rearranged MLN. Off-label use: ruxolitinib, fedratinib, pacritinib, and momelotinib for JAK2-rearranged MLN; midostaurin and ponatinib for FGFR1-rearranged MLN; midostaurin, sorafenib, sunitinib, and gilteritinib for FLT3-rearranged MLN; and imatinib, dasatinib, nilotinib, bosutinib, ponatinib, and asciminib for ETV6::ABL1-rearrranged MLN. Correspondence Jason Gotlib, Stanford Cancer Institute, 875 Blake Wilbur Drive, Room 2324, Stanford, CA 94305-6555, USA; e-mail: jason.gotlib@ stanford.edu. 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Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness. Blood. 2003;101(12):4660-4666. 61. Pardanani A, Brockman SR, Paternoster SF, et al. FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia. Blood. 2004;104(10):3038-3045. 62. Metzgeroth G, Walz C, Score J, et al. Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma. Leukemia. 2007;21(6):1183-1188. 63. Pardanani A, Ketterling RP, Brockman SR, et al. CHIC2 deletion, a surrogate for FIP1L1-PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy. Blood. 2003;102(9):3093-3096. 64. Olsson-Arvidsson L, Norberg A, Sjögren H, Johansson B. Frequent falseneg a tive FIP1L1-PDGFRA FISH ana ly ses of bone mar row sam ples from clonal eosinophilia at diagnosis. Br J Haematol. 2020;188(5):e76-e79. 65. Klion AD, Robyn J, Akin C, et al. Molecular remission and reversal of mye lo fi bro sis in response to imatinib mesylate treat ment in patients with the myeloproliferative variant of hypereosinophilic syndrome. Blood. 2004;103(2):473-478. 66. Baccarani M, Cilloni D, Rondoni M, et al. The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRalpha-positive hypereosinophilic syndrome. Results of a multicenter prospective study. Haematologica. 2007;92(9): 1173-1179. 67. Jovanovic JV, Score J, Waghorn K, et al. Low-dose imatinib mesylate leads to rapid induction of major molecular responses and achievement of com plete molecular remission in FIP1L1-PDGFRA-positive chronic eosinophilic leukemia. Blood. 2007;109(11):4635-4640. 68. Gotlib J, Cools J. Five years since the discovery of FIP1L1-PDGFRA: what we have learned about the fusion and other molecularly defined eosinophilias. Leukemia. 2008;22(11):1999-2010. 69. Helbig G, Stella-Hołowiecka B, Majewski M, et al. A single weekly dose of imatinib is sufficient to induce and maintain remission of chronic eosin ophilic leukaemia in FIP1L1-PDGFRA-expressing patients. Br J Haematol. 2008;141(2):200-204. 70. Pardanani A, Ketterling RP, Li C-Y, et al. FIP1L1-PDGFRA in eosin o philic disorders: prevalence in routine clinical practice, long-term experience with imatinib therapy, and a critical review of the literature. Leuk Res. 2006;30(8):965-970. 71. Klion AD, Robyn J, Maric I, et al. Relapse fol low ing dis con tin u a tion of imatinib mesylate ther apy for FIP1L1/PDGFRA-pos i tive chronic eosin o philic leukemia: implications for optimal dosing. Blood. 2007;110(10):35523556. 72. Pardanani A, D’Souza A, Knudson RA, Hanson CA, Ketterling RP, Tefferi A. Long-term follow-up of FIP1L1-PDGFRA-mutated patients with eosinophilia: survival and clinical outcome. Leukemia. 2012;26(11):2439-2441. 73. Helbig G, Kyrcz-Krzemień S. Cessation of imatinib mesylate may lead to sustained hematologic and molecular remission in FIP1L1-PDGFRA-mutated hypereosinophilic syndrome. Am J Hematol. 2014;89(1):115. 46 | Hematology 2022 | ASH Education Program 102. Shomali W, Damnernsawad A, Theparee T, et al. A novel activating JAK1 mutation in chronic eosinophilic leukemia. Blood Adv. 2021;5(18):35813586. 103. Helbig G, Soja A, Bartkowska-Chrobok A, Kyrcz-Krzemień S. Chronic eosinophilic leukemia-not otherwise specified has a poor prognosis with unresponsiveness to conventional treatment and high risk of acute trans formation. Am J Hematol. 2012;87(6):643-645. 104. Kuang FL, Fay MP, Ware J, et al. Long-term clinical outcomes of high-dose mepolizumab treatment for hypereosinophilic syndrome. J Allergy Clin Immunol Pract. 2018;6(5):1518-1527.e51527e5. 105. Kuang FL, Legrand F, Makiya M, et al. Benralizumab for PDGFRA-negative hypereosinophilic syndrome. N Engl J Med. 2019;380(14):1336-1346. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000368 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/34/2021847/34gotlib.pdf by guest on 09 December 2022 97. Wang SA, Hasserjian RP, Tam W, et al. Bone marrow morphology is a strong discriminator between chronic eosinophilic leukemia, not other wise specified and reactive idiopathic hypereosinophilic syndrome. Haematologica. 2017;102:1352-1360. 98. Morsia E, Reichard K, Pardanani A, Tefferi A, Gangat N. WHO defined chronic eosinophilic leukemia, not otherwise specified (CEL, NOS): a contempo rary series from the Mayo Clinic. Am J Hematol. 2020;95(7):E172-E174. 99. Wang SA, Tam W, Tsai AG, et al. Targeted next-generation sequencing identifies a subset of idiopathic hypereosinophilic syndrome with fea tures similar to chronic eosinophilic leukemia, not otherwise specified. Mod Pathol. 2016;29(8):854-864. 100. Cross NCP, Hoade Y, Tapper WJ, et al. Recurrent acti vat ing STAT5B N642H mutation in myeloid neoplasms with eosinophilia. Leukemia. 2019;33(2):415-425. 101. Patel AB, Franzini A, Leroy E, et al. JAK2 ex13InDel drives oncogenic trans formation and is associated with chronic eosinophilic leukemia and poly cythemia vera. Blood. 2019;134(26):2388-2398. ANXIETY PROVOKING CONSULTATIONS: MAST CELLS AND EOSINOPHILS Approach to the patient with suspected hypereosinophilic syndrome Human Eosinophil Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD Hypereosinophilic syndromes (HES) are a heterogenous group of rare disorders with clinical manifestations ranging from fatigue to life-threatening endomyocardial fibrosis and thromboembolic events. Given the broad differential diagnosis of HES, a comprehensive approach is needed to identify potential secondary (treatable) causes and define end-organ manifestations. Classification by clinical HES subtype is also useful in terms of assessing prognosis and guiding therapy. Corticosteroids remain the mainstay of initial therapy in the setting of acute, life-threatening PDGFR mutation-negative HES. Whereas the recent availability of eosinophil-targeted therapies with extraordinary efficacy and little apparent toxicity is changing the treatment paradigm, especially for idiopathic HES and overlap syndromes, questions remain unanswered regarding the choice of agent, impact of combination therapies, and long-term effects of eosinophil depletion. This review provides a case-based discussion of the differential diagnosis of HES, including the classification by clinical HES subtype. Treatment options are reviewed, including novel eosinophil-targeted agents recently approved for the treatment of HES and/or other eosinophil-associated disorders. Primary (myeloid) disorders associated with hypereosinophilia are not be addressed in depth in this review. LEARNING OBJECTIVES • To describe the heterogeneity of clinical presentations of hypereosinophilia • To discuss the approach to targeted therapy of hypereosinophilic syndrome CLINICAL CASE A 38yearold previously healthy woman presented with intermittent but intense pruritus without rash. The pruritus initially involved only her ankles but subsequently spread up her legs with accompanying angioedema of the thighs and buttocks that prevented her from being able to wear pants. Antihistamines were ineffective, and a short course of solumedrol prescribed for sinusitis did not relieve the pruritus or swelling. A dermatologist prescribed oral and topical antibiotics for presumed folliculitis without improvement. One year after the onset of the symptoms, a complete blood count was performed and revealed ane mia, thrombocytopenia (platelets 34 000), and eosinophilia (14.0×109/L). She was referred to hematology. Differential diagnosis and initial evaluation Eosinophilia, defined as an absolute eosinophil count (AEC) >0.45 × 109/L, is quite common, occurring in 1% to 2% of the general population.1 In contrast, hypereosino philia (HE; AEC ≥1.5 × 109/L) is extremely rare, with an esti mated incidence of 0.315 to 6.3 per 100 000 in the United States.2 The potential etiologies of eosinophilia (including HE) are varied and include allergic, infectious, neoplas tic, genetic, and immune disorders (Table 1). Moreover, clinical symptoms are extremely heterogeneous. Derma tologic, pulmonary, and gastrointestinal manifestations are most frequently reported, but any organ system can be affected, and progression can occur over time with out effective therapy.3 A careful and complete history and physical examination, including prior complete blood counts (if available), medication and travel history, assess ment of cancer risk factors, and family history, is essential to narrow the differential. Initial laboratory and diagnos tic testing should include complete blood count with differential, routine chemistries, serum immunoglobulin levels, B12 and tryptase, and assessment of lymphocyte clonality and phenotype. If there is a possible history of Strongyloides exposure, no matter how remote, serologic testing should be performed and/or empiric ivermectin (150 µg/kg × 1 dose) administered to prevent potentially Hypereosinophilia and hypereosinophilic syndromes | 47 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 Amy D. Klion Table 1. Disorders associated with marked eosinophilia Examples Comments Asthma; atopic dermatitis; chronic rhinosinusitis Typically cause mild to moderate eosinophilia Drug hypersensitivity Varied; drug rash, eosinophilia, and systemic symptoms; eosinophilia-myalgia syndrome Can occur with any drug or supplement; manifestations range from asymptomatic eosinophilia to life-threatening complications40 Infection and infestation Helminth infection (especially those with a tissue invasive phase) Fungal infections Viral infection (HIV, COVID-19) Ectoparasite infestation Protozoal infection (limited to Sarcocystis and Cystoisospora) Tuberculosis (rare) Most common etiology worldwide; Strongyloides infection should always be considered due to worldwide distribution and the potential for fatal dissemination with steroid therapy41 Autoimmune and immunodysregulatory disorders Inflammatory bowel disease, sarcoidosis, IgG4 disease Clinical sequelae of eosinophilia may or may not be present and can be difficult to distinguish from the manifestations of the underlying disorder Neoplasia Leukemia, lymphoma, solid tumors (especially adenocarcinoma) Although any leukemia/lymphoma can present with HE/HES, pre–B-cell acute lymphoblastic leukemia can be particularly difficult to diagnose Inborn errors of immunity Omenn syndrome, DOCK8 deficiency, Loeys-Dietz syndrome Usually diagnosed in childhood, recurrent or unusual infections common42 Rare hypereosinophilic syndromes Eosinophilic myeloid neoplasms, lymphocytic variant HE/HES, idiopathic HE/HES, familial HE/HES, single-organ HE/HES, and other overlap disorders See Figure 2 for additional details Other Radiation, hypoadrenalism, cholesterol emboli, administration of IL-2 COVID-19, coronavirus disease 2019; HIV, human immunodeficiency virus. fatal hyperinfection syn drome. Bone mar row biopsy and chest/abdo men/pel vis imag ing should be strongly con sid ered in any patient with AEC ≥1.5 × 109/L and no clear secondary cause of the HE. Additional testing, including testing for para sitic infections other than Strongyloides, should be guided by the clinical history and disease manifestations. CLINICAL CASE (Cont inu ed) A bone mar row biopsy spec i men was hypercellular with increased eosinophils and plasma cells. Testing for FIP1L1::PDGFRA was negative. She was treated with prednisone 60mg daily for presumed idiopathic thrombocytopenic purpura with clinical and hematologic improvement. However, as the prednisone was tapered, the eosinophilia, pruritus, and edema returned. She was referred to Mayo Clinic for further evaluation, which included an indeterminate serologic test for Strongyloides. She was treated with 2 doses of ivermectin and a 2-week course of albendazole. During this time, the prednisone was slowly tapered despite a rising AEC, peaking at 26.0×109/L on prednisone 5mg every other day, and recurrent symptoms. The prednisone dose was increased to 25mg daily, and hydroxyurea therapy (500mg twice daily) was initiated. This was ineffective, and she was referred to the National Institutes of Health for further evaluation. 48 | Hematology 2022 | ASH Education Program At the time of referral, she complained of fatigue, swelling, and extreme pruritus. Physical examination revealed symmet ric nonpitting edema of the thighs and exco ri a tions pre dom i nantly on the lower legs. Laboratory testing was notable for AEC 3.01×109/L; platelets 114 000; markedly elevated IgG, IgM, and IgE; and normal serum B12 and tryptase. Computed tomography (CT) scan was notable for borderline splenomegaly and minimal dif fuse lymphadenopathy. Repeat bone marrow again showed only increased eosinophils. Mast cells were not increased, and testing for D816V KIT was negative. T-cell receptor testing by polymerase chain reaction showed a clonal pattern, and flow cytometry was notable for an aberrant CD3–CD4+CD10+ T-cell population, con sistent with a diagnosis of lymphocytic variant hypereosinophilic syndrome (HES). B-cell clonality studies were negative. She was started on interferon α 1mU daily with resolution of eosinophilia, platelets >50 k and symptomatic improvement. Definition and classification of HES The definition of HES has evolved over time since Chusid’s land mark description of 14 patients with idiopathic HE and varied clin ical manifestations in 1975.4 Whereas the current World Health Organization definition uses the term HES to describe only idi opathic HE with clinical manifestations,5 a recently updated consensus definition provides a broader approach that recog nizes the overlap in clinical presentation between idiopathic and other types of HES, the imperfect sensitivity and specificity of Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 Category Atopic disorders Table 2. Definitions of hypereosinophilic syndrome WHO definition of HES Consensus definition AEC >1.5×109/L for greater than 6 months AEC >1.5×109/L on 2 examinations at least 1 month apart and/or tissue HE Evidence of end-organ manifestations Organ damage and/or dysfunction attributable to tissue HE and exclusion of other conditions as a major reason for organ damage “Idiopathic”—exclusion of reactive HE, lymphocytic variant HES, CEL-NOS, WHO-defined malignancies, eosinophilia-associated MPNs, or AML/ALL with rearrangements of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 Includes multiple clinical classifications, including idiopathic, primary (clonal/neoplastic), secondary (presumed cytokine driven—including lymphocytic variant), and hereditary ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; CEL-NOS, chronic eosinophilic leukemia-not otherwise specified; MPN, myeloproliferative neoplasm; WHO, World Health Organization. the peripheral blood. Expanded surface phenotyping is often necessary to demonstrate and/or confirm the aberrant popula tion.12,13 It is important to recognize that the surface phenotype of the clonal population in LHES can be indistinguishable from that seen in T-cell malignancies (especially angioimmunoblastic T-cell lymphoma and cutaneous T-cell lymphoma).14 Thus, LHES is a diagnosis of exclusion. Moreover, progression of LHES to a lymphoid malignancy occurs in approximately 10% of patients, sometimes after many years of stable disease.15-17 Consequently, at a minimum, patients with LHES should undergo assessment for occult lymphoma at diagnosis and in the setting of an increase in size of the clonal T-cell population or development of resistance to previously effective therapy. Approach to therapy Despite the differences in definitions, the general approach to HE is very similar between World Health Organization and the consensus group (Figure 1). Since secondary causes of eosin ophilia, such as helminth infection, typically require a different therapeutic approach, these should be considered early in the diagnostic process. If an underlying etiology is identified or highly suspected, specific treatment should be initiated. Primary (clonal/neoplastic) eosinophilia is also important to identify early due to prognostic and therapeutic implications.18 Finally, the presence and severity of clinical manifestations should be assessed as this will affect both the nature and urgency of ther apeutic intervention. For example, careful monitoring without therapy may be appropriate for asymptomatic HE without evi dence of end-organ involvement (hypereosinophilia of undeter mined significance),19 whereas urgent intervention is needed in the context of myocarditis or thromboembolism. Prednisone remains the mainstay of therapy in the acute setting for severe and/or life-threatening manifestations of HES. If the eosin o philia does not dra mat i cally decrease within 24 to 48 hours, additional therapy should be consid ered depending on the suspected clinical subtype (ie, imati nib for patients with clinical findings suggestive of a myeloid neoplasm, cyclophosphamide for patients with manifestations suggestive of eosinophilic granulomatosis with polyangiitis). The use of eosin o phil-targeting bio log ics in the acute set ting remains controversial but is supported by case reports and small series.20 Once the patient is stable, further evalua tion should focus on the identification of the most likely clin i cal sub type (Table 3). With the excep tion of patients with myeloid HES,18 most patients with symptomatic HES respond Hypereosinophilia and hypereosinophilic syndromes | 49 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 available diagnostic testing, and the identification of new eti ologies of HES over time6 (Table 2). In this consensus definition, a diagnosis of HE requires AEC >1.5×109/L on 2 examinations at least 1 month apart (to exclude laboratory error but allow diag nosis without a delay of 6 months) and/or tissue HE (to recog nize the arbitrary nature of the AEC cutoff in the setting of clear eosinophil-mediated disease). HES is defined as HE with evi dence of end-organ dysfunction attributable to the eosinophilia, irrespective of the cause. To address the heterogeneity of disor ders included in this umbrella definition of HES and help guide diagnostic procedures and therapeutic choices, the following clin i cal sub types have been pro posed (Table 3)7: (1) mye loid HE/HES (suspected or proven eosinophilic myeloid neoplasm, including those associated with rearrangements of PDGFRA and other recurrent molecular abnormalities), (2) lymphocytic variant HE/HES (presence of a clonal or phenotypically aberrant T-cell population that produces cytokines that drive the eosinophilia), (3) overlap HES (single-organ-restricted eosinophilic disorders and clin i cally defined eosin o philic syn dromes that over lap in presentation with idiopathic HES; ie, eosinophilic gastrointesti nal disorders, eosinophilic granulomatosis with polyangiitis), (4) associated HE/HES (in the context of a defined disorder, such as a helminth infection, neoplasm, immunodeficiency, or hyper sensitivity reaction), (5) familial HE/HES (occurrence in >1 fam ily member excluding associated HE/HES), and (6) idiopathic HE/HES (unknown cause and exclusion of other subtypes). Lymphocytic variant HES (LHES) was first described in 1994 in a 30-year-old man with pruritus and cough and a large CD2+CD3– CD4+ T-cell population that produced interleukin (IL) 4 and IL-5 in response to stimulation.8 Since that time, other surface phe no types have been described, and there have been sev eral informative case series describing the clinical and laboratory findings of patients with LHES.9-11 Equally frequent in males and females, LHES most often presents with dermatologic manifes tations. That said, any organ system can be involved, and some patients with asymptomatic HE have clonal aberrant T-cell pop ulations indistinguishable from those with symptomatic LHES. Serum IgM, IgE, and serum and thymus and activation-regulated chemokine levels are elevated in most patients with LHES and can provide useful diagnostic clues.12 Whereas the gold standard for diagnosis of LHES is identification of a clonal and/or aber rant population of T cells producing type 2 cytokines, intracel lular flow cytometry is not available at most centers, and the diagnosis most often relies on a compatible clinical picture and demonstration of a clonal and/or aberrant T-cell population in Table 3. Initial assessment of the patient with hypereosinophilia Comments Comprehensive history and physical examination Including prior eosinophil counts, medications, travel/exposure history Complete blood count with differential and smear* Dysplastic eosinophils, other lineage involvement, and/or presence of myeloid precursors are suggestive of (but not diagnostic for) MHES Routine chemistries, including liver function tests* To assess end organ involvement Quantitative serum immunoglobulin levels IgE levels are typically elevated in a variety of conditions (ie, LHES, EGPA, parasitic infections, and some immunodeficiencies); IgM levels are elevated in LHES and episodic angioedema and eosinophilia Serum tryptase and B12 levels Elevated serum B12 levels can be seen in many myeloid neoplasms; elevated serum tryptase is near universal in PDGFRA and KIT-associated disease T- and B-cell receptor rearrangement studies*; lymphocyte phenotyping by flow cytometry* (see Carpentier et al11)† Clonal and/or aberrant T-cell populations are characteristic of LHES and some types of lymphoma. Clonal B cells are suspicious for B-cell neoplasm, including pre–B-cell acute lymphoblastic leukemia in children/adolescents. Serum troponin,* electrocardiogram, and echocardiogram If abnormal, cardiac MRI should be considered Chest/abdomen/pelvis CT* To assess for splenomegaly, lymphadenopathy, asymptomatic pulmonary involvement, and occult neoplasms Biopsy of affected tissues (if possible)* Cardiac tissue involvement can be patchy, limiting the utility of cardiac biopsy Selected patients with HE/HES Pulmonary function tests* Any patient with suspected pulmonary involvement or abnormal findings on chest CT Bone marrow aspirate and biopsy* All patients with AEC >5.0×109/L and/or features suggestive of LHES or MHES; patients with clear diagnoses, such as EGPA or parasitic infection, may not need bone marrow testing despite AEC >5.0×109/L Testing for BCR::ABL1, FIP1L1::PDGFRA, and translocations/mutations involving PDGFRB, JAK2, FGFR1, and KIT Testing should be guided by results of initial testing and bone marrow examination; allpatients with elevated serum tryptase and/or B12 levels should be tested for FIP1L1::PDGFRA NGS myeloid panel; targeted or whole-exome sequencing; other genetic testing Depending on initial evaluation PET scan,* EBV viral load Particularly in patients with suspected LHES Other testing for secondary causes As indicated by clinical history and physical examination *Can be dramatically affected by corticosteroid therapy. †Not allpatients with LHES will have clonal or aberrant T-cell populations detectable by routine testing. CT, computed tomography; EBV, Epstein-Barr virus; EGPA, eosinophilic granulomatosis and polyangiitis; MHES, myeloid variant hypereosinophilic syndrome; MRI, magnetic resonance imaging; NGS, next-generation sequencing; PET, positron emission tomography. rapidly to corticosteroid therapy, although toxicity and resis tance limit the utility of this therapy in the long term.3 Conven tional second-line agents, including hydroxyurea and interferon α, are fraught with similar issues but have advantages in select populations/clinical HES subtypes (Tables 3 and 4). CLINICAL CASE (Cont inu ed) Over the next 4 years, she remained relatively stable on inter feron α therapy with partially controlled symptoms, AEC <1.0 to 2.0 × 109/L, but was unable to taper prednisone below 12.5 mg daily without significant worsening. Her clonal T-cell popu lation increased to approximately 30% of total lymphocytes, prompting repeat CT scan and bone mar row, which were unchanged, and positron emission tomography (PET)/CT scan, 50 | Hematology 2022 | ASH Education Program which showed no evidence of lymphoma. She was enrolled on a phase 2 placebo-controlled trial of benralizumab. Eosinophil-targeted therapies and HES The avail abil ity of bio log ics targeting IL-5 (mepolizumab and reslizumab) and its recep tor (benralizumab), allof which are approved by the US Food and Drug Administration for the treatment of asthma, has profoundly altered the approach to the treatment of idiopathic, lymphocytic, and overlap variants of HES. Whereas corticosteroids are still recommended as ini tial therapy in most cases, eosinophil-targeting biologics have shown excellent safety and efficacy profiles in the treatment of HES,21-23 leading to the recent approval of mepolizumab for HES and eosinophilic granulomatosis with polyangiitis and the initi ation of phase 3 trials of benralizumab for the same indications. Of note, mepolizumab and reslizumab cause maturational arrest Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 All patients with confirmed HE in the bone marrow with dramatic but incomplete reduction of blood and tissue eosinophilia. In contrast, benralizumab, an afu cosylated monoclonal antibody to IL-5 receptor α, targets eosin ophils, basophils, and their precursors for antibody-dependent cell-medi ated cyto tox ic ity, resulting in com plete (or near- com plete) deple tion in alltis sues stud ied to date. Although definitive data are lacking, what little information is available concerning the efficacy of these agents in myeloid forms of HES is discouraging.22,24,25 Other agents that directly or indirectly decrease blood and tissue eosinophilia are in development for HES and/or approved for select eosinophilic indications (Table 4). These include liren telimab (an afucosylated anti body to Siglec-8 that depletes eosin o phils and baso phils and inhib its mast cell acti va tion),26 dupilumab (a monoclonal antibody to IL-4 receptor α approved for asthma, atopic dermatitis, chronic rhinosinusitis, and eosin o philic esophagitis that blocks IL-4 and IL-13 sig nal ing and eotaxin-mediated tissue migration of eosinophils),27 and dex pramipexole (an orally available small molecule that causes mat urational arrest and eosinophil depletion through an unknown mechanism).28 CLINICAL CASE (Cont inu ed) After an initial response to benralizumab, interferon α was dis continued. Two weeks later, eosin o philia and severe symp toms returned. Benralizumab was discontinued, and she was started on prednisone 60 mg in addition to interferon α. She subsequently developed acute sensorineural hearing loss that resolved with cessation of interferon α and a prednisone burst. Cyclosporine was added. Due to persistent symptoms and inability to taper prednisone below 20 mg daily, she was enrolled on a phase 3 placebo-controlled trial of mepolizumab. Her symptoms improved, and she was able to taper prednisone to 9 mg daily, at which point she developed cough and short ness of breath requir ing hos pi tal admis sion. Evaluation was notable for ground-glass infiltrates, diffuse lymphadenopa thy, and spleno meg aly. Lymph node biopsy (approx i ma tely 10 years after her initial diagnosis) revealed angioimmunoblastic T-cell lymphoma. She was treated with CHOP-R (cyclophospha mide, doxorubicin hydrochloride, vincristine sulfate [Oncovin], prednisone, and rituximab) with transient response. Romidep sin was added, but she developed right-sided heart failure and died of respiratory failure. Predictors of response to targeted therapy There is currently little available information to guide the initial choice of biologic for a patient with HES. Although AEC has been shown to predict response to IL-5/IL-5 receptor targeting agents in patients with asthma, neither serum IL-5 levels nor eosinophil count at initiation of treatment were found to predict response to mepolizumab in the phase 3 trial in patients with PDGFRAnegative, corticosteroid-responsive HES,29 and neither historic peak nor baseline AEC predicted response to benralizumab in a phase 2 trial in patients with PDGFRA-negative, treatmentrefractory HES.22 The only consistent finding across trials has been differences across clinical HES subtypes, with decreased response rates and/or increased relapse rates in patients with lymphocytic variant HES22,25,30-32 (Figure 2). That said, responses to the different biologics targeting the IL-5 axis are variable, and a lack of response to one agent does not preclude success with another.32 Potential risks of eosinophil depletion Over the past 10 to 15 years, it has become increasingly appar ent that eosin o phils play an impor tant role in homeo static processes, including tissue remodeling, tumor surveillance, Hypereosinophilia and hypereosinophilic syndromes | 51 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 Figure 1. Initial approach to the patient with hypereosinophilic syndrome. Table 4. Selected therapeutic agents for the treatment of hypereosinophilic syndromes Selected agents Target Comments Oral prednisone and equivalents Topical CS (ie, swallowed budesonide) NA First-line therapy in most types of HES; although most patients respond initially, toxicity and resistance are limiting long term May be sufficient in single-organ eosinophilic disorders, such as eosinophilic esophagitis, and eosinophilic dermatitis Cytotoxic Hydroxyurea Methotrexate Cyclophosphamide Dividing cells Conventional second-line therapy for idiopathic HES* and some MHES; can be used at high dose to rapidly lower counts; inexpensive but significant toxicity Steroid-sparing agent most commonly used in EGPA and other rheumatologic overlap disorders* Mostly used for steroid-refractory or life-threatening EGPA Immunomodulatory Interferon α Cyclosporine Biologics Mepolizumab* Reslizumab Benralizumab Dupilumab Lirentelimab IL-5 IL-5 IL-5 receptor IL-4 receptor Siglec-8 Approved for the treatment of HES and EGPA at 300mg monthly19,21 Likely to be comparable to mepolizumab; approved for asthma with weight-based dosing In phase 3 trials for HES; approved for asthma Blocks tissue eosinophilia but may cause peripheral eosinophilia and, rarely, eosinophilic compli cations; approved for chronic rhinosinusitis, atopic dermatitis, and, most recently, eosinophilic esophagitis Depletes eosinophils and prevents mast cell degranulation; in clinical development Tyrosine kinase inhibitors Imatinib Ruxolitinib Multiple JAK Approved for the treatment of HES; near 100% efficacy in PDGFRassociated myeloid neoplasms; some efficacy in other HES with myeloid features17 Approved for several myeloid disorders typically associated with JAK2 mutations; case reports suggest that it may be useful in JAK2-associated HES and LHES Novel targeted Dexpramipexole Causes maturational arrest at eosinophilic promyelocyte stage via unknown mechanism In clinical development for HES; phase 2 study promising Conventional second-line therapy for LHES and some idiopathic HES; substantial toxicity* Alternative second-line agent, especially in LHES; renal toxicity is a significant problem* *Mepolizumab, which is approved for the treatment of HES, has better efficacy and lower toxicity but is expensive and not available in allcountries. Primary outcome of the phase 3 trial of mepolizumab for PDGFRA-negative, steroid-responsive HES in adults: reduction of disease flares over a 32-week period in patients receiving mepolizumab and stable background therapy compared with those receiving placebo and stable background therapy.21 Dual primary outcomes of the phase 3 trial of mepolizumab for relapsing or treatment-refractory EGPA in adults: total accrued weeks of remission, defined as a Birmingham vasculitis score of 0 on less than 4mg prednisone daily for 52 weeks, and proportion of participants in remission at weeks 36 and 48.23 CS, corticosteroid; EGPA, eosinophilic granulomatosis and polyangiitis; MHES, myeloid variant hypereosinophilic syndrome. † 52 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 Class CS confirm the safety of these agents over the long term and to assess the impact of blocking multiple lineages and/or pathways on homeostatic processes. Acknowledgment This work was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Conflict-of-interest disclosure Amy D. Klion: no competing financial interests to declare. Off-label drug use Figure 2. Clinical subtypes of hypereosinophilic syndrome: frequency distribution of 554 patients referred to the National Institutes of Health for unexplained eosinophilia. EGID, eosino philic gastrointestinal disease; EGPA, eosinophilic granulomato sis and polyangiitis; EO FASCIITIS, eosinophilic fasciitis. Correspondence Amy D. Klion, Human Eosinophil Section, Deputy Chief, Labo ratory of Parasitic Diseases, National Institute of Allergy and In fectious Diseases, National Institutes of Health, Building 4, Room B1-27, 4 Memorial Drive, Bethesda, MD 20892; e-mail: aklion@ nih.gov. References metabolic function, and immunoregulation.33 Although this has led to theoretical concerns about the effects of long-term ther apy with biologics and other agents that significantly reduce eosinophils in blood and tissue, data to date suggest that eosin ophil depletion in humans is safe.25,34-37 Importantly, there have been no reports of an increased inci dence of malig nancy or autoimmune disease related to eosinophil depletion, and even subtle immunologic consequences demonstrated in murine models, such as impaired vaccine responses, have not been rep licated in human studies.22,34,38,39 This lack of significant toxicity is likely due, at least in part, to the redundancy and complexity of the human immune system, which raises potential concerns as the number of targeted therapies and biologics increases and the use of combination therapies becomes more common. It is also important to note that toxicities may be restricted to spe cific populations or clinical settings (ie, patients exposed to hel minth infection, infected with coronavirus disease 2019, or at high risk of autoimmune disease), for which there are little to no prospective data to date. Conclusions Whereas eosinophilia is common in the general population, HES are a heterogeneous and complex group of rare disorders with clinical manifestations that span the range of medical subspe cialties. Comprehensive clinical evaluation is necessary both to assess end-organ man i fes ta tions and deter mine the most likely etiology and/or clinical subtype of HES, as this informa tion has important therapeutic and prognostic implications. Although cor ti co ste roids con tinue to be first-line ther apy in most situations, novel targeted therapies are rapidly replacing conventional cytotoxic and broad immunosuppressive agents as second-line agents of choice for the treatment of eosinophilassociated clinical manifestations. Despite the lack of safety sig nals to date, vigilance and prospective studies are needed to 1. Sunadome H, Sato S, Matsumoto H, et al. Similar distribution of periph eral blood eosinophil counts in European and East Asian populations from investigations of large-scale general population studies: the Nagahama Study. Eur Respir J. 2021;57(1):2004101. 2. Crane MM, Chang CM, Kobayashi MG, Weller PF. Incidence of myelopro liferative hypereosinophilic syndrome in the United States and an esti mate of allhypereosinophilic syndrome incidence. J Allergy Clin Immunol. 2010;126(1):179-181. 3. Ogbogu PU, Bochner BS, Butterfield JH, et al. Hypereosinophilic syn drome: a multicenter, retrospective analysis of clinical characteristics and response to therapy. J Allergy Clin Immunol. 2009;124(6):1319-1325.e3. 4. Chusid MJ, Dale DC, West BC, Wolff SM. The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature. Medicine (Baltimore). 1975;54(1):1-27. 5. Shomali W, Gotlib J. World Health Organization-defined eosinophilic disor ders: 2022 update on diagnosis, risk stratification, and management. Am J Hematol. 2022;97(1):129-148. 6. Valent P, Klion AD, Horny H-P, et al. Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes. J Allergy Clin Immunol. 2012;130(3):607-612.e9. 7. Klion AD. How I treat hypereosinophilic syndromes. Blood. 2015;126(9):10691077. 8. Cogan E, Schandené L, Crusiaux A, Cochaux P, Velu T, Goldman M. Brief report: clonal proliferation of type 2 helper T cells in a man with the hyper eosinophilic syndrome. N Engl J Med. 1994 Feb 24;330(8):535-538. 9. Simon HU, Plötz SG, Dummer R, Blaser K. Abnormal clones of T cells produc ing interleukin-5 in idiopathic eosinophilia. N Engl J Med. 1999;341(15):11121120. 10. Carpentier C, Verbanck S, Schandené L, et al. Eosinophilia associated with CD3-CD4+ T cells: characterization and outcome of a single-center cohort of 26 patients. Front Immunol. 2020;11:1765. 11. Lefèvre G, Copin M-C, Staumont-Sallé D, et al. The lymphoid variant of hypereosinophilic syndrome: study of 21 patients with CD3-CD4+ aberrant T-cell phenotype. Medicine. 2014;93(17):255-266. 12. Carpentier C, Schandené L, Dewispelaere L, Heimann P, Cogan E, Roufosse F. CD3-CD4+ lymphocytic variant hypereosinophilic syndrome: diagnostic tools revisited. J Allergy Clin Immunol Pract. 2021;9(6):2426-2439.e72439e7. 13. Ledoult E, Groh M, Kahn J-E, et al; CEREO—French National Reference Cen ter for Hypereosinophilic Syndromes. Assessment of T-cell polarization on the basis of surface marker expression: diagnosis and potential therapeutic implications in lymphocytic variant hypereosinophilic syndrome. J Allergy Clin Immunol Pract. 2020;8(3):1110-1114.e21114e2. Hypereosinophilia and hypereosinophilic syndromes | 53 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 Amy D. Klion: all of the drugs discussed, with the exception of imatinib and mepolizumab, are considered off-label for the treat ment of hypereosinophilic syndromes. 54 | Hematology 2022 | ASH Education Program 29. Rothenberg ME, Roufosse F, Faguer S, et al. Mepolizumab reduces hyper eosinophilic syndrome flares irrespective of blood eosinophil count and IL-5 [published online ahead of print 12 May 2022]. J Allergy Clin Immunol Pract. 30. Williams AK, Dou C, Chen LYC. Treatment of lymphocyte-variant hypereo sinophilic syndrome (L-HES): what to consider after confirming the elusive diagnosis. Br J Haematol. 2021;195(5):669-680. 31. Roufosse F, de Lavareille A, Schandené L, et al. Mepolizumab as a cortico steroid-sparing agent in lymphocytic variant hypereosinophilic syndrome. J Allergy Clin Immunol. 2010;126(4):828-835.e3. 32. Chen MM, Roufosse F, Wang SA, et al. An international, retrospective study of off-label biologic use in the treatment of hypereosinophilic syndromes. J Allergy Clin Immunol Pract. 2022;10(5):1217-1228.e31228e3. 33. Wen T, Rothenberg ME. The regulatory function of eosinophils. Microbiol Spectr. 2016;4(5):10.1128/microbiolspec.MCHD-0020-2015. 34. Kuang FL, Bochner BS. Lessons learned from targeting eosin o phils in human disease. Semin Immunopathol. 2021;43(3):459-475. 35. Khatri S, Moore W, Gibson PG, et al. Assessment of the long-term safety of mepolizumab and durability of clinical response in patients with severe eosinophilic asthma. J Allergy Clin Immunol. 2019;143(5):1742-1751.e71751e7. 36. Korn S, Bourdin A, Chupp G, et al. Integrated safety and efficacy among patients receiving benralizumab for up to 5 years. J Allergy Clin Immunol Pract. 2021;9(12):4381-4392.e44392e4. 37. Beck LA, Deleuran M, Bissonnette R, et al. Dupilumab provides accept able safety and sustained efficacy for up to 4 years in an open-label study of adults with moderate-to-severe atopic dermatitis. Am J Clin Dermatol. 2022;23(3):393-408. 38. Zeitlin PL, Leong M, Cole J, et al; ALIZE study investigators. Benralizumab does not impair antibody response to seasonal influenza vaccination in adolescent and young adult patients with moderate to severe asthma: results from the phase IIIb ALIZE trial. J Asthma Allergy. 2018;11:181-192. 39. Manetz S, Maric I, Brown T, et al. Successful pregnancy in the setting of eosinophil depletion by benralizumab. J Allergy Clin Immunol Pract. 2021;9(3):1405-1407.e31407e3. 40. Hama N, Abe R, Gibson A, Phillips EJ. Drug-induced hypersensitivity syn drome (DIHS)/drug reac tion with eosin o philia and sys temic symp toms (DRESS): clinical features and pathogenesis. J Allergy Clin Immunol Pract. 2022;10(5):1155-1167.e51167e5. 41. O’Connell EM, Nutman TB. Eosinophilia in infectious diseases. Immunol Allergy Clin North Am. 2015;35(3):493-522. 42. Olbrich P, Ortiz Aljaro P, Freeman AF. Eosinophilia associated with immune deficiency. J Allergy Clin Immunol. 2022;10(5):1140-1153. DOI 10.1182/hematology.2022000367 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/47/2021769/47klion.pdf by guest on 09 December 2022 14. Moerman-Herzog A, Mehdi SJ, Wong HK. Gene expression comparison between Sézary syndrome and lymphocytic-variant hypereosinophilic syndrome refines biomarkers for Sézary syndrome. Cells. 2020;9(9):1992. 15. Lefèvre G, Copin M-C, Roumier C, et al; French Eosinophil Network. CD3-CD4+ lym phoid var i ant of hypereosinophilic syn drome: nodal and extranodal histopathological and immunophenotypic features of a periph eral indolent clonal T-cell lymphoproliferative disorder. Haematologica. 2015;100(8):1086-1095. 16. Roufosse F, de Leval L, van Krieken H, van Deuren M. Lymphocytic vari ant hypereosinophilic syndrome progressing to angioimmunoblastic T-cell lymphoma. Leuk Lymphoma. 2015;56(6):1891-1894. 17. Shi Y, Wang C. What we have learned about lymphocytic variant hyper eosinophilic syndrome: a systematic literature review. Clin Immunol. 2022;237:108982. 18. Gotlib J. Available and emerging therapies for bona fide advanced sys temic mastocytosis and primary eosinophilic neoplasms. Hematology Am Soc Hematol Educ Program. 2022;2022:34-46. 19. Chen Y-Y, Khoury P, Ware J-M, et al. Marked and per sis tent eosin o philia in the absence of clinical manifestations. J Allergy Clin Immunol. 2014;133(4):1195-1202. 20. Dellon ES, Simon D, Wechsler ME. Controversies in allergy: the potential role of biologics as first-line therapy in eosinophilic disorders. J Allergy Clin Immunol Pract. 2022;10(5):1169-1176. 21. Roufosse F, Kahn J-E, Rothenberg M-E, et al; HES Mepolizumab Study Group. Efficacy and safety of mepolizumab in hypereosinophilic syndrome: a phase III, randomized, placebo-controlled trial. J Allergy Clin Immunol. 2020;146(6):1397-1405. 22. Kuang FL, Legrand F, Makiya M, et al. Benralizumab for PDGFRA-negative hypereosinophilic syndrome. N Engl J Med. 2019;380(14):1336-1346. 23. Wechsler ME, Akuthota P, Jayne D, et al; EGPA Mepolizumab Study Team. Mepolizumab or placebo for eosinophilic granulomatosis with polyangiitis. N Engl J Med. 2017;376(20):1921-1932. 24. Klion AD, Law MA, Noel P, Kim Y-J, Haverty T-P, Nutman T-B. Safety and efficacy of the monoclonal anti-interleukin-5 antibody SCH55700 in the treat ment of patients with hypereosinophilic syn drome. Blood. 2004; 103(8):2939-2941. 25. Kuang FL, Fay MP, Ware J, et al. Long-term clinical outcomes of high-dose mepolizumab treat ment for hypereosinophilic syn drome. J Allergy Clin Immunol Pract. 2018;6(5):1518-1527.e51527e5. 26. Dellon ES, Peterson KA, Murray JA, et al. Anti-Siglec-8 antibody for eosino philic gastritis and duodenitis. N Engl J Med. 2020;383(17):1624-1634. 27. Hirano I, Dellon ES, Hamilton JD, et al. Efficacy of dupilumab in a phase 2 randomized trial of adults with active eosinophilic esophagitis. Gastroenterology. 2020;158(1):111-122.e10122e10. 28. Panch SR, Bozik ME, Brown T, et al. Dexpramipexole as an oral steroidsparing agent in hypereosinophilic syndromes. Blood. 2018;132(5):501-509. ANXIETY PROVOKING CONSULTATIONS: MAST CELLS AND EOSINOPHILS Cem Akin Division of Allergy and Clinical Immunology, Department of Medicine, University of Michigan, Ann Arbor, MI Mast cell disorders include mastocytosis and mast cell activation syndromes. Mastocytosis is a rare clonal disorder of the mast cell, driven by KIT D816V mutation in most cases. Mastocytosis is diagnosed and classified according to World Health Organization criteria. Mast cell activation syndromes encompass a diverse group of disorders and may have clonal or nonclonal etiologies. Hematologists may be consulted to assist in the diagnostic workup and/or management of mast cell disorders. A consult to the hematologist for mast cell disorders may provoke anxiety due to the rare nature of these diseases and the management of nonhematologic mast cell activation symptoms. This article presents recommendations on how to approach the diagnosis and management of patients referred for common clinical scenarios. LEARNING OBJECTIVES • Review the diagnostic criteria and classification of mastocytosis • Identify which patients with mast cell activation symptoms and elevated tryptase levels need further workup for a mast cell disorder CLINICAL CASE A 35yearold man is referred for evaluation of a mast cell disorder. He was admitted to the hospital for hypoten sive syncope after sustaining a wasp sting while biking. He felt flushed and lightheaded and collapsed within 10 minutes of the sting. When emergency medical ser vices arrived, his systolic blood pressure was noted to be 50 mmHg, with a heart rate of 120/min. There were no hives or angioedema, and the skin exam otherwise was unremarkable. His past medical history is significant for occasional flushing and lightheadedness with vigorous exercise. A serum tryptase level obtained 1 hour after the episode in the emergency department was 120 ng/mL. Another tryptase level 1 week later when he is at his base line was 28 ng/mL. Introduction Consults with a hematologist for a “mast cell disorder” can provoke anxiety for a number of different reasons. First, these disorders are rare, and the provider may not have enough practical experience to direct a diagnostic workup, choose the right tests, and interpret them correctly. Sec ond, treatment guidelines for many disease categories are evolving. Third, many patients with mast cell disorders have no abnormalities in other hematologic lineages, and therefore the hematologist may feel the treatment options to be out of their range of expertise. Likewise, there may be questions about when and how to initiate a workup for a patient referred to rule out a mast cell disorder due to symptoms of mast cell activation or elevated tryptase. The hematologist may receive consultations or referrals for mast cell disorders due to a number of different clinical scenarios: 1. Patient has an established diagnosis of advanced systemic mastocytosis (advSM) and is in need of cytoreductive ther apy of the mast cell disease and any associated non–mast cell neoplastic component such as a myelodysplastic syn drome (MDS) or myeloproliferative neoplasm (MPN). 2. Patient has skin lesions of maculopapular mastocytosis, and a bone marrow biopsy is needed to confirm or rule out systemic mastocytosis and categorize the disease. 3. Patient has a diagnosis of cutaneous or nonadvanced (indolent) systemic mastocytosis, and recommendations for management and followup are needed. Dr Prakash Singh Shekhawat Mast cell disorders | 55 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 How to evaluate the patient with a suspected mast cell disorder and how/when to manage symptoms 4. Patient has an elevated tryptase level. 5. Patient has symptoms of mast cell activation, and an opinion is needed regarding whether the patient is in need of further hematologic workup. Before discussing each of these scenarios, a primer on the diagnosis and classification of mastocytosis and mast cell activa tion disorders would be helpful. Mast cell disorders Mast cell disorders can be broadly categorized into those involv ing mast cell activation and those involving proliferation, with a significant overlap between the 2 categories (Figure 1).1 One can also encounter the terminology of “clonal” and “nonclonal” mast cell disorders in the literature, the former referring to mastocyto sis and the latter referring to patients presenting with recurrent idiopathic anaphylaxis or other mast cell activation disorders without evidence of mastocytosis.2 The prototypical proliferative mast cell disorder is mastocytosis.3,4 Although patients with rare myelomastocytic leukemia or reactive mast cell hyperplasia pres ent with increased mast cells in bone marrow biopsies, these are not “disorders of the mast cell” and are not discussed here.5 Diagnosis and classification of mastocytosis Mastocytosis is a clonal dis ease of the mast cell pro gen i tor, most often driven by a somatic gain-of-function mutation in KIT, resulting in the pathologic accumulation and activation of mast cells in tissues. It can be diagnosed in children and adults. The driver mutation is KIT D816V in more than 90% of adults and in about 30% of children.6 Pediatric-onset disease Pediatric-onset mastocytosis usually presents in the first year of life with typical maculopapular skin lesions, also known as urticaria pigmentosa. Pediatric mastocytosis generally has a self-limited course, with spontaneous resolution or significant regression by adolescence.7 Systemic involvement can be diagnosed in about 10% of cases. These children present with progressively increas ing tryptase levels, liver or spleen enlargement, lymphadenop athy, or unexplained abnormalities in the complete blood count with differential. The peripheral blood KIT D816V mutation may 56 | Hematology 2022 | ASH Education Program be positive. Children with typical self-limited pediatric mastocy tosis have polymorphic skin lesions, while children with systemic disease have monomorphic, smaller lesions resembling adultonset skin lesions. Other rare types of skin involve ment in children include mastocytomas and diffuse cutaneous masto cytosis (CM).8 A bone marrow biopsy is usually not necessary in children with typical polymorphic skin lesions and a tryptase level within normal range unless the child presents with increas ing tryptase levels or one of the red flags mentioned above, or the skin lesions fail to improve by adolescence. It is very unusual for pediatric patients with mastocytosis to present with symp toms of mast cell activation without skin lesions (as opposed to some adult patients). Therefore, in a pediatric patient referred for mast cell activation symptoms without skin lesions, a bone marrow biopsy is usually not necessary unless there is another indication. If such a patient has an elevated baseline tryptase (nor mal range is con sid ered <11.5 ng/mL in most com mer cial assays), hereditary alpha tryptasemia (HaT) should be consid ered first (see below for more detailed discussion). If HaT is not found, a bone marrow biopsy can be considered. Adult-onset disease Adult-onset mastocytosis can be diagnosed in the third decade of life or later.9 Patients may present with maculopapular CM lesions, symptoms of mast cell activation such as recurrent ana phylaxis, signs and symptoms of a hematologic disorder, or skel etal abnormalities on imaging raising suspicion for a metastatic disease or myeloma. Adult-onset mastocytosis is almost always systemic, and a bone marrow biopsy is needed to establish the diagnosis. Systemic disease refers to the presence of neoplastic mast cells in extracutaneous tissue. The World Health Organization (WHO) diagnostic criteria for systemic mastocytosis are shown in Table 1.10,11 A major criterion plus 1 minor criteria or 3 minor criteria are required to establish the diagnosis. Patients fulfilling only 1 or 2 minor clonality cri te ria (KIT mutation and/or CD25 expression) with symptoms of mast cell activation are termed to have monoclonal mast cell activation syndrome (MMAS).11 MMAS represents a low-burden clonal mast cell disease and is managed similarly to systemic mastocytosis. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 Figure 1. Mast cell disorders can be separated into those involving proliferation and activation. Mastocytosis is a clonal proliferative disorder of the mast cell and its progenitor. Reactive mast cell hyperplasia can be seen in a number of inflammatory and neoplastic conditions. Mast cell activation disorders (MCADs) include a broad range of conditions stemming from primary (mastocytosis/mono clonal MCAS), secondary (IgE and non–IgE-mediated mast cell activation due to allergic and nonallergic inflammatory and neoplastic diseases), and idiopathic origins. MCAS is a subgroup of MCADs usually presenting with severe episodic symptoms, which may be similar to anaphylaxis (see text for explanation). There may be overlap in patients with mastocytosis presenting with recurrent ana phylaxis symptoms or in patients with monoclonal MCAS. Table 1. WHO diagnostic criteria for systemic mastocytosis Major criterion Comments Multifocal compact infiltrates of mast cells (>15 cells per infiltrate) in a tissue biopsy other than skin (such as bone marrow). Mast cells are best visualized by tryptase or CD117 immunohistochemical stains in biopsy sections. These infiltrates are generally found in perivascular and paratrabecular locations. Minor criteria CD25 is the most specific neoplastic mast cell marker. CD2 may be absent in some advanced SM cases. CD30 has been recently added as a marker in the latest WHO document. These markers can be assessed by serial sections by immunohistochemistry or by flow cytometry. However, mast cell flow cytometry should be treated as a rare event analysis with acquisition of ideally 1 million or more events. Typical leukemia/lymphoma FC panels do not contain enough cells to gate on mast cells. Morphological abnormalities in mast cells, such as spindle-shaped, elongated mast cells with hypogranulation, cytoplasmic projection, and an off-centric or multilobated nucleus. More than 25% of mast cells in the infiltrate should be morphologically aberrant. A bone marrow aspirate smear is the best sample to evaluate for these aberrant mast cell forms. Mast cells are usually found embedded in or in close proximity to spicules. There is insufficient data on other tissue biopsies to assess mast cell morphology. A detailed photographic guide to these abnormalities is presented in Sperr et al.42 Detection of KIT D816V mutation or another gain of function KIT mutation in blood, bone marrow, or another noncutaneous tissue. Mutation detection should be done by a high-sensitivity test such as allele specific PCR or droplet digital PCR with a sensitivity to detect mutated allele frequency of <0.1%. NGS panels or sequencing-based assays lack this sensitivity and are often falsely negative. Baseline serum or plasma tryptase level of >20 ng/ml. Tryptase is a highly specific marker for mast cell burden and activation. It should be measured when the patient is at baseline and not after an anaphylactic or mast cell activation event, during which it may be found elevated regardless of mastocytosis. This criterion is not valid if the patient has another myeloid neoplasm as tryptase can be found in smaller quantities in myeloid progenitor cells. An important point has to be made about KIT D816V mutation detection: next generation sequencing (NGS) or sequencingbased mutation detection methods are often not adequate, and KIT D816V allele-specific polymerase chain reaction (PCR) or dig ital droplet PCR are required, especially if peripheral blood is used for screening (see scenario 4 below). However, NGS would be useful for evaluating for additional mutations that one would typically see in advSM, especially hematologic non–mast cell neoplasm (SM-AHN), as these mutations may have further prog nostic significance. Bone marrow biopsy and aspirate is the gold standard to establish the diagnosis by checking for the molecular and his to path o logic mark ers below, as the bone mar row is almost uniformly involved in SM. However, working with a gastroenter ologist may often be required in the further workup of symptoms such as abdominal pain and diarrhea. In this regard, immuno his to chem i cal stains for tryptase, CD117, and CD25 should be employed on endo scopic biop sies to deter mine whether abdominal symptoms may be due to mast cell infiltration or mediator release or both. Some mast cells in gastrointestinal (GI) tract biopsies may be negative for tryptase and positive for CD117. It should be noted that an increased number of mast cells alone in the GI tract are not diagnostic of mastocytosis and that bands or clusters of aberrant mast cells fulfilling WHO pathology criteria are required to prove GI involvement.12 Well-differentiated systemic mastocytosis (WDSM) is a rare histologic subtype in which mast cells have a mature, round mor phology, do not express aberrant CD25 or CD2, and u sually lack the typical KIT D816V mutation, making it a challenging diagno sis.13,14 Patients with WDSM can have a history of pediatric-onset cutaneous disease that may have resolved or been persistent. Mast cells in WDSM express CD30, which should not be present in normal mast cells. Classification of mastocytosis The most recent WHO classification of mastocytosis is shown in Table 2. CM is the most common category in children with involve ment limited to the skin. Patients with CM most often present with polymorphic skin lesions in the first year of life. Other less common skin manifestations of CM include mastocytomas and diffuse CM. These children do not require a bone marrow biopsy unless one of the red flag signs discussed above is present. In con trast, adult patients with skin lesions almost always have bone marrow involvement. If an adult patient with skin lesions does not have a bone marrow biopsy, that patient should be classified as “mastocytosis in the skin” rather than CM, as the probability of systemic disease is high. Systemic mastocytosis means the pres ence of neo plas tic mast cells meeting WHO diagnostic criteria in noncutaneous tissue, characteristically bone marrow. Systemic mastocytosis can be fur ther subdivided into nonadvanced SM (bone mar row mastocytosis [BMM], indolent SM [ISM], and smoldering SM [SSM]) and advanced SM (SM-AHN, aggressive SM [ASM], and mast cell leukemia [MCL]) based on histopathologic criteria, end organ dysfunction, and the presence of another associated Dr Prakash Singh Shekhawat Mast cell disorders | 57 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 Mast cells co-express CD25, CD2, and CD30. Table 2. WHO classification of mastocytosis Category Comment Cutaneous mastocytosis Most common category in children. Subtypes include MPCM, mastocytoma, and diffuse CM. Systemic mastocytosis Presence of neoplastic mast cell infiltrates in extracutanous tissues. Most common in adults. The most common category of SM, representing >80% of patients with SM. Patients in this category have mast cell collections meeting WHO criteria for systemic disease in the bone marrow but do not have an associated hematologic neoplasm or advanced disease findings or 2 or more B findings of SSM. Patients with ISM have a life expectancy that is comparable to the general age-matched population with less than 5% risk of progression to advanced disease. Bone marrow mastocytosis (BMM) This category was added in the most recent WHO document and was formerly included in ISM. BMM is diagnosed in a patient without skin lesions, tryptase level <125, no mast cell infiltrates in extramedullary tissue, and otherwise fits the criteria for ISM. This category is believed to have a lower risk of progression than ISM and SSM. Smoldering systemic mastocytosis (SSM) This is a rare category of nonadvanced SM with higher mast cell burden as evidenced by 2 or more B findings: i. tryptase level ≥200 ng/mL or bone marrow biopsy infiltration by mast cells of ≥30% or KIT D816V vari ant allele fraction of ≥10% in bone marrow or peripheral blood; ii. splenomegaly without hypersplenism and/or hepatomegaly without liver dysfunction and/or lymphadenopathy >2 cm; iii. myeloproliferation or subtle morphologic abnormalities in myeloid cells without meeting the criteria for a WHO-classified neoplasm. Patients with SSM may have a higher rate of progression to advSM, which still remains <10%. Systemic mastocytosis with associated hematologic non–mast cell neoplasm (SM-AHN) These patients have an AHN (usually an MPN or MDS) meeting the WHO criteria in addition to SM. Prognosis is determined by the AHN but is generally poor, with a median survival time of about 2 years after diagnosis. Aggressive systemic mastocytosis (ASM) Patients with ASM have high-level mast cell burden and tissue dysfunction due to infiltrating mast cells (also known as C findings). One or more C findings attributable to MC infiltration are required for diagnosis: cytopenias (hemoglobin <10 g/dL, platelets <100 000, and neutropenia <1000), liver dysfunction with portal hypertension, elevated liver function tests, ascites, malabsorption, and diarrhea with extensive GI infiltrates, splenomegaly with hypersplenism, large (≥2 cm) lytic bone lesions with pathological bone fractures. It should be noted that osteoporosis and smaller lytic and sclerotic bone lesions are common in allcategories of SM and are not considered a C finding. Patients with ASM have a reduced life expectancy, with <3 years of average survival after diagnosis. Mast cell leukemia (MCL) MCL is diagnosed when ≥10% mast cells are found in peripheral circulation or ≥20% in bone marrow aspirate smears in a nonspicular area. It should be noted that 20% infiltration grade refers to bone marrow aspirate and not to bone marrow biopsy. Patients without circulating mast cells are referred to as aleukemic MCL. Patients with typical MCL also have C findings similar to ASM and carry a very poor prognosis. A chronic form of MCL without C-findings or cytopenias have recently been recognized with more favorable survival rates. Mast cell sarcoma Rare invasive solid mast cell tumor with poor prognosis. non–mast cell clonal disease).11 Patients with advanced SM usu ally have other myeloid mutations detected in NGS, in addition to KIT D816V.15 As mentioned in Table 2, osteoporosis or sclerotic lesions should not be misinterpreted as advanced disease as they are common findings in ISM. To that end, a routine dual-energy x-ray absorptiometry bone density scan is recommended for each patient diagnosed with SM. Furthermore, radiographic imaging of areas with bone pain may be considered in individual patients. A bone scan may show increased uptake focally or appear as a 58 | Hematology 2022 | ASH Education Program superscan, although it is not routinely recommended. We would recommend treatment of osteoporosis in collaboration with an endocrinologist. Bisphosphonates or denosumab has been suc cessfully used in these patients.16 Mast cell sarcoma is an exceedingly rare subtype that does not meet the diagnostic criteria for SM but is characterized by a highgrade invasive solid MC tumor that carries a poor prognosis.17 Categories of systemic mastocytosis include ISM (most com mon), BMM, SSM, SM-AHN, ASM, and MCL. See Table 2 for impor tant comments about these categories. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 Indolent systemic mastocytosis (ISM) Mast cell activation syndromes in this scenario is fairly straightforward. If the patient is an adult with skin lesions, the likelihood of systemic disease is high, and a bone marrow biopsy and aspiration is indicated to establish the diagnosis and categorize the disease. In pediatric patients pre senting with typical CM, a bone marrow biopsy is generally not indicated due to the low risk of systemic disease unless the child has liver or spleen enlargement, has unexplained persistent or progressive lymphadenopathy, has peripheral blood abnormal ities not explainable by another process, demonstrates consis tently increasing tryptase levels in repeated measurements, has a positive peripheral blood KIT D816V mutation, or has persistent or progressive skin disease after adolescence. 3. The patient has a diagnosis of cutaneous or nonadvanced (indolent) systemic mastocytosis, and recommendations for management and follow-up are needed. In this scenario, patients do not have a hematologic abnormality but present for manage ment of various mast cell activation symptoms, such as flushing, abdominal pain, diarrhea, peptic symptoms, tachycardia, hypo tensive recurrent anaphylaxis, neurocognitive problems such as brain fog, musculoskeletal pain, and fatigue. Symptoms vary greatly from patient to patient; while some have minimal or no symptoms, others may have severe and disabling presentations. Symptom burden does not correlate with the extent of bone marrow infiltration by mast cells. Up to 50% of adult patients may experience anaphylaxis during the course of their disease.28 A prescription of multiple doses of self-injectable epinephrine is therefore recommended for allpatients diagnosed with SM. Approximately 1 of 3 patients have clinically significant osteo porosis,16 and about 10% may have an associated IgE-mediated hymenoptera (bee or vespid) venom allergy, which can be life threatening.29 In this scenario, hematologists may feel they lack the experience or expertise to manage these symptoms, and it is reasonable to refer these patients to an allergist with special expertise in treating mast cell activation symptoms. Common management strategies include the avoidance of triggers known to cause symptoms for each specific patient and the use of anti–mast cell mediator–targeting therapies such as H1 and H2 antihistamines, antileukotriene drugs, and mast cell stabilizers such as cromolyn.30,31 The anti-IgE monoclonal anti body omalizumab has been shown to reduce recurrent anaphy lactic symptoms in patients who do not respond to first-line antimediator therapies.32 Patients with SM may be more susceptible to perioperative MC activation events and anaphylaxis, although most patients undergo suc cess ful sur ger ies with premedication and the selection of agents less likely to cause mast cell activation and the avoidance of those known to cause symptoms in individual patients.33 Multidisciplinary management involving surgeons, allergists, and anesthesiologists is crucial to mitigate MC acti vation in patients needing surgery. Pregnancy is generally well tolerated in SM, and the management of SM in pregnancy is discussed elsewhere.34 KIT D816V–targeting TKIs are cur rently approved for the treatment of advanced SM and are in the clinical trial stage for patients with nonadvanced SM who do not respond to optimized antimediator management.35 Hematological expertise may be required for the treatment and monitoring of these patients with TKIs in the future if they are approved for nonadvanced SM indi cations. Avapritinib, currently approved for advSM, was evalu ated in a multicenter placebo-controlled clinical trial. According Dr Prakash Singh Shekhawat Mast cell disorders | 59 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 Mast cell activation syndromes (MCASs) represent a heteroge neous group of disorders characterized by (1) the episodic pres ence of mast cell activation symptoms in more than 2 organ systems, such as cutaneous, cardiovascular, GI, pulmonary, and naso-ocular; (2) response of symptoms to mast cell mediator– targeting drugs; and (3) the detection of a validated marker of mast cell activation during the symptomatic phase.2,18 The best validated surrogate marker of mast cell activation is tryptase.19 Tryptase should be checked at baseline and within 4 hours of a suspected mast cell activation event. A formula of 20% of baseline plus 2ng/mL is used to calculate the minimal increase required to diagnose mast cell activation.20 Urinary metabolites of other mast cell mediators such as histamine, prostaglandin D2, and leukotri ene C4 measured in a 24-hour or spot collection can also be used to document mast cell activation if tryptase levels are not avail able; however, the sensitivity and specificity of these markers as well as the minimal increases and cutoff levels diagnostic for mast cell activation have not been established.21 A patient with recur rent anaphylaxis is a prototypical presentation of MCAS; however, less severe manifestations can meet the above criteria. MCAS can be primary or clonal when it is associated with mastocytosis or MMAS, secondary when associated with immu noglobulin (Ig) E or non–IgE-mediated mast cell activation trig gers, or idiopathic when no underlying cause has been found. There is quite an extensive controversy surrounding the diag nosis of MCAS, and alternative diagnostic schemes with broader inclusion criteria may result in diagnosing nearly 17% of the general population with MCAS.22 However, this can become problematic for patients whose symptoms may be caused by another entity that may simply be associated with reactive mast cell activation because focusing on mast cell activation may leave the underlying entity undiagnosed. Contributing to this controversy somewhat is the International Classification of Diseases ICD-10-CM classifi cation of diagnostic codes that currently includes additional cat egories of mast cell activation, including “Mast cell activation, not otherwise specified,” which until recently did not have diagnostic guidance.23 A detailed discussion of mast cell activation disorders is outside the scope of this article, as hematologists are generally not expected to diagnose and treat these disorders. With this background information in mind, let us examine specific consultation scenarios mentioned at the beginning of the text. 1. Patients with advSM. This group of patients represents a classical indication for referral to a hematologist. Patients with advanced SM are in need of cytoreductive therapy and treat ment of non–mast cell hematologic neoplasias such as MPNs and MDSs.4,24 The recent approval of KIT D816V–targeting tyrosine kinase inhibitors (TKIs) revolutionized the treatment of patients with advSM, who typically have a reduced life expectancy.25-27 Since most of these patients present with mutations in addition to KIT D816V, additional therapies in those who do not respond or lose their response to TKIs may be needed, and targeted or palliative treatment options for the associated AHN regardless of mastocytosis should be considered. These options are discussed in a separate article by Gotlib in this educational program.43 2. The patient has skin lesions of maculopapular mastocytosis, and a bone marrow biopsy is needed to confirm or rule out sys temic mastocytosis and categorize the disease. This is another common scenario for referral to the hematologist. The approach tition, skin rashes, and venom allergies.37 Subsequent studies, however, failed to consistently confirm many of the nonallergic phenotypes.38 HaT alone is not a mast cell activation disorder; how ever, some reports indi cate that HaT may be a dis easemodifying factor in those with concurrent hymenoptera venom allergy, idiopathic anaphylaxis and mastocytosis accounting for more severe mast cell activation symptoms in HaT carriers, although prospective studies with no referral bias are needed to confirm these associations.39 Interestingly, HaT is 2 to 3 times more prevalent in patients with systemic mastocytosis than in the general population.37 Patients with SM and concurrent HaT tend to present more often with mast cell activation symptoms rather than skin lesions or hematologic abnormalities.40 Whether this association is due to a mechanistic link between HaT and SM or selection bias needs to be investigated in future studies. There is no specific treatment available (or needed) for HaT, other than the treatment of underlying mastocytosis symptoms or management of anaphylaxis. Testing for HaT (TPSAB1 gene copy number) is currently com mercially available in the US. In this test, copy numbers of alpha and beta alleles are reported, and their sum should be 4 in most individuals. Possible combinations include 4 beta and 0 alpha, 3 beta and 1 alpha, and 2 beta and 2 alpha. A quick interpreta tion would be to consider any number above 4 to be an extra alpha allele, provided there are at least 2 alpha alleles. For exam ple, in a patient with 3 beta and 2 alpha alleles, there is 1 extra alpha allele. However, as stated above, a positive test for HaT does not rule out concurrent mastocytosis. Patients with HaT and mastocytosis tend to have higher tryptase levels than what might be expected for HaT alone. A correction of tryptase level according to HaT genotype has been proposed as tryptase level divided by the extra alpha allele number plus 1. For example, if the serum tryptase is 15 ng/mL and the patient has an extra copy Figure 2. Hereditary alpha tryptasemia. 60 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 to part 1 data available from the PIONEER study (clinicaltrials.gov, NCT03731260), a dose of 25 mg/d was selected to move forward in an expanded part 2 cohort based on safety and efficacy data showing an approximately 30% reduction in patient-reported symp tom scores and no seri ous adverse events.36 Avapritinib has also been shown to be highly effective in improving skin lesions in SM, and these improvements were associated with objective reductions in mast cell disease burden. The PIONEER trial is currently closed to new patient enrollment. Other clinical trials currently open for enrollment to evaluate D816V-selective KIT inhibitors in patients with ISM involve BLU-263 (HARBOR, NCT04910685) and bezuclastinib (SUMMIT, NCT05186753). 4. The patient has an elevated tryptase level. In this scenario a patient with or without mast cell activation symptoms and no skin lesions is referred due to an elevated tryptase level to rule out mastocytosis. The most common cause of an elevated trypt ase level in the general population is HaT, which is seen in up to 7% of the population in the US (Figure 2).37 HaT is an auto somal dominantly transmitted genetic polymorphism of uncer tain clinical significance due to copy number variations of the TPSAB1 gene encoding the alpha tryptase gene. Alpha tryptase, a proenzyme with no proteolytic activity, accounts for the bulk of measurable serum tryptase in baseline conditions. A median normal tryptase level is around 4.5 to 5 ng/mL. Patients with HaT are generally measured to have tryptase levels higher than 8 ng/mL. The elevation in tryptase level is proportionate to the number of alpha-encoding TPSAB1 genes. Due to the autosomaldominant mode of transmission, patients with HaT are expected to have at least 1 parent with an elevated tryptase level and have a 50% chance of passing it on to their children. The initial description of HaT associated this genetic event with a number of seemingly unrelated phenotypic features, such as irritable bowel syndrome, skeletal abnormalities, retained primary den The clinical case presented at the beginning of this article is an example of a patient with an elevated baseline tryptase and severe hypotensive anaphylaxis. Even though he is fairly asymptomatic other than the anaphylaxis episode, his elevated baseline tryptase and high REMA score indicate the need for a bone marrow biopsy. While one can also check HaT status, a positive HaT test does not necessarily rule out mastocytosis in this case. The patient should also be referred to an allergist for venom allergy testing and consideration for venom immuno therapy and should be prescribed self-injectable epinephrine for as-needed use. 5. The patient has symp toms of mast cell acti va tion with a nor mal base line tryptase level, and an opin ion is needed regard ing whether the patient is in need of fur ther hema to logic workup. This scenario is probably the most confusing for the hematologist, as most of these patients present without an obvious hematologic abnormality, and hematologists may feel they lack sufficient expertise to evaluate and treat mast cell acti vation or anaphylaxis. While a normal tryptase level does not necessarily rule out systemic mastocytosis, SM is exceedingly rare in patients with a baseline tryptase lower than 4 ng/mL. Further guidance can be obtained by assessing the patient for REMA criteria (Figure 3).41 These criteria were developed by the Span ish Network of Mastocytosis and have a high pre dic tive value for determining underlying mastocytosis in patients pre senting with anaphylactic symptoms. Patients with hypotensive Figure 3. REMA score to predict clonal mast cell disease (mastocytosis) in patients presenting with mast cell activation symptoms and/or anaphylaxis. Reproduced with permission from Alvarez-Twose et al.41 Dr Prakash Singh Shekhawat Mast cell disorders | 61 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 of the alpha tryptase allele, the corrected tryptase level would be 7.5 ng/mL. Our approach to a patient with an elevated tryptase level higher than 8 ng/mL without skin lesions of mastocytosis or any other reason to explain elevated tryptase (such as mye loid neoplasia, chronic renal failure, etc) is as follows: If the patient has a history of severe hypotensive anaphylaxis and a score of 2 points or greater in REMA score (see below),41 or if the corrected tryptase is greater than 8 ng/mL, we consider a bone marrow biopsy and aspiration to rule out mastocyto sis. Some experts also propose a high-sensitivity peripheral blood KIT D816V test in patients reluctant to have a bone mar row biopsy; however, a negative result in this test would not necessarily rule out mastocytosis. If the patient, however, is asymptomatic or has nonspecific symptoms such as fatigue, musculoskeletal pain, abdominal pain, or multiple food intol erances, HaT testing is done. If this test shows positive results and the corrected tryptase is lower than 8 ng/mL, a bone mar row biopsy is optional, and the patient can be followed yearly with symptomatic assessment and a complete blood count with differential and tryptase levels. If the patient shows an increasing trend in tryptase levels or develops new symptoms such as hypotensive anaphylaxis, a bone marrow biopsy is considered. If HaT testing is negative in a patient with an ele vated tryptase level, a bone marrow biopsy is recommended regardless of symptomatic status. Conclusions Mast cell disorders encompass mastocytosis and MCASs. Due to their rarity and various forms of presentation without a hemato logic disease, hematologists may feel they lack sufficient exper tise to diagnose and treat these disorders. However, using the approach outlined above for different scenarios, most of these patients can be triaged to an appropriate diagnostic workup. A hematopathologist with experience in reviewing bone marrow and other tissue biopsy samples as well as an allergist knowl edgeable about the symptomatic management of these patients can be helpful resources during this process. A referral to a cen ter with more expertise can be considered for management rec ommendations and evaluation for clinical trials. A list of these academic centers is available through the American Initiative on Mastocytosis (www.aimcd.net). Finally, the patient support group the Mast Cell Disease Society (www.tmsforacure.org) has many useful informational materials for patients diagnosed with a mast cell disorder. Conflict-of-interest disclosure Cem Akin: consultancy: Blueprint Medicines, Cogent; research funding: Blueprint Medicines, Cogent. Off-label drug use Cem Akin: omalizumab is discussed. In addition, avapritinib, BLU-263, and bezuclastinib are in clinical trials for ISM. Correspondence Cem Akin, Division of Allergy and Clinical Immunology, Depart ment of Medicine, University of Michigan, 24 Frank Lloyd Wright Dr, Lobby H-2100, Ann Arbor, MI 48106; e-mail: cemakin@umich.edu. References 1. Theoharides TC, Valent P, Akin C. Mast cells, mastocytosis, and related dis orders. N Engl J Med. 2015;373(2):163-172. 2. Valent P, Akin C, Arock M, et al. Definitions, criteria and global classifica tion of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal. Int Arch Allergy Immunol. 2012;157(3): 215-225. 3. Valent P, Akin C, Gleixner KV, et al. Multidisciplinary challenges in mastocy tosis and how to address with personalized medicine approaches. Int J Mol Sci. 2019;20(12):2976. 62 | Hematology 2022 | ASH Education Program 4. Pardanani A. Systemic mastocytosis in adults: 2021 update on diagnosis, risk stratification and management. Am J Hematol. 2021;96(4):508-525. 5. Horny HP, Sotlar K, Reiter A, Valent P. Myelomastocytic leukemia: histo pathological features, diagnostic criteria and differential diagnosis. Expert Rev Hematol. 2014;7(4):431-437. 6. Hoermann G, Sotlar K, Jawhar M, et al. Standards of genetic testing in the diagnosis and prognostication of systemic mastocytosis in 2022: recom mendations of the EU-US Cooperative Group. J Allergy Clin Immunol Pract. 2022;10(8):1953-1963. 7. Lange M, Hartmann K, Carter MC, et al. Molecular background, clinical fea tures and management of pediatric mastocytosis: status 2021. Int J Mol Sci. 2021;22(5):2586. 8. Hartmann K, Escribano L, Grattan C, et al. Cutaneous manifestations in patients with mastocytosis: consensus report of the European Compe tence Network on Mastocytosis; the American Academy of Allergy, Asthma and Immunology; and the European Academy of Allergology and Clinical Immunology. J Allergy Clin Immunol. 2016;137(1):35-45. 9. Brockow K. Epidemiology, prog no sis, and risk fac tors in mastocytosis. Immunol Allergy Clin N Am. 2014;34(2):283-295. 10. Valent P, Horny HP, Escribano L, et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res. 2001;25(7):603-625. 11. Valent P, Akin C, Hartmann K, et al. Updated diagnostic criteria and clas sification of mast cell disorders: a consensus proposal. Hemasphere. 2021;5(11):e646. 12. Doyle LA, Sepehr GJ, Hamilton MJ, Akin C, Castells MC, Hornick JL. A clin icopathologic study of 24 cases of systemic mastocytosis involving the gastrointestinal tract and assessment of mucosal mast cell density in irri ta ble bowel syn drome and asymp tom atic patients. Am J Surg Pathol. 2014;38(6):832-843. 13. Akin C, Fumo G, Yavuz AS, Lipsky PE, Neckers L, Metcalfe DD. A novel form of mastocytosis asso ci ated with a trans mem brane c-kit muta tion and response to imatinib. Blood. 2004;103(8):3222-3225. 14. Álvarez-Twose I, Jara-Acevedo M, Morgado JM, et al. Clinical, immuno phenotypic, and molecular characteristics of well-differentiated systemic mastocytosis. J Allergy Clin Immunol. 2016;137(1):168-178.e1178e1. 15. Schwaab J, Schnittger S, Sotlar K, et al. Comprehensive mutational profil ing in advanced systemic mastocytosis. Blood. 2013;122(14):2460-2466. 16. Greene LW, Asadipooya K, Corradi PF, Akin C. Endocrine manifestations of systemic mastocytosis in bone. Rev Endocr Metab Disord. 2016;17(3):419431. 17. Ryan RJ, Akin C, Castells M, et al. Mast cell sarcoma: a rare and potentially under-recognized diagnostic entity with specific therapeutic implications. Mod Pathol. 2013;26(4):533-543. 18. Akin C, Valent P, Metcalfe DD. Mast cell activation syndrome: proposed diagnostic criteria. J Allergy Clin Immunol. 2010;126(6):1099-1104.e4. 19. Schwartz LB. Diagnostic value of tryptase in anaphylaxis and mastocytosis. Immunol Allergy Clin North Am. 2006;26(3):451-463. 20. Valent P, Bonadonna P, Hartmann K, et al. Why the 20% +2 tryptase formula is a diagnostic gold standard for severe systemic mast cell activation and mast cell activation syndrome. Int Arch Allergy Immunol. 2019;180(1):44-51. 21. Butterfield JH. Nontryptase urinary and hematologic biomarkers of mast cell expansion and mast cell activation: status 2022. J Allergy Clin Immunol Pract. 2022;10(8):1974-1984. 22. Afrin LB, Ackerley MB, Bluestein LS, et al. Diagnosis of mast cell activation syndrome: a global “consensus-2.” Diagnosis (Berl). 2021;8(2):137-152. 23. Valent P, Hartmann K, Bonadonna P, et al. Global classification of mast cell activation disorders: an ICD-10-CM-Adjusted Proposal of the ECNM-AIM Consortium. J Allergy Clin Immunol Pract. 2022;10(8):1941-1950. 24. Reiter A, George TI, Gotlib J. New devel op ments in diag no sis, prog nos ti ca tion, and treat ment of advanced sys temic mastocytosis. Blood. 2020;135(16):1365-1376. 25. Gotlib J, Kluin-Nelemans HC, George TI, et al. Efficacy and safety of mido staurin in advanced systemic mastocytosis. N Engl J Med. 2016;374(26):25302541. 26. DeAngelo DJ, Radia DH, George TI, et al. Safety and efficacy of avapritinib in advanced systemic mastocytosis: the phase 1 EXPLORER trial. Nat Med. 2021;27(12):2183-2191. 27. Gotlib J, Reiter A, Radia DH, et al. Efficacy and safety of avapritinib in advanced systemic mastocytosis: interim analysis of the phase 2 PATH FINDER trial. Nat Med. 2021;27(12):2192-2199. 28. Brockow K, Jofer C, Behrendt H, Ring J. Anaphylaxis in patients with masto cytosis: a study on history, clinical features and risk factors in 120 patients. Allergy. 2008;63(2):226-232. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 anaphylaxis episodes (particularly after bee/wasp stings or idio pathic) without urticaria or angioedema are at particular risk. It should be noted that while patients with several unexplainable symptoms such as chronic fatigue; fibromyalgia; headaches; irri table bowel syndrome-like symptoms; dysautonomia, including postural orthostatic tachycardia syndrome; Ehlers-Danlos syn drome; and multiple food, drug, and environmental intolerances are considered for a diagnosis of mast cell disorder, there are no mechanistic studies proving a causal link between a primary mast cell disorder, such as mastocytosis or MCAS, and these enti ties, and such patients, especially those with normal tryptase levels, are not candidates for a hematologic workup. Some of these patients may have other localized manifestations of mast cell activation common to the general population, such allergic rhinitis, urticaria, and even flushing, and these patients could be evaluated for MCAS with the help of an allergist for these specific entities. Patients with a history of anaphylaxis should be evalu ated and managed by an allergist. 38. Chollet MB, Akin C. Hereditary alpha tryptasemia is not associated with specific clinical phenotypes. J Allergy Clin Immunol. 2022;149(2):728-735.e2. 39. Lyons JJ, Chovanec J, O’Connell MP, et al. Heritable risk for severe anaphy laxis associated with increased α-tryptase-encoding germline copy num ber at TPSAB1. J Allergy Clin Immunol. 2021;147(2):622-632. 40. Greiner G, Sprinzl B, Górska A, et al. Hereditary α tryptasemia is a valid genetic biomarker for severe mediator-related symptoms in mastocytosis. Blood. 2021;137(2):238-247. 41. Alvarez-Twose I, González de Olano D, Sánchez-Muñoz L, et al. Clinical, bio logical, and molecular characteristics of clonal mast cell disorders present ing with systemic mast cell activation symptoms. J Allergy Clin Immunol. 2010;125(6):1269-1278.e2. 42. Sperr WR, Escribano L, Jordan JH, et al. Morphologic properties of neo plastic mast cells: delineation of stages of maturation and implication for cytological grading of mastocytosis. Leuk Res. 2001;25(7):529-536. 43. Gotlib J. Available and emerging therapies for bona fide advanced systemic mastocytosis and primary eosinophilic neoplasms. Hematology Am Soc Hematol Educ Program. 2022;2022:34-46. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000366 Dr Prakash Singh Shekhawat Mast cell disorders | 63 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/55/2021719/55akin.pdf by guest on 09 December 2022 29. Bonadonna P, Scaffidi L. Hymenoptera anaphylaxis as a clonal mast cell disorder. Immunol Allergy Clin North Am. 2018;38(3):455-468. 30. Castells M, Butterfield J. Mast cell activation syndrome and mastocytosis: initial treatment options and long-term management. J Allergy Clin Immunol Pract. 2019;7(4):1097-1106. 31. Gotlib J, Gerds AT, Bose P, et al. Systemic Mastocytosis, Version 2.2019, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2018;16(12):1500-1537. 32. Jendoubi F, Gaudenzio N, Gallini A, Negretto M, Paul C, Bulai Livideanu C. Omalizumab in the treatment of adult patients with mastocytosis: a sys tematic review. Clin Exp Allergy. 2020;50(6):654-661. 33. Dewachter P, Castells MC, Hepner DL, Mouton-Faivre C. Perioperative man agement of patients with mastocytosis. Anesthesiology. 2014;120(3):753759. 34. Lei D, Akin C, Kovalszki A. Management of mastocytosis in pregnancy: a review. J Allergy Clin Immunol Pract. 2017;5(5):1217-1223. 35. Akin C, Arock M, Valent P. Tyrosine kinase inhibitors for the treatment of indolent systemic mastocytosis: are we there yet? J Allergy Clin Immunol. 2022;149(6):1912-1918. 36. Akin C, Sabato V, Gotlib J, et al. PIONEER: a randomized, double-blind, placebo-controlled, phase 2 study of avapritinib in patients with indolent or smoldering systemic mastocytosis (SM) with symptoms inadequately controlled by standard therapy. J Allergy Clin Immunol. 2020;145(2):AB336. 37. Lyons JJ, Yu X, Hughes JD, et al. Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number. Nat Genet. 2016;48(12):1564-1569. ARE ALTERNATIVE DONORS NOW MAINSTREAM IN ALLOGENEIC TRANSPLANT ? In 2022, which is preferred: haploidentical or cord transplant? Division of Hematology, Bone Marrow Transplantation, and Hemato-Oncology Center, Chaim Sheba Medical Center, Tel HaShomer, Ramat Gan, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3Acute Leukemia Working Party, European Society for Blood and Marrow Transplantation, Paris, France; 4Department of Clinical Hematology and Cellular Therapy, Saint-Antoine Hospital, Assistance Publique-Hȏpitaux de Paris, Sorbonne University, Paris, France; and 5Sorbonne University, INSERM, Saint-Antoine Research Center, Paris, France 1 Allogeneic hematopoietic stem cell transplantation is the treatment of choice for high-risk hematological malignancies such as acute myeloid and lymphocytic leukemia, myelodysplastic syndrome, and myeloproliferative disorders. Alternative donor transplantation from either haploidentical (haplo-SCT) or cord blood donor (CBT) is an established therapeutic alternative for patients who need transplants but lack a human leukocyte antigen–matched donor. Although haplo-SCT (mainly non–T-cell-depleted haplo-SCT with posttransplant cyclophosphamide) is increasing while CBT is decreasing worldwide (Figure 1), recent developments in CBT, especially cord blood expansion and other strategies to improve engraftment and immune reconstitution post-CBT, make CBT still a valuable option. This article discusses the 2 options based on the currently available data, focusing on adults, and tries to give some clues to help the transplant physician choose a haploidentical vs a cord blood donor. Given the limited numbers of published or ongoing well-designed randomized controlled trials comparing haplo-SCT to CBT and the overall similar clinical results in the available, mostly registrybased, and single-center studies, with substantial heterogeneity and variability, the decision to perform haplo-SCT or CBT in a given patient depends not only on the patient, disease, and donor characteristics and donor availability (although most if not all patients should have in principle an alternative donor) but also on the transplant physician’s discretion and, most importantly, the center’s experience and preference and ongoing protocols and strategies. LEARNING OBJECTIVES • Review the available published literature on haploidentical vs cord blood transplantation • Discuss the available literature that should guide decisions regarding haploidentical vs cord blood transplantation for an adult patient with a high-risk hematological malignancy in need of allogeneic transplantation CLINICAL CASE 1 A 50-year-old man hospitalized because of acute myeloid leukemia (AML) presented with subfebrile fever, flu-like symptoms, bleeding tendency and leukocytosis, neutropenia, anemia, and thrombocytopenia. Bone marrow (BM) aspiration revealed 70% myeloblasts. Cytogenetic and mutational analysis disclosed monosomy 7 and fmslike tyrosine kinase 3 internal tandem duplication positivity while nucleophosmin 1 was negative. Conventional induction chemotherapy with cytarabine and daunorubicin (7 + 3) in combination with midostaurin yielded the first complete remission (CR1; by morphology and cytogenetics), but molecular remission was achieved only post highdose cytarabine-midostaurin consolidation. The patient had no human leukocyte antigen (HLA)-matched brothers or sisters, and a search for an unrelated donor that 64 | Hematology 2022 | ASH Education Program began at the time of diagnosis failed to allocate a 10 of 10 HLA-matched donor. The patient had 4 brothers and sisters (2 of them haploidentical [haplo] and 1 with an HLA DRB1 mismatch), 3 children, and parents with no comorbidities. In parallel, the search for unrelated donors yielded few potential cord blood units (CBUs) with a 6 out of 8 HLA match (considering HLA loci A, B, C, and DRB1). Two of the potential CBUs contained a class II mismatch on HLA DRB1. The total nucleated cell dose of the units ranged between 1 to 2 × 107/kg with 1 to 1.2 × 105 CD34+/kg. Screening by Luminex assay for anti-HLA donor-specific antibodies (DSAs) was positive for 2 of the potential CBUs, with a mean fluorescence intensity level greater than 1000. The patient had no DSAs to the potential haplo donors. The patient was referred by his primary hematologist to a transplant expert for a consultation about allogeneic transplantation, which is strongly indicated for Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 Arnon Nagler1–3 and Mohamad Mohty3–5 Haplo and cord blood transplantaon in Europe (EBMT) A. Haplo&CBT 2000-2020 B. Haplo&CBT 2000-2020 Haplo vs CB 2011-2020 Haplo vs CB 2000-2010 CB allo 2006-2010 46% N - 4641 CB allo 2011-2020 19% N - 4779 Haplo 2000-2010 54% Haplo 2011-2020 81% N - 20207 N - 5535 EBMT Survey 2020 Haplo CBT Haplo&CBT 2000-2020 in acute leukemia AML CBT 8% N - 311 Haplo vs CB Haplo vs CB in adults CBT 4% N - 117 ALL Haplo vs CB in peds CBT 19% N - 194 HAPLO 81% N - 825 HAPLO 96% N - 2537 HAPLO 92% N - 3362 Haplo vs CB in AML CBT 7% Haplo vs CB in ALL CBT 9% N - 70 N - 98 Haplo CBT Haplo CBT Haplo CBT Haplo CBT HAPLO 91% N - 676 HAPLO 93% N - 1346 Haplo vs CB in NHL Haplo vs CB in MDS/MPN CBT 8% N - 31 HAPLO 92% N - 354 CBT 3% N-8 HAPLO 97% N - 248 Figure 1. Haploidentical and cord blood transplantation activities in Europe, the US, and China. (A) Haplo-SCT and CBT for AML and ALL in Europe (Acute Leukemia Working Party of the EBMT). Data include non–T-cell- depleted and T-cell depleted haplo-SCT, single CTB, and double CBT in 2000-2020. (B) Haplo-SCT and CBT in Europe in adult and pediatric populations in 2000-2020. Data include haplo-SCT and CBT for various hematological malignancies (AML, ALL, MDS, MPN [myeloproliferative neoplasms], NHL) in 2020. (C) Haplo-SCT and CBT in the US (CIBMTR) in 2000-2020 for various hematological malignancies (AML, ALL, MDS/MPN, NHL, HL) and nonmalignant diseases, excluding SAA, modified from CIBMTR 2021 summary slides. Data include adults (>18 years) and children and adolescents (<18 years) and BM and PB (peripheral blood) grafts, with MAC or RIC/NMA (nonmyeloablative conditioning). (D) HaploSCT (HID) and CBT in China (Chinese Blood and Marrow Transplantation Registry Group) in 2008-2021. Data are for haplo-SCT and CBT in various hematological malignancies (AML, ALL, MDS, NHL) in 2021. peds, pediatric patients; SCBT; single cord blood transplantation; URD, unrelated donor transplant. high-risk AML. As no HLA-matched sibling or unrelated donor was available, the potential relevant donors included a haplo donor,1,2 CB with 2 CBUs or ex vivo expanded CB in a clini cal trial, or a combination of a graft from a haplo donor and a cord (haplo-cord).3-5 The theoretical considerations, advan tages, and disadvantages of haplo transplants (haplo-SCT) vs CB transplants (CBT) were discussed (Table 1). The remainder of this article focuses mainly on a comparison of non–T-cell-depleted haplo-SCT with posttransplant cyclophos phamide (PTCy) and CBT. Theoretical considerations for haplo vs CB grafts As depicted in Table 1, CB and haplo donors as alternative hema topoietic stem cell sources have potential strengths as well as lim itations.1-4 Some of these historical advantages and disadvantages have subsequently been overcome with recent developments in the field, including double-unit CBT (DCBT) and the ex vivo expan sion of CBUs, overcoming the cell-dose limitation that resulted in slow and low engraftment as well as slow immune reconstitution and lead to the high infection rates and early transplant-related mortality rate associated with CBT.3,4 In par al lel, the non–Tcell-depleted haplo plat form, espe cially with PTCy, improves engraft ment rates and immune recon sti tu tion posttransplant, significantly reducing the historical limitations of T-cell-depleted haplo-SCT, which are mainly nonengraftment, high infection rates, high transplant-related complications and mortality, and high relapse incidence (RI).1,2 Still, haplo grafts possess some advan tages in comparison to CBU grafts, including a shorter time for donor identification, the possibility for a repeat stem cell dona tion, donor lymphocyte infusion (DLI), and lower cost (Table 1). Dr Prakash Singh Shekhawat Haploidentical vs cord transplantation | 65 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 Haplo&CBT by me period Haplo CBT 2500 2000 Haplo CB Haplo and cord blood transplantaon in the US (CIBMTR) 1500 1000 500 Number of Transplants 300 Haplo > 18 Years BM PB BM PB 1200 900 600 300 0 250 Haplo < 18 Years 200 150 100 50 0 700 600 500 AML ALL MDS / MPN NHL/HL Non-malignant disease* Number of Transplants Number of Transplants 800 400 300 200 100 0 500 450 400 350 300 250 200 150 100 50 0 CB > 18 Years 400 CB < 18 Years 350 300 250 200 150 100 50 0 2000 1800 1600 1400 1200 1000 800 600 400 200 0 MAC RIC/NMA Figure 1. Continued Haplo-STC vs CBT: registry-based studies Several registry-based studies have compared alternative donor transplants, including haplo-SCT, to CBT (Supplemental Table 1). Ruggeri et al,6 on behalf of the European Society for Blood and Marrow Transplantation (EBMT), compared non–T-cell-depleted haplo-SCT to CBT in 1446 patients with AML (n=918) and lympho cytic leukemia (ALL; n=528). CBT was associated with delayed engraftment and higher graft failures in both AML and ALL. CBT resulted in a lower incidence of chronic graft-versus-host dis ease (cGVHD) in both AML and ALL. The main take-home mes sage of this large-cohort registry-based analysis was that there were no statistical differences in main outcomes after haplo-SCT and CBT. Specifically, RI did not differ between the two graft sources. Nonrelapse mortality (NRM) and leukemia-free survival 66 | Hematology 2022 | ASH Education Program (LFS) were comparable as well (Supplemental Table 1).6 A sub sequent analysis, by Versluis et al,7 assessed the outcomes of poor-risk AML in CR1 from various donor categories. The study included 193 patients who underwent haplo-SCT and 333 under going CBT. The 2-year OS was significantly lower for umbilical cord blood (UCB) grafts compared to haplo-SCT, with no differ ences in 2-year NRM. The results were confirmed by multivari able analysis (MVA). The author concludes that especially haplo but also CB donors are alternative graft sources for high-risk AML patients who need hematopoietic stem cell transplantation (HSCT) and do not have an HLA-matched donor. However, suffi cient numbers and follow-up to define a hierarchy are lacking.7 In a more recent EBMT analysis that included 106 188 patients with different hematological malignancies and assessed longitudinal Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 1500 Number of Transplants 1800 Number of Transplants 0 Number of Transplants Number of Transplants C. outcomes of HSCT from various graft sources comparing the results of trans plants performed in 2006-2010 to those performed in 2011-2015, the authors demonstrated improved 3-year OS for both haplo-SCT and CBT. Interestingly, the improvement in OS was mostly driven by a reduction in NRM, including for haplo-SCT, with the excep tion of CBT recip i ents, who had a lower RI.8 Both haplo-SCT and CBT demonstrated a greater haz ard for NRM, with overlapping risk between the 2 donor types. The leading cause of NRM in both haplo-SCT and CBT was infec tions.8 A combined EBMT-Eurocord study focused on patients with AML receiv ing thio tepa, busul fan, and fludarabine (TBF) con di tion ing before both non–T-cell-depleted haplo-SCT and single CBT. It demonstrated increased NRM, delayed engraft ment, and higher graft failure in the CBT vs haplo-SCT, which translated into reduced OS and GVHD-free, relapse-free survival (GRFS) in the CBT group compared to the haplo group.9 The Adult AML and the Donor/Source Working Group of the Japan Society for Hematopoietic Cell Transplantation also performed a registry-based study retrospectively comparing the results of haplo-SCT to CBT in 1313 adult patients with intermediate- or poor-risk AML in CR (Supplemental Table 1).10 Between 2007 and 2018 in Japan, 211 patients received haplo-SCT and 1102, sin gle CBT. The cumulative incidences of neutrophil and platelet recovery were significantly lower in CBT when compared with haplo-SCT. Grade 2 to 4 acute GVHD (aGVHD) was significantly higher in CBT, while extensive cGVHD and cytomegalovirus antigenemia were higher in haplo-SCT. In the MVA and propensity scores matching grade 3 to 4, aGVHD as well as RI, NRM, LFS, OS, and GRFS did not differ between the two graft sources.10 The Kyoto Stem Cell Transplantation Group retrospectively com Dr Prakash Singh Shekhawat Haploidentical vs cord transplantation | 67 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 Figure 1. Continued Table 1. Haplo-SCT vs CBT: advantages and disadvantages Donor CBT HAPLO-transplant SCBT DCBT TCD TCR Indications Nonmalignant and malignant Nonmalignant and malignant Mainly malignant Mainly malignant Age Mainly pediatric Adult and pediatric Adult and pediatric Adult and pediatric Donor availability High High High High 1-2 mismatches 1-2 mismatches 2-3 mismatches 2-3 mismatches Important Important Important Important Time for donor identification Slower <1 month Slower compared to SCBT Faster (immediate) Faster (immediate) Time of graft acquisition 15-30 days 15-30 days 15-30 days 15-30 days Type of graft Cryopreserved CBU 2 cryopreserved CBUs Mobilized PB Mobilized PB and/or BM Limitation of graft acquisition Cell dose Improved compared to SCBT Poor mobilization Improved compared to TCD haplo Graft manipulation Ex vivo expansion None In vivo TCD None Donor safety (risk to the donor) No risk No risk Very low Very low Easy to repeat stem cell donation Not possible Not possible Possible Possible Conditioning MAC and RIC MAC and RIC MAC MAC and RIC Engraftment Delayed Improvement com pared to SCBT Comparable to MRD or MUD Comparable to MRD or MUD Use of ATG Determinantal omitted Determinantal omitted Indicated (can be replaced by PTCy) Indicated (can be replaced by PTCy) GVHD Lower incidence with less stringent HLA matching needed Incidence increased compared to SCBT Low incidence Low incidence with BM and PTCy; higher incidence with PB NRM High due to graft failure and infections Improved compared to SCBT High Improved compared to TCD-haplo RI Comparable to MSD or MUD Low compared to SCBT High Comparable to MSD or MUD (higher in initial report) with BM and RIC DLI Not possible Not possible Possible Possible Immune reconstitution Slow Improved compared to SCBT Slow Improved compared to TCD-haplo Infections incidence High Improved compared to SCBT High Improved compared to TCD-Haplo OS Lower than other stem cell sources Improved compared to SCBT Lower than other stem cell sources Improved compared to TCD-Haplo Cost associated with donor search and graft procurement High Very high Lower Lower SCBT, single cord blood transplantation; TCD, T-cell depletion; TCR, T-cell repletion. pared 57 non–T-cell-depleted haplo-SCT patients to 460 patients receiving single CBT between 2013 and 2019 and demonstrated similar results: significantly delayed neutrophil and platelet engraftment for CBT recipients but similar OS, NRM, GVHD, and RI. Interestingly, in the AML subgroup RI was lower with CBT in com par i son to the haplo-SCT group (Supplemental Table 1).11 The Center for International Blood and Marrow Transplant Research (CIBMTR) has also recently compared 375 non–T-celldepleted haplo-SCT with PTCy to 333 CBT performed between 2012 and 2017 with myeloablative conditioning (MAC) in patients (adults and pediatric) with different hematological malignan 68 | Hematology 2022 | ASH Education Program cies. Three-year OS and LFS were similar. RI was lower and NRM was higher in fewer than or equal to 5 out of 8 HLA-matched CBT patients vs 6 to 8 out of 8 HLA-matched CBT and haplo-SCT patients. The engraft ment of both neu tro phils and plate lets was delayed with CBT vs haplo-SCT. aGVHD was higher with CBT, while cGVHD did not differ between the 2 graft sources. Of note, the authors did not find a center effect, and no differ ence was observed between cen ters that con trib uted more than 10 vs 6 to 10 cases to the study (Supplemental Table 1).12 In a recent analysis by the CIBMTR focusing on ALL and com paring 393 non–T-cell-deleted haplo-SCT to other graft sources, Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 Degree of HLA mismatch DSA Haplo-SCT vs CBT: randomized controlled trials The 2 randomized controlled trials (RCTs) comparing haplo-SCT to CBT are rather limited. The CTN1101 trial (NCT01597778) was performed by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) and published in Blood in 2021 (Supplemental Table 1 and Table 2).15 This was a phase 3 trial that ran between 2012 to 2018, with a rather long recruiting period, and included 368 patients aged 18 to 70 years with various hemato logical malignancies in remission (CR; it also included patients beyond the first CR). Of these, 182 patients underwent haplo with BM graft, while 186 underwent CBT with 2 CBUs. Both groups received low-dose TBI/fludarabine/Cy as a condition ing regimen, and haplo recipients received PTCy as GVHD pro phylaxis. The trial demonstrated similar 2-year progression-free survival (PFS) (the study primary end point) between haplo-SCT and CBT. However, the 2-year NRM was significantly higher in the CBT group compared to the haplo group (Supplemental Table 1). This resulted in significantly better 2-year overall sur vival (OS) for haplo-SCT vs CBT (Supplemental Table 1). Both were secondary end points in the study, with the limitation that, usually, studies are not powered on the basis of second ary end points, and rarely are sta tis ti cal adjust ments made for the issue of multiple comparisons. In agreement with the previous literature, neutrophil recovery was lower in the CBT group compared to the haplo group. Platelet recovery was no different between CBT and haplo-SCT. Of note, the incidence of both aGVHD and cGVHD, including the severe and extensive forms, was similar between the 2 study arms. As for the graftversus-leukemia effect (GVL) initially believed to be somewhat separated from GVHD in CBT, interestingly, the 2-year RI was almost identical post haplo and CBT (Supplemental Table 1). Also, the main cause of death in both study arms was the recur rence of the malignancy. The trial did not assess immunologi cal reconstitution. Notably, mortality from GVHD and infections was comparable in haplo-SCT and CBT. As the most impor tant RTC in the field, even this study faced severe limitations, including a rather long running time and, most importantly, early termination as the trial failed to complete accrual, high lighting the main hurdles in running this type of randomized study. A second analysis of the trial demonstrated that trans plant center experience matters and that centers with previous experience in haplo BM transplants (≥10 transplants) had better results, while those with no such experience (≤10 transplants) showed similar results for haplo-SCT and CBT. Therefore, cen ters with no experience in performing DCBT may do better performing BM haplo with PTCy.16 Moreover, the CIBMTR recently reported on an extended follow-up of trial participants beyond 2 years. A comparison of trial double-unit UCB (dUCB) and nontrial haplo-BM transplantation recipients demonstrated higher PFS and OS at 5 years after haplo-BM transplantation. A com parison to real-world transplants disclosed similar 5-year OS in registry-based and nontrial DCBT and between trial and nontrial haplo-BM transplants, respectively.17 Notably, this addi tional registry-based study comparing real-world transplantation outcomes in substantially larger numbers of nontrial haplo-BM transplants to trial results with longer follow-up revealed that 5-year sur vival was sig nifi cantly higher after nontrial haplo BM trans plants com pared with trial dUCB, while the lon ger follow-up of the original trial cohorts failed to demonstrate a difference in 5-year PFS and OS between the treatment groups. Further, the type of graft mat ters, as nontrial patients who underwent haplo transplants using mobilized PBSC transplants demonstrated significantly higher 5-year survival compared to trial haplo BM and nontrial haplo BM. Similarly, survival was bet ter after haplo-PBSC compared to trial UCB and nontrial UCB.17 The second RCT to randomize haplo-SCT vs CBT was performed by Sanz et al (NCT02386332) (Supplemental Table 1 and Table 2).18 The study prospectively randomized CBT with single CBUs and non–T-cell-depleted haplo with PBSCs, both follow ing MAC consisting of TBF. Of note, single UCB is not a stan dard of care in North America. Anti-GVHD prophylaxis included antithymocyte globulin (ATG) in the CBT group and PTCy in the haplo-SCT group. The study report includes 22 patients who underwent haplo-SCT and 23 who underwent CBT.18 Interestingly, 4 patients allocated to the haplo arm crossed over to the CBT arm because no haplo donor could be allocated (in the CTN1101 trial, CB was used for 11 of 182 patients random ized to the haplo arm due to donors being ineligible or declin ing to donate, whereas only 1 of 186 patients randomized to receive CB crossed over to the haplo study arm, due to patient preference). In this rather small study, neutrophil and plate let recovery was slower and lower with CBT compared to the haplo-SCT. Acute and chronic GVHD, as well as RI, was similar between the haplo-SCT and the CBT groups. NRM was higher in CBT in comparison to haplo-SCT. Two-year disease-free sur vival (DFS), OS, and GRFS were significantly better in haplo-SCT compared to CBT. This study emphasizes once again the techni cal difficulties of running donor-based RTC studies and also the heterogeneity, even in RTC studies, in comparing haplo-SCT to CBT not just in patients and disease characteristics but also in the transplant platform, including single vs double CB grafts, BM vs PBSC graft, MAC vs reduced-intensity conditioning (RIC), and GVHD prophylaxis. Haplo-SCT vs CBT: meta-analysis Several meta-ana ly ses have com pared haplo to other graft sources, including CBT, as stem cell sources for allogeneic trans plantation.19 In 1 such meta-analysis recently reported by Owattanapanich et al comparing haplo-SCT and CBT in patients with ALL in mostly registry-based studies, the pooled odds ratios of OS and LFS in the haplo-SCT and CBT groups were not statis tically different. Similarly, the NRM, RI, and aGVHD grade 2 to 4 outcomes of the 2 groups did not statistically differ. Another meta-anal y sis included 7 stud ies with 1311 adults under go ing Dr Prakash Singh Shekhawat Haploidentical vs cord transplantation | 69 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 including 305 CBTs in the years 2013 to 2017, CBT had inferior OS and LFS before 18 months and similar OS and LFS after 18 months. The rates of RI and cGVHD after 18 months were no different between CBT and haplo-SCT, but CBT had increased incidences of NRM and grade 3 to 4 aGVHD (Supplemental Table 1).13 Finally, the health care burden, especially the long-term health care bur den, is becoming a very relevant issue with modern, sophisti cated, and complex therapies. Recently, the Minnesota group ana lyzed the 5-year health care bur den in 1077 con sec u tive adults undergoing HSCT from various graft sources, including BM, peripheral blood stem cells (PBSCs), and CBUs. Notably, adults undergoing CBT had a lower long-term health care bur den compared with the BM and PBSC graft sources, probably due to a lower incidence of cGVHD, as well as other potential unknown factors, which translated into a better quality of life.14 Dr Prakash Singh Shekhawat Ohio State University Comprehensive Cancer Center, Columbus, OH, US Algemeen Ziekenhuis Sint-Jan, Brugge, Belgium Institut Jules Bordet, Brussels, Belgium Universitair Ziekenhuis Gasthuisberg, Leuven, Belgium Hamilton Niagara Regional Haemophilia Centre, Hamilton, Ontario, Canada (and 5 more) NCT02188290 Data used with permission from ClinicalTrials.gov. ATIR, haploidentical, naive cell-enriched T-cell product, depleted of recipient-alloreactive T cells.40 178 adult, older adult (18y to 65y) 368 adult, older adult (18y to 70y) CIBMTR, Medical College of Wisconsin, US University of Alabama at Birmingham, Birmingham, AL, US Arizona Cancer Center, Phoenix, AZ, US City of Hope National Medical Center, Duarte, CA, US (and 35 more) NCT01597778 A Multi-Center, Phase III, Randomized Trial of RIC and Transplantation of (dUCB) Versus HLA-Haplo Related Bone Marrow for Patients With Hematologic Malignancies An Observational Cohort Study on Transplant-Related Mortality in Patients Receiving Either a Hematopoietic Stem Cell Transplantation Without ATIR or an Umbilical Cord Blood Transplantation 206 adults, ≥18 and ≤55y Instituto de Investigacion Sanitaria La Fe, Valencia, Spain NCT02386332 A Randomized Multicenter Study Comparing Unrelated Umbilicalcord Blood Transplant Versus Human Leukocyte Antigen (HLA)Haploidentical Related Hematopoietic Stem Cell Transplant for Adult Patients With Hematologic Malignancies Retrospective Randomized Horowitz M Completed AML ALL Burkitt lymphoma Follicular lymphoma Hodgkin lymphoma Mantle cell lymphoma Non-Hodgkin lymphoma AML ALL MDS Percentage of participants with PFS (time frame: 2y) Transplantrelated mortality (time frame: up to 12 mo after transplantation) September 2014-6 October 2015 12 May 2012-11 September 2020 Sanz MA Unknown Hematologic malignancies DFS (time frame: 2y) Completed Rovers J Jia Chen M. 11 March 2015-March 2020 Recruiting Hematologic diseases Estimated OS at 3y post SCT Primary investigator 1 June 2017- 31 December 2022 Randomized 100 child, adult First Affiliated Hospital of Soochow University Suzhou, Jiangsu, China NCT03719534 Haplo SCT vs Haplo-cord SCT for Patients With Hematological Disorders Status Indication Primary outcome measures Study period Allocation Patients, N Institution/ country Clinical trial identifier Study title Table 2. Randomized controlled trials of haplo-SCT vs CBT Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 70 | Hematology 2022 | ASH Education Program Fuchs et al15 Brunstein et al16 Sanz et al18 References CBT and 915 adults undergoing haplo-SCT and showed no major difference in aGVHD and cGVHD and no difference in 2-year RI, NRM, and DFS (measures of survival could not be pooled because of the different definitions used in the different studies included). The authors concluded that CBTs and haplo-SCT can be consid ered equally effective options for adult patients without HLAmatched donors.20 A third meta-analysis included 12 studies that compared haplo-SCT to CBT in patients with various hematologi cal malignancies that differed from the above 2 meta-analyses. It reported superior OS and lower NRM and aGVHD with haplo-SCT compared to the CBT group, while RI was similar and cGVHD higher, respectively.21 CBT.29 More recently, Childs et al conducted a prospective phase 2 trial of haplo-cord (co-infusing a single UCB unit with CD34+selected cells from a haplo relative) in 29 patients, including 10 with hypoplastic MDS post severe aplastic anemia (SAA) and 10 SAA patients. Neutrophil recovery occurred with a median of 10 days and platelet recovery, with a median of 32 days. Grade 2 to 4 aGVHD was 21% while cGVHD was 41%. The 7.5-year OS was 83% and GRFS was 69%. The authors concluded that haplo-cord transplants provide an alternative option for patients with hypo plastic MDS and refractory SAA who lack HLA-matched donors.30 CLINICAL CASE 1 (Cont inu ed) The patient was informed, after going over the available litera ture, including RCTs, registry-based studies, and meta-analysis, that both haplo trans plan ta tion and CBT were legit i mate options. However, the potential CBUs allocated to him were below the recommended cell dose. Additionally, given the patient’s DSAs and the referred trans plant cen ter’s lack of experience in CBT and the lack of an ongoing clinical trial with ex vivo expanded or haplo-cord, the center recommended a non–T-cell-depleted haplo transplant with TBF conditioning and PTCy as anti-GVHD pro phy laxis.3,22 The pre ferred haplo donor was his brother with the HLA-DRB1 mismatch in view of the emerging data demonstrating that HLA-DRB1 mismatches in haplo-SCT may be associated with a lower risk of disease recur rence and that HLA-DRB1 mismatching with HLA-DQB1 matching and HLA-B leader matching in special scenarios correlates with improved DFS and OS.23 Furthermore, the patient was told that due to harboring monosomy 7 and fms-like tyrosine kinase 3 internal tandem duplication mutation defining high-risk leu kemia, which has been shown to be a poor prognostic factor in alternative donor transplants,24,25 the risk of posttransplant recurrence was substantial. As per recent publications, HLA-B leader might also have implications for NK immunotherapy,26 which may be important for the GVL effect. For the same rea son, posttransplant sorafenib was recommended.27 Haplo-SCT vs CBT: myelodysplastic syndrome and secondary AML Few studies have compared haplo-SCT and CBT in myelodysplastic syn drome (MDS) and sec ond ary AML. The EBMT performed a registry-based study comparing non–T-cell-depleted haplo-SCT with PTCy to CBT and mismatched unrelated trans plants (MMUDs) in patients with MDS (Supplemental Table 1).28 The haplo-SCT group consisted of 222 patients while the CBT group comprised 168 patients. Engraftment was lower with CBT compared to haplo-SCT, while GVHD was lower in haplo-SCT, and 3-year cGVHD and RI did not differ. OS and NRM were bet ter in haplo-SCT compared to CBT (Supplemental Table 1). Additional analysis was performed for 409 secondary AML patients receiv ing either CBT (n=163) or haplo-SCT (n=246). CBT was associated with a higher risk of grade 2 to 4 aGVHD and lower GRFS compared to haplo-SCT. Of note, cGVHD, RI, NRM, LFS, and OS were not statistically different between haplo-SCT and A 32-year-old man presented with clinically meaningful lymph adenopathies. A morphological, immunohistochemical, and molecular evaluation of a lymph node biopsy disclosed highrisk lymphoma with MYC translocation and TP53 mutation. The disease was refractory to multiple lines of immune chemother apy as well as HSCT from an HLA-matched sibling donor (MSD) following MAC. Within 3 weeks of transplant, the disease progressed to a leukemic phase refractory to high-dose cytarabine and mitoxantrone and DLI. The patient was admitted and received chimeric antigen receptor (CAR) CD19 T-cell therapy. One month after cell infusion, positron emission tomography/ computed tomography showed significant improvement, with the resolution of previous F-fluorodeoxyglucose-avid lesions consistent with a complete metabolic response. In view of the ultra-high-risk disease and the short persistence of the CAR T cells and the previous relapse 3 weeks post HLA-identical sib ling transplant, a second transplant from an alternative donor was considered. As no HLA-compatible unrelated donor was available, the alternatives were haplo-SCT or CBT. Haplo-SCT vs CBT: non-Hodgkin, Hodgkin lymphoma, and other lymphatic malignancies Although the num ber of HSCTs in malig nant lym pho mas is decreas ing due to the emer gence of novel drug-con ju gated and bispecific novel mono clo nal antibodies and CAR T cells, HSCT still has a potential role in a selected group of young lym phoma patients. Few single-center and registry-based studies have compared haplo-SCT to CBT for non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). A joint study by the EBMT, Eurocord, and the CIBMTR analyzed 740 patients with NHL and HL who received either haplo-SCT or CBT between 2009 and 2016. Haplo transplants with either BM or PBSCs as a graft source resulted in significantly lower NRM and better PFS and OS (Supplemental Table 1).31 Cornillon et al compared haplo-SCT to CBT in 95 patients with T-cell lymphoma, demonstrating similar PFS and OS and showing that the donor source, whether haplo or CBU, had no major impact on the transplant outcome (Supplemental Table 1).32 Messer et al systematically reviewed some of the early studies on alternative and unrelated donor transplan tation in HL. In the 2 or 3 studies that included haplo-SCT and CBT, outcomes did not differ at large; however, the group was very het ero ge neous, and some of the out come param e ters were missing.33 Additional studies combined the 2 graft sources in high-risk lymphoma and chronic lymphocytic leukemia with interesting results.34,35 Another addresses haplo-SCT and CBT as Dr Prakash Singh Shekhawat Haploidentical vs cord transplantation | 71 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 CLINICAL CASE 2 well as haplo-cord transplants in pediatric ALL, which is beyond the scope of this review.36-38 Conflict-of-interest disclosure CLINICAL CASE 2 (Cont inu ed) Future directions The future optimization of CBT should include a better under standing of the clinical significance of the HLA barrier, hope fully leading to improvements in the current selection of the opti mal CBU, increas ing CBT cell dose, improv ing engraft ment by ex vivo expansion, and omitting in vivo T-cell deple tion (ATG) from the pre par a tive pro to cols. Some of these strategies have already been implemented, leading to signif icant improvements in hematopoietic engraftment post CBT and a substantially reduced nonengraftment rate, shortening the length of posttransplant pancytopenia, reducing infection rates, improv ing immune recon sti tu tion post CBT, impres sively reducing day-100 mortality post CBT, and improving outcomes.39 As for haplo transplants, future directions must focus on optimizing conditioning protocols, defining the optimal dose and schedule of PTCy in non–T-cell-depleted transplants, and facilitating posttransplant immune reconsti tution.40 Optimizing conditioning protocols with compounds such as treosulfan and especially using pharmacokineticsbased conditioning improves haplo-SCT success rates in the advanced-age patient population.2 The shift from BM grafts, the orig i nal stem cell source in non–T-cell- depleted haplo-SCT with PTCy, to PBSC grafts may speed engraftment and immune reconstitution and reduce the relapse rate, with the cost of higher incidences of both GVHD and cytokine release syndrome (CRS).2,41 Notably, patients who developed CRS fol lowing a non–T-cell-depleted haplo-SCT with PBSC grafts and PTCy demonstrated a higher incidence of grade 2 to 4 acute GVHD compared to those who did not.41 Finally, the optimal dose and schedule of PTCy are yet to be defined, and it is conceivable that lower doses reduce some of the reported transplant-associated toxicity, including cardiotoxicity and CRS.41,42 Based on the promising results of the ongoing stud ies aiming to improve engraftment in the setting of CBT and to fasten immune reconstitution post haplo-SCT,39,40 future out comes with both transplant options look encouraging. Acknowledgments The authors thank Annalisa Ruggeri for helpful discussion and crit ical reading of the manuscript; Jacob Passweg, Helen B aldomero, the Acute Leukemia Working Party (ALWP) office, Xiao Jun Huang, and Marcelo Pasquini for providing transplant activities in the EBMT, Chinese Blood and Marrow Transplantation Registry, and Arnon Nagler: no competing financial interests to declare. Mohamad Mohty: no competing financial interests to declare. Off-label drug use Arnon Nagler: nothing to disclose. Mohamad Mohty: nothing to disclose. Correspondence Arnon Nagler, Division of Hematology, Bone Marrow Transplantation, and Hemato-Oncology Center, Chaim Sheba Medical Center, Tel HaShomer, 52621 Ramat Gan, Israel; e-mail: a .nagler@sheba.health.gov.il. References 1. McCurdy SR, Luznik L. How we perform haploidentical stem cell transplan tation with posttransplant cyclophosphamide. Blood. 2019;134(21):18021810. 2. Nagler A, Ruggeri A. Haploidentical stem cell transplantation (HaploSCT) for patients with acute leukemia—an update on behalf of the ALWP of the EBMT. Bone Marrow Transplant. 2019;54(suppl 2):713-718. 3. Politikos I, Davis E, Nhaissi M, et al; American Society for Transplantation and Cellular Therapy Cord Blood Special Interest Group. Guidelines for cord blood unit selection. Biol Blood Marrow Transplant. 2020;26(12):21902196. 4. Baron F, Ruggeri A, Nagler A. Methods of ex vivo expansion of human cord blood cells: challenges, successes and clinical implications. Expert Rev Hematol. 2016;9(3):297-314. 5. van Besien K, Artz A, Champlin RE, et al. Haploidentical vs haplo-cord transplant in adults under 60 years receiving fludarabine and melphalan conditioning. Blood Adv. 2019;3(12):1858-1867. 6. Ruggeri A, Labopin M, Sanz G, et al; Eurocord, Cord Blood Committee of Cellular Therapy and Immunobiology Working Party—European Society for Blood and Marrow Transplantation; ALWP-EBMT Study Investigators. Comparison of outcomes after unrelated cord blood and unmanipulated haploidentical stem cell transplantation in adults with acute leukemia. Leukemia. 2015;29(9):1891-1900. 7. Versluis J, Labopin M, Ruggeri A, et al. Alternative donors for allogeneic hematopoietic stem cell transplantation in poor-risk AML in CR1. Blood Adv. 2017;1(7):477-485. 8. Shouval R, Fein JA, Labopin M, et al. Outcomes of allogeneic haematopoietic stem cell transplantation from HLA-matched and alternative donors: a European Society for Blood and Marrow Transplantation registry retro spective analysis. Lancet Haematol. 2019;6(11):e573-e584. 9. Giannotti F, Labopin M, Shouval R, et al. Haploidentical transplantation is associated with better overall survival when compared to single cord blood transplantation: an EBMT-Eurocord study of acute leukemia patients conditioned with thiotepa, busulfan, and fludarabine. J Hematol Oncol. 2018;11(1):110. 10. Konuma T, Kanda J, Yamasaki S, et al. Single cord blood trans plan ta tion versus unmanipulated haploidentical transplantation for adults with acute myeloid leukemia in complete remission. Transplant Cell Ther. 2021;27(4):334.1-334334.e11. 11. Wada F, Kanda J, Yoshioka S, et al; Kyoto Stem Cell Transplantation Group. Single cord blood transplantation versus HLA-haploidentical-related donor transplantation using posttransplant cyclophosphamide in patients with hematological malignancies. Transplantation. 2022;106(6):1279-1287. 12. Wagner JE, Ballen KK, Zhang MJ, et al. Comparison of haploidentical and umbilical cord blood transplantation after myeloablative conditioning. Blood Adv. 2021;5(20):4064-4072. 13. Wieduwilt MJ, Metheny L, Zhang MJ, et al. Haploidentical vs sibling, unre lated, or cord blood hematopoietic cell transplantation for acute lympho blastic leukemia. Blood Adv. 2022;6(1):339-357. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 Following broad con sul ta tion with the patient and his fam ily evaluating the limited therapeutic alternatives in a highly aggressive TP53-pos i tive lym phoma and the course of the patient’s disease, haplo-SCT with TBF conditioning and PTCy was performed. The donor was a haplo brother because it was felt that a haplo donor, rather than a CB donor, offered the fast engraftment needed. Unfortunately, the patient died early after the second transplant from transplant-related toxicity. 72 | Hematology 2022 | ASH Education Program Center for International Blood and Marrow Transplant Research, respectively; Ivetta Danylesko and Katia Beider for helping with the manuscript tables and illustrations, and Myriam Labopin and other colleagues from the ALWP of the EBMT. with secondary acute myeloid leukemia, a comparative study from Eurocord and the ALWP EBMT. Bone Marrow Transplant. 2019;54(12):1987-1994. 30. Childs RW, Tian X, Vo P, et al. Combined haploidentical and cord blood transplantation for refractory severe aplastic anaemia and hypoplastic myelodysplastic syndrome. Br J Haematol. 2021;193(5):951-960. 31. Fatobene G, Rocha V, St Martin A, et al. Nonmyeloablative alter na tive donor transplantation for Hodgkin and non-Hodgkin lymphoma: from the LWP-EBMT, Eurocord, and CIBMTR. J Clin Oncol. 2020;38(14):1518-1526. 32. Cornillon J, Daguenet E, Tournilhac O, et al. Allogeneic hema to poi etic stem cell transplantation from unmanipulated haploidentical donor and unrelated cord blood for T-cell lymphoma: a retrospective study from the Société Francophone de Greffe de Moelle et de Therapie Cellulaire. Bone Marrow Transplant. 2021;56(11):2849-2856. 33. Messer M, Steinzen A, Vervölgyi E, et al. Unrelated and alternative donor allo ge neic stem cell trans plant in patients with relapsed or refrac tory Hodgkin lymphoma: a systematic review. Leuk Lymphoma. 2014;55(2):296306. 34. Zhou B, Xu M, Lu S, et al. Clinical outcomes of B cell acute lymphoblastic leukemia patients treated with haploidentical stem cells combined with umbilical cord blood transplantation. Transplant Cell Ther. 2022;28(3):173.1173173.e6. 35. Hsu J, Artz A, Mayer SA, et al. Combined haploidentical and umbil i cal cord blood allogeneic stem cell transplantation for high-risk lymphoma and chronic lymphoblastic leukemia. Biol Blood Marrow Transplant. 2018;24(2):359-365. 36. Ponvilawan B, Owattanapanich W, Charoenngam N, et al. Effects of differ ent types of allogeneic hematopoietic stem cell transplantation donors on Philadelphia chromosome-positive acute lymphoblastic leukemia during the tyrosine kinase inhibitor era: a systematic review and meta-analysis [published online ahead of print 2 June 2022]. Hematol Oncol Stem Cell Ther.doi:10.1016/j.hemonc.2021.09.007. 37. Gómez-Santos C, González-Vicent M, Molina B, et al. Comparison of clinical outcomes between unrelated single umbilical cord blood and “ex-vivo” T-cell depleted haploidentical transplantation in children with hematolog ical malignancies. World J Pediatr. 2021;17(6):609-618. 38. Mo XD, Tang BL, Zhang XH, et al. Comparison of outcomes after umbili cal cord blood and unmanipulated haploidentical hematopoietic stem cell transplantation in children with high-risk acute lymphoblastic leukemia. Int J Cancer. 2016;139(9):2106-2115. 39. Horwitz ME, Stiff PJ, Cutler C, et al. Omidubicel vs standard myeloablative umbilical cord blood transplantation: results of a phase 3 randomized study. Blood. 2021;138(16):1429-1440. 40. Roy DC, Walker I, Maertens J, et al. ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia. Leukemia. 2020;34(7):1907-1923. 41. Solán L, Landete E, Bailén R, et al. Cytokine release syndrome after allo geneic stem cell transplantation with posttransplant cyclophosphamide. Hematol Oncol. 2020;38(4):597-603. 42. Duléry R, Mohty R, Labopin M, et al. Early cardiac toxicity associated with post-transplant cyclophosphamide in allogeneic stem cell transplantation. JACC CardioOncol. 2021;3(2):250-259. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000327 Dr Prakash Singh Shekhawat Haploidentical vs cord transplantation | 73 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/64/2022007/64nagler.pdf by guest on 09 December 2022 14. Garcia JG, Grillo S, Cao Q , et al. Low 5-year health care burden after umbil ical cord blood transplantation. Blood Adv. 2021;5(3):853-860. 15. Fuchs EJ, O’Donnell PV, Eapen M, et al. Double unrelated umbilical cord blood vs HLA-haploidentical bone marrow transplantation: the BMT CTN 1101 trial. Blood. 2021;137(3):420-428. 16. Brunstein CG, O’Donnell PV, Logan B, et al; Blood and Marrow Transplant Clinical Trials Network. Impact of center experience with donor type on outcomes: a secondary analysis, Blood and Marrow Transplant Clinical Trials Network 1101. Transplant Cell Ther. 2022;28(7):406.e1-406.e6. 17. O’Donnell PV, Brunstein CG, Fuchs EJ, et al. Umbilical cord blood or HLA-haploidentical transplantation: real-world outcomes versus random ized trial outcomes. Transplant Cell Ther. 2022;28(2):109.1-109109.e8. 18. Sanz J, Montoro J, Solano C, et al. Prospective randomized study compar ing myeloablative unrelated umbilical cord blood transplantation versus HLA-haploidentical related stem cell transplantation for adults with hema tologic malignancies. Biol Blood Marrow Transplant. 2020;26(2):358-366. 19. Owattanapanich W, Leelakanok N, Sanpakit K, Buaboonnam J. A compar ison of the clinical outcomes of haploidentical transplantation and other graft sources in acute lymphoblastic leukemia: a systematic review and meta-analysis. Clin Lymphoma Myeloma Leuk. 2022;22(3):174-191. 20. Li D, Li X, Liao L, Li N. Unrelated cord blood transplantation versus haploidentical transplantation in adult and pediatric patients with hematolog ical malignancies—a meta-analysis and systematic review. Am J Blood Res. 2020;10(1):1-10. https://pubmed.ncbi.nlm.nih.gov/32206440/. Accessed 1 February 2020. 21. Wu R, Ma L. Haploidentical hematopoietic stem cell transplantation ver sus umbilical cord blood transplantation in hematologic malignancies: a systematic review and meta-analysis. Cell Transplant. 2020;29(JanDec):963689720964771. 22. Ciurea SO, Cao K, Fernandez-Vina M, et al. The European Society for Blood and Marrow Transplantation (EBMT) consensus guidelines for the detec tion and treatment of donor-specific anti-HLA antibodies (DSA) in haploidentical hematopoietic cell transplantation. Bone Marrow Transplant. 2018;53(5):521-534. 23. Fuchs EJ, McCurdy SR, Solomon SR, et al. HLA informs risk predictions after haploidentical stem cell transplantation with posttransplantation cyclo phosphamide. Blood. 2022;139(10):1452-1468. 24. Canaani J, Labopin M, Huang XJ, et al. T-cell replete haploidentical stem cell transplantation attenuates the prognostic impact of FLT3-ITD in acute myeloid leukemia: a report from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation. Am J Hematol. 2018;93(6):736-744. 25. Ustun C, Giannotti F, Zhang MJ, et al. Outcomes of UCB transplantation are comparable in FLT3+ AML: results of CIBMTR, EUROCORD and EBMT collab orative analysis. Leukemia. 2017;31(6):1408-1414. 26. Petersdorf EW, Gooley T, Volt F, et al. Use of the HLA-B leader to optimize cord blood transplantation. Haematologica. 2021;106(12):3107-3114. 27. Bazarbachi A, Bug G, Baron F, et al. Clinical practice recommendation on hematopoietic stem cell transplantation for acute myeloid leukemia patients with FLT3-internal tandem duplication: a position statement from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation. Haematologica. 2020;105(6):1507-1516. 28. Robin M, Porcher R, Ruggeri A, et al. HLA-mismatched donors in patients with myelodysplastic syndrome: an EBMT registry analysis. Biol Blood Marrow Transplant. 2019;25(1):114-120. 29. Ruggeri A, Labopin M, Savani B, et al. Hematopoietic stem cell transplanta tion with unrelated cord blood or haploidentical donor grafts in adult patients ARE ALTERNATIVE DONORS NOW MAINSTREAM IN ALLOGENEIC TRANSPLANT ? New strategies for mismatched unrelated donor (MMUD) hematopoietic cell transplant (HCT) City of Hope National Medical Center, Duarte, CA With increasing numbers of patients with hematologic malignancies requiring allogeneic hematopoietic cell transplant (HCT), including minority racial and ethnic groups, the limited availability of matched related donors and matched unrelated donors remains a significant obstacle. Hence, the use of alternative donors such as haploidentical and mismatched unrelated donors (MMUDs) is on the rise. Herein, we present case studies to outline a rational and stepwise approach with a focus on the use of MMUD for HCT in patients with hematologic malignancies. We also review novel approaches used to reduce the incidence of severe graft-versus-host disease and improve HCT outcomes in patients undergoing MMUD HCT. LEARNING OBJECTIVES • Describe the need for mismatched unrelated donors (MMUDs) for hematopoietic cell transplant (HCT) when matched donors are not available for patients with hematologic malignancies • Illustrate the impact of different human leukocyte antigen locus mismatch on HCT outcomes • Defne novel strategies used to improve HCT outcomes in the MMUD setting Introduction MMUDs Allogeneic hematopoietic cell transplantation (HCT) offers a potentially curative treatment for patients with most hematologic malignancies.1 Historically, HCT from a human leukocyte antigen (HLA) matched sibling donor (MSD) is considered the gold standard with the lowest nonrelapse mortality (NRM) and graft-vs-host disease (GVHD)-related mortality and morbidity.2 Comparable transplant outcomes have been reported with matched unrelated donors (MUDs), which became a widely acceptable alternative for patients with no MSD.3,4 The likelihood of identifying an 8/8 MUD varies with ethnicity and race; for example, this could be as high as 70% for Caucasians but falls to below 20% for African American and other ethnic minorities and even more challenging for mixed-race individuals.5 Other factors affecting donor availability include typing issues, medical deferral, and donor or donor center availability.5 With the recent advancement and improvement in outcomes, alternative donors have been increasingly used in close to 36% of cases where match donors are not available (Figure 1). These include mismatched unrelated donors (MMUDs), haploidentical donors (Haplo), and umbilical cord blood. Matching at HLA loci plays an integral role in selecting donors for HCT. When a matched donor (MSD or MUD) is not available, alternative donors are sought such as haploidentical donors, MMUDs, and cord blood units. 74 | Hematology 2022 | ASH Education Program Outcomes using standard (conventional) GVHD prophylaxis for MMUD transplants Large single-center and registry retrospective comparative studies have shown worse outcome with higher degree of mismatch in the setting of conventional GVHD prophylaxis, regardless of disease type or risk, HCT conditioning intensity, or stem cell source (Table 1). Conventional GVHD prophylaxis usually includes a calcineurin inhibitor (ie, tacrolimus or cyclosporine) combined with mycophenolate mofetil, methotrexate, or sirolimus. In 1 study, mostly in patients undergoing myeloablative conditioning (MAC) for bone marrow (BM) HCT,6 difference in survival is less when disease risk is higher, but this was not confrmed in other studies when expanded to other conditioning intensities and stem cell source. For example, patients with poor-risk acute myeloid leukemia (AML) in complete remission 1 who Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Shukaib Arslan and Monzr M. Al Malki Table 1. Studies using conventional GVHD prophylaxis for MMUD HCTs Study Population Design Comparison Key findings Gupta et al (2010)7 Poor-risk AML in CR1 underwent HCT from MSD (n=226), 8/8 MUD (n=254), and MMUD (n=104) CIBMTR—retrospective NA 1. Similar LFS and OS for MSD and 8/8 MUD transplants but LFS and OS were worse for MMUD HCT Al Malki et al (2020)9 482 patients with hematologic malignancies underwent MUD/MMUD HCT with tacrolimus and sirolimus combination for GVHD prophylaxis Single-institution retrospective study NA 1. 5-year OS: 39% for MMUD vs 50.7% for MUD, P = .022 2. NRM: 33.5% for MMUD vs 21.7% for MUD, P = .038 3. Severe GVHD: 20.8% for MMUD vs 12.8% for MUD, P = .22 4. Infection 33.0% for MMUD vs 18.1% for MUD, P < .01 5. No improvement in rates of relapse with MMUD HCT Kornblit et al (2020)10 77 patients who underwent RIC PBSC HCT from MMUD for hematologic malignancies (76 evaluable patients) Multicenter phase 2 trial Historical 1. Day 100 CI of grade II to IV aGVHD was 36% 2. OS at 4 years=62% 3. NRM=18% 4. Relapse/progression=30% CR1, complete remission 1; NA, not available. underwent HCT from MSD (n=226), 8/8 MUD (n=254), and MMUD (n=104) had similar leukemia-free survival (LFS) and overall sur vival (OS) to MSD and 8/8 MUD transplants, but LFS and OS were worse for MMUD HCT.7 Also, in patients with other diseases, such as myelodysplastic syndrome, similar results were reported with worse outcomes in MMUD HCT.8 A large single-center retrospective study compared out comes of MUD and MMUD with tacrolimus and sirolimus as GVHD prophylaxis in 482 peripheral blood stem cell (PBSC) HCTs. With a long-term follow-up (median = 6.2 years), 5-year OS was sig nifi cantly worse in patients under go ing MMUD HCT (39% vs 50.7%, P = .022), mostly due to higher risk of NRM (33.5% vs 21.7%, P = .038), with more severe GVHD (20.8% vs 12.8%, P = .22) and infection (33.0% vs 18.1%, P < .01). There was no improvement in rates of relapse with MMUD HCT.9 A multicenter phase 2 trial used a triplet of cyclospor ine, mycophenolate mofetil, and sirolimus as GVHD pro phylaxis for reduced intensity conditioning (RIC) transplant using MMUD for hematologic malignancies.10 A combina tion of fludarabine and total body irradiation (2-3 Gy) was used as conditioning therapy. The study included 76 evaluable patients with a median age of 63 years. With a median follow-up of 47 months (4-94 months), the cumulative inci dence of day 100 grade II to IV acute GVHD (aGVHD) was 36%. The study reported a cumulative incidence of NRM (18%), relapse/progression (30%), and OS (62%) after 4 years. Mismatch type, number, and location Multiple studies have attempted to correlate HCT outcomes with mismatch at certain HLA loci (Table 2). In 1 study, outcomes of 1874 donor-recip i ent pairs under go ing myeloablative BM HCT were analyzed with respect to high-resolution typing at HLA-A, -B, -C, -DRB1, -DQ , and -DP.11 Survival was worse with higher number of mismatched loci regardless of the mismatched locus location Dr Prakash Singh Shekhawat MMUD transplant PTCy | 75 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Figure 1. CIBMTR-reported relative proportion of allogeneic HCTs in the United States by donor type. Table 2. Landmark studies for MMUD transplant (HCT) Population Study design Comparison Key findings 1874 patients in NMDP who underwent HCT for HMs and nonmalig nant disorders Retrospective 1. Mismatches at loci HLA-A (n=374), -B (n=477), -C (n=749), or -DRB1 (n=311), -DQ (n=415), and -DP (n=1648) 2. Study included 108 patients with 8/8 match 1. Reduced OS after MMUD HCT with mis matches at loci HLA-A, -B, -C, or -DRB1 2. Survival was worse with increasing number of mismatches. 3. Mismatches at loci HLA-A, -B, -C, or -DRB1 all showed more frequent aGVHD 4. The incidence of grade III/IV aGVHD was much higher with mismatch at HLA-A as com pared to mismatch at HLA-B, -C, or -DRB1. 5. Mismatches at HLA-DQ and -DP had little effect on HCT outcomes. 6. Class I HLA mismatches with or without HLADQ mismatch had similar outcomes. Lee et al (2007)6 3857 patients with HMs (early stage, intermediate stage, and advanced/higher stage) who under went MAC HCT • 94% received BMSCs • 78% received T-cell replete grafts • GVHD prophylaxis included calcineurin inhibitor Retrospective (CIBMTR) 1. 8/8 MUD (n=1840) 2. Single (n=985) or multiple (n=1032) MMUD 1. Higher-risk disease at the time of HCT had a higher association with poor survival as compared to increased number of HLA mis matches. 2. No statistically significant difference in sur vival outcomes between single-antigen mis match vs allele mismatch on the same locus. No statistically significant difference between 8/8 and 7/8 with respect to engraftment, relapse, and chronic GVHD. 3. A single mismatch at HLA-A, -C, or -DRB1 was associated with significantly reduced OS compared to 8/8 matches. 4. Multiple mismatches were associated with poorer survival outcomes. 5. Single mismatch at HLA-DQ was not associ ated with any effect on survival. 6. HLA-DP mismatch did not affect survival but was associated with increased risk of aGVHD. Saber et al (2012)4 2223 adult patients with AML who underwent alloHCT between 2002 and 2006 Retrospective (CIBMTR) 1. MSD (n=624) 2. 8/8 MUD (n=1193) 3. 7/8 MUD (MMUD) (n=406) 1. Significantly lower 100-day cumulative inci dence of grade II to IV aGVHD in MSD HCT recipients than in 8/8 MUD and 7/8 MUD HCT recipients (33%, 51%, and 53%, respectively; P<.001). 2. 8/8 MUD HCT recipients had a similar survival rate compared with MSD HCT recipients (RR, 1.03; P=.62). 3. 7/8 MUD HCT recipients had higher early mortality than MSD HCT recipients (RR, 1.40; P<.001), but beyond 6 months after HCT, their survival rates were similar (RR, 0.88; P=.30). Pidala et al (2014)12 8003 patients with HMs (early stage, intermediate stage, and advanced/higher stage) who under went MUD HCT with MAC between 1999 and 2011 Retrospective (CIBMTR) 1. 8/8 MUD (n=5449) 2. 7/8 MMUD (n=2071) 3. 6/8 MMUD (n=483) 1. 6/8-7/8 led to significantly increased risk of grade II to IV and III to IV aGVHD, chronic GVHD, TRM, and reduced OS as compared to 8/8 matched pairs. 2. For 8/8 HCTs, mismatch at both HLA-DQB1 and HLA-DPB1 loci led to an increased inci dence of aGVHD and mismatch at HLA-DPB1 resulted in reduced relapse. Both PR and no-PR HLA-DPB1 allele mismatches led to significantly increased incidence of grade II to IV and grade III to IV aGVHD and reduced risk of relapse. 3. Significantly increased risk of TRM in no-PR pairs as compared to matched pairs or pairs with PR DPB1 allele mismatches. 4. Adjusted OS for 8/8 matched HCT was best among the HLA-DPB1 allele mismatched pairs followed by fully matched HLA-DPB1, with worst adjusted OS for nonpermissive mis matches (P=.015). 5. No difference in outcomes for 1 or double allele mismatches at HLA-DPB1 for permissive or nonpermissive mismatches. 76 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Study Flomenberg et al (2004)11 Table 2. Landmark studies for MMUD transplant (HCT) (Continued) Population Study design Comparison Key findings 310 patients RIC HCT Retrospective 1. 12/12 MUD 2. 11/12 MUD (DPB1mismatched) 3. 10/12 MUD (DPB1mismatched) 1. Better OS and relapse in 11/12 when com pared to 12/12 and better OS as compared to 10/12. 2. Among the 11/12 pairs, when compared with permissive DP mismatch (PR), nonpermis sive DP mismatch (no-PR) is associated with increased risk of grade II to IV aGVHD and NRM. 3. When DP expression was considered, using high DP expressors as donors for low-expressor recipients was associated with increased risk of mortality. DP PR 11/12 mismatch was associated with higher OS when both donor and patient were DP low expressors when compared with other combinations. BMSC, bone marrow stem cell; HM, hematological malignancies; NMDP, National Marrow Donor Program; PR, permissive match; no-PR, nonpermis sive match; TRM, transplant-related mortality. (A, B, C, or DR) or level of testing (antigen vs allele).11 Although mismatches at loci HLA-A, -B, -C, or -DRB1 allshowed more fre quent aGVHD, the inci dence of grade III/IV GVHD was much higher with mismatch at HLA-A (in this particular study) compared with mismatch at HLA-B, -C, or -DRB1. Mismatches at HLA-DQ and -DP showed little effect on HCT outcomes. Another study showed that for the selection of an appropriate MMUD, preference should always be to match at high resolution with HLA-A, -B, -C, and -DR, allowing for HLA-DQ and/or -DP mismatch.11 HLA-DPB1 mismatch.13 Adjusted OS for 8/8 matched HCT was best among patients with permissive HLA-DPB1 allele mismatched pairs followed by fully matched HLA-DPB1 with worst adjusted OS for nonpermissive mismatches (P = .015). Same results were obtained in a smaller cohort with RIC undergoing PBSC MUD HCT.14 In addition, since risk of GVHD associated with HLA-DPB1 mismatching is influ enced by the HLA-DPB1 rs9277534 expres sion marker, patients with higher HLA DPB1 expression allele have a high risk for GVHD when receiving transplants from donors with the low-expression allele, regardless of the conditioning intensity.15 HLA-B leader match in HLA-B mismatch CLINICAL CASE 1 A 65-year-old woman in complete remission 1 after induction therapy for FLT3+ acute myeloid leukemia, with no suitable sib ling donors, presented in consultation. A MUD search revealed four 8/8 or 10/10 HLA matched donors. Patient’s high-resolution HLA typing showed that HLA-DPB1 was homozygous at 04:01, which are low expresser HLA-DPB1 alleles. Review of the HLA typing of the 3 potential donors revealed one 30-year-old donor with 11/12 match with permissive HLA-DPB1 at 02:01, which is a low expressor allele that was activated. Patient underwent HCT with con di tion ing ther apy consisting of fludarabine and mel phalan, as well as GVHD prophylaxis of tacrolimus and sirolimus. Patient is at 2.5 months posttransplant, in continued complete remission (CR) and no evidence of GVHD. Role of HLA-DPB1 mismatch HLA-DPB1 mismatch was studied in multiple different large studies. In a study by Pidala et al,12 8003 donor-recipient pairs underwent MUD HCT with MAC between 1999 and 2011. Mismatch at A, B, C, or DRB1 led to significantly increased risk of grade II to IV and III to IV aGVHD, chronic GVHD, transplant-related mortality, and reduced OS compared with 8/8 matched pairs, confirming prior results. GVHD was higher with no impact on OS/NRM in patients with HLADQB1 and/or HLA-DPB1 mismatch. However, mismatch at HLA-DPB1 resulted in reduced rate of relapse. Based on T-cell epitope matching algorithm, risk of NRM was higher in pairs with nonpermissive HLA-B has high poly mor phism at the pep tide bind ing site. Mismatch at HLA-B leads to increased risk of GVHD and lower disease-free survival.16 Leader pep tides have methi o nine (M) or thre o nine (T) at posi tion 2 containing sequence dimor phisms in exon 1 of HLA-B.16 These leader peptides are bound by HLA-E, which serves as a ligand for the natural killer recep tors. The 2 HLA-B allotypes determine the leader genotype in a person. T- and M-leaders can affect the T-cell and natural killer–cell responses by changing the expression of HLA-E.16-19 These changes could affect alloimmune responses. A large retrospec tive study reported outcomes of 33 982 patients who underwent alloHCT from a MUD/MMUD between 1988 and 2016. The study included 1457 transplants with mismatch at HLA-B and evalu ated outcomes based on B-leader match/mismatch. Presence of M-leader in recipients or donors increases the risk of grade III to IV aGVHD in patients who underwent HCT from an HLA-B mismatched donor. GVHD risk is also increased with the presence of different leaders at the mismatched HLA-B a llotype.16 In this setting, if available donors are mismatched at HLA-B, the selection of the most suitable donor is based on the recipi ent’s leader genotype. For single-locus HLA-B mismatched hematopoietic cell transplants, 94.6% of the recipients had the MT or TT leader genotype. Patients with the MT leader genotype had 2 leader-matched donor possibilities: a donor matched at the M-leader allotype and mismatched for the T-leader allotype (TTM) or a donor mismatched at the M-leader allotype and matched for the T-leader allotype (MMT). Authors reported that TTM trans plants had a higher risk of severe aGVHD compared with MMT Dr Prakash Singh Shekhawat MMUD transplant PTCy | 77 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Study Malki et al (2020)14 (odds ratio, 1.99; 95% confidence interval [CI], 1.10-3.59; P=.022), preferring MMT over TTM to improve HCT outcomes.16 The donor choices for recipients with the TT leader genotype include leader matched (TTT) and leader mismatched (TMT), and TTT pairing is preferred over TMT for improved GVHD outcomes.16 HLA-C mismatch and role of permissive C mismatch CLINICAL CASE 2 A 69-year-old Mediterranean woman in complete remission 2 after induction therapy for NPM1-positive AML with hypomethylating agents with venetoclax was seen in consultation. HLA typing for patient and 2 siblings did not reveal MSD. No MUD was found in the registry. She had multiple high titers (Mean Fluorescent Intensity >5000-10 000) and donor-specific antibodies (DSAs) against HLA-A, -B, and -DRB1 loci in her chil dren. Unrelated donor search revealed a young 7/8 donor with HLA-C mismatch that was permissive, for which the patient had no DSAs. Patient underwent PBSC MMUD HCT with HLA-C mismatched donor using GVHD prophylaxis of tacrolimus and sirolimus. The patient developed grade II skin GVHD, which responded to steroid. Day +100 BM biopsy specimen showed minimal residual disease CR and full donor chimerism. Patient is at >6 months post-HCT in continued remission and no evidence of GVHD. Donor selection for patients with high-risk HLA antibodies Antibodies to HLA can be detected at ele vated fre quen cies in patients with prior sensitizing events such as pregnancy or transfusion,23 but HLA antibodies are also sometimes present in otherwise healthy individuals in the absence of known sensitiz ing events. Although the impact of humoral sensitization in HCT out comes remains poorly under stood com pared with the sig nifi cance of DSAs devel op ing in solid organ trans plan ta tion,24 recent studies of HLA antibodies pre- and early post-HCT have been shown to exert a negative impact on engraftment and graft function.25,26 To avoid graft failure or graft dysfunction, screening patients for HLA antibodies when undergoing workup prior to HLA 78 | Hematology 2022 | ASH Education Program Novel approaches to improve outcomes in MMUD HCT The role of posttransplant cyclo phos pha mide (PTCy) in haploidentical transplant is well established.30 Alloreactive T cells are sensitive to the killing effect of cyclophosphamide, while memory and regulatory T cells, with expression of aldehyde dehydrogenase, are resistant, making PTCy a highly effective therapy to prevent GVHD while preserving T-cell regulatory and memory function.31-34 A phase 2 prospective multicenter trial was conducted to study outcomes of MMUD HCT using PTCy.35 All patients (n=80) received fresh BM grafts on day 0, PTCy on days 3 and 4 after HCT, and sirolimus with mycophenolate starting on day 5. The study included 48% of the patients who were racial or ethnic minorities, and 39% of the patients received a BM graft with more than 1 HLA allele mismatch (4-6/8). One-year OS for the whole group was 76%, as well as 72% and 79% for MAC and RIC, respectively. Degree of HLA mismatch did not affect the OS (75% for 7/8 and 77% for 4-6/8) in this small cohort, which was later confirmed in a longer follow-up presentation. The incidence of day 100 grade II to IV aGVHD, III to IV aGVHD, and 1-year chronic GVHD was 43%, 18%, and 36% for patients receiving MAC and 33%, 0%, and 18% for patients who received RIC, respectively.35 These results were comparable to the out come of a historical control undergoing Haplo HCT and reported to the Center for International Blood, and Marrow Transplant Research (CIBMTR) around the same time.35 Similar retrospec tive but timely comparison was also published by the European Society for Blood and Marrow Transplantation36 in patients with AML in CR with patients undergoing MMUD HCT (n=155) com pared with Haplo BM (n=647) and Haplo PB (n=949) in the setting of PTCy-based GVHD prophylaxis. Haplo BM and Haplo PB had a higher NRM compared with MMUD (hazard ratio, 2.28; 95% CI, 1.23-4.24; P<.01 and hazard ratio, 2.65; 95% CI, 1.46-4.81; P<.01, respectively), with lower LFS and OS. All raised the need for a randomized study comparing the 2 graft sources in a prospec tive and systemic manner. A concurrently conducted, single-center, prospective study of 38 patients who underwent PBSC MMUD HCT (Figure 2) with a similar design showed similar outcomes.29 Median number of HLA mismatches was 2 (range, 1-4) of 12. Racial and ethnic minor ities made up 61% of the study population. The 1-year OS and GVHD-free/relapse-free survival was 87% and 68%, respectively. The incidences of grade II to IV and III to IV aGVHD at day +100 and chronic GVHD at 1 year were 50%, 18%, and 48%, respec tively.29 Combined together, these studies showed that MMUD HCT with PTCy is feasible and well tolerated with promising results using either BM or PBSCs. A large National Marrow Donor Program-sponsored study (ACCESS) is ongoing to confirm these results using PBSC in adults and BM in children (NCT04904588). The European Society for Blood and Marrow Transplantation conducted a retrospective analysis on 272 patients with AML who underwent 9/10 HLA matched HCT with GVHD prophylaxis consisting of PTCy-based (n = 93) or antithymocyte globulin–based Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 For HCTs involving mismatch at HLA-C, the immunogenicity is affected by the level of expression of mismatched HLA-C in the patient.20 A large retrospective study compared outcomes of 1933 unre lated donor-recip i ent pairs who under went PBSC alloHCT for hematologic malignancies. This study included 189 pairs mismatched at the HLA-C antigen. HLA-C mismatch at high resolution was associated with significantly lower leukemia-free survival (rel ative risk [RR], 1.36; 95% CI, 1.13-1.64; P = .0010) and significantly increased risk for mortality (RR, 1.41; 95% CI, 1.16-1.70; P = .0005), transplant-related mortality (RR, 1.61; 95% CI, 1.25-2.08; P = .0002), and grade III to IV aGVHD (RR, 1.98; 95% CI, 1.50-2.62; P < .0001).21 Single-allele mismatch at HLA-C, C*03:03/C*03:04, was very fre quent in a reg is try study of patients with Cau ca sian ances try (68.7%).22 When compared with 8/8 matched HCTs, 7/8 allele level match with C*03:03/C*03:04 mismatch was associated with comparable outcomes such as OS, NRM, disease-free survival, and risk of aGVHD.22 Due to this finding, C*03:03/C*03:04 mismatch is referred to as a permissive mismatch and is preferred at least in the setting of myeloablative BM HCT. mismatched (related or unrelated) HCT has become a standard practice.23 Patients with clinically significant DSAs may undergo DSA desensitization to mitigate risk and facilitate engraftment in most cases but not all.27 With recent advances in GVHD prophy laxis and improvement in outcome of MMUD,27-29 avoiding the mismatched loci with DSAs and mismatching to a non-DSA locus has become one of the advantages of this donor type.28 (n=179) regimens. HLA mismatch involved class I in 74% and class II in 26%; half of the patients received MAC and other half received RIC. Use of PTCy was associated with a lower incidence of severe aGVHD and higher LFS and GVHD-free/relapse-free survival.37 Abatacept (ABA) is a CTLA-4-IgG1 fusion protein that binds to CD80/CD86 on the surface of antigen presenting cells and blocks the CD28-mediated costimulation.38 A single-arm fea sibility study of ABA as prophylaxis of GVHD was conducted enrolling 10 patients with hematologic malignancies using unrelated donors.39 Four patients underwent HCT from 8/8 HLA matched donors, and 6 patients received 7/8 HLA matched donor grafts. Two patients received a BM graft, and 8 patients received PBSCs. Preparative regimen consisted of MAC in 7 and RIC in 3 patients. GVHD prophylaxis was abatacept, cyclospor ine, and methotrexate.39 This helped establish proof of concept and prepared for a larger phase 2 study evaluating the use of ABA in combination with calcineurin inhibitor (CNI) and meth o trex ate in HLA matched unre lated donors for hema to logic malignancies.40 Thirty-eight patients in this study reported to have received MMUD, mostly MAC, with 48% receiving PBSCs. This was reported in comparison to a historical cohort from CIBMTR with or without antithymocyte globulin. Abatacept in combination with CNI and methotrexate (MTX) and in compari son to a historical cohort was shown to be effective in decreas ing day 100 grade III to IV and day 180 severe aGVHD-free survival with a minimal effect on the incidence and severity of chronic GVHD.40 A large multicenter prospective study is ongo ing to confirm these results and to test if extended exposure to ABA would affect incidence and severity of chronic GVHD (NCT04380740). A recent abstract, presented at an Amer i can Society of Hematology meet ing in 2021, com pared the real-world out come of 7/8 HLA MMUD HCTs for hema to logic malig nan cies reported to CIBMTR between 2011 and 2018 using either CNI + MTX with (n=54) or without ABA (n=162).41 The OS at day +180 for the cohort receiving ABA was 98% compared with 75% for CNI + MTX alone (P=.0028). In an exploratory analysis focused on short-term end point (OS at 180 days), outcome of patients undergoing MMUD HCT with ABA with CNI + MTX was compara ble to patients undergoing PTCy.41 Further long-term analysis will be needed to define the effect of this novel GVHD prophylaxis regimen on chronic GVHD and ultimately on OS and the efficacy of this regimen in a higher degree of mismatch (<7/8). Other novel GVHD prophylaxis approaches in the MMUD HCT setting, used recently, with a short-term follow-up include graft manipulation, such as CD34+ cells with add back of mem ory CD45RA+ T cells, α/β T-cell receptor depletion, and CD34+ cell depletion with add back of a conventional T cells and regulatory T cells approach. Those promising approaches are still under investigation (Table 3). Dr Prakash Singh Shekhawat MMUD transplant PTCy | 79 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Figure 2. Study schema. (A) RIC regimen. Fludarabine was administered at the daily dose of 25 mg/m2 from days −7 to −3 before HCT. Melphalan was given on day −2 at 140 mg/m2 or 100 mg/m2 for patients ≥60 years old. (B) MAC regimen consisted of daily fludarabine at 30 mg/m2, from days −7 to −5 before HCT. Total body irradiation was administered in 8 fractions of 150 cGy, 2 times a day, from days −4 to −1, for a total of 1200 cGy. Graft source was PBSCs for both strata. ANC, absolute neutrophils count; G-CSF, granulocyte colony stimulating factor; TBI, total body irradiation. Table 3. Studies using PTCy or abatacept for GVHD prophylaxis for MMUD HCT Population Design Comparison Key findings Shaw et al (2021)35 80 patients who underwent RIC/MAC BMSC MMUD HCT with PTCY/MMF/sirolimus-based GVHD prophylaxis Multicenter, prospective, phase 2 trial NA 1. The 1-year OS was 76% (72% for MAC and 79% for RIC) 2. Degree of HLA mismatch did not affect the OS (75% for 7/8 and 77% for 4-6/8). 3. The day +100 incidence of grade II to IV and grade III to IV aGVHD was 43% and 18% for MAC and 33% and 0% for RIC. 4. The 1-year incidence of chronic GVHD MAC and RIC was 36% and 18%, respectively. Al Malki et al (2021)29 38 patients who underwent PBSC MMUD HCT Single-center, prospective study Battipaglia et al (2022)36 Patients with AML in CR undergoing MMUD HCT (n=155) compared to Haplo BM (n=647) and Haplo PB (n=949) with PTCy-based GVHD prophylaxis Retrospective study Battipaglia et al (2019)37 272 patients with AML underwent 9/10 HLA matched HCT with GVHD prophylaxis consisting of PTCy-based (n=93) or ATG-based (n=179) regimens. HLA mismatch involved class I in 74% and class II in 26%. Half of the patients received MAC, and the other half received RIC. Retrospective study Watkins et al (2021)40 38 received MMUD, mostly MAC, with 48% receiving PBSCs and 52% received BMSCs. GVHD prophylaxis with abatacept in combi nation with CNI and MTX Phase 2 Kean et al (2021)41 7/8 HLA MMUD HCTs for hematologic malignancies between 2011 and 2018 using either CNI + MTX with (n=54) or without ABA (n=162) CIBMTR retrospective 1. The 1-year OS was 87%. 2. The 1-year GRFS 68%. 3. The day +100 incidence of grade II to IV and III to IV aGVHD was 50% and 18%, respectively. 4. The 1-year incidence of chronic GVHD was 48%. Haplo HCT 1. Haplo BMSC and Haplo PBSC had a higher NRM com pared to MMUD (HR, 2.28; 95% CI, 1.23-4.24; P<.01 and HR, 2.65; 95% CI, 1.46-4.81; P<.01, respectively) with lower LFS and OS. 1. Use of PTCy was associated with lower incidence of severe aGVHD and higher LFS and GRFS. Historical cohort from CIBMTR with or without ATG 1. The incidence of grade II to IV aGVHD was 2.3% (CNI/MTX plus abatacept, intention-to-treat popula tion), which compared favor ably with a nonrandomized matched cohort of CNI/MTX (30.2%, P<.001), and the SGFS was better (97.7% vs 58.7%, P<.001). 1. The OS at day +180 for the cohort receiving ABA was 98% compared to 75% for CNI + MTX alone (P=.0028). 2. In an exploratory analy sis focused on short-term end point (OS at 180 days), outcome of patients under going MMUD HCT with ABA with CNI + MTX was compa rable to patients undergoing PTCy. ATG, antithymocyte globulin; GRFS, GVHD free relapse-free survival; HR, hazard ratio; MMF, mycophenolate mofetil; SGFS, severe aGVHD-free survival. 80 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 Study Conclusion Recent advances in GVHD prophylaxis and donor selection have reignited interest in MMUD as a feasible and suitable option for patients without a matched donor for life-saving HCT. Careful selection of optimal MMUDs involving high-resolution HLA typing and HLA antibody measurement to detect DSAs helps guide in the selection of HLA mismatches to improve outcomes. Novel GVHD pro phy laxis approaches such as PTCy, abatacept, and graft manipulation seem to improve GVHD outcomes, although prospective studies are under way. There is constant need for novel therapies to improve the outcomes using MMUDs. Acknowledgments Conflict-of-interest disclosure Shukaib Arslan: no competing financial interests to declare. Monzr M. Al Malki: no competing financial interests to declare. Off-label drug use Shukaib Arslan: cyclophosphamide is discussed. Monzr M. Al Malki: cyclophosphamide is discussed. Correspondence Monzr M. Al Malki, Department of Hematology and HCT, City of Hope National Medical Center, 1500 E. Duarte Rd, Duarte, CA 91010, USA; e-mail: malmalki@coh.org. References 1. Copelan EA. Hematopoietic stem-cell trans plan ta tion. N Engl J Med. 2006;354(17):1813-1826. 2. Beatty PG, Clift RA, Mickelson EM, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med. 1985;313(13):765771. 3. Hows JM, Passweg JR, Tichelli A, et al; IMUST Study Participating Centers; Late Effects Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Comparison of long-term outcomes after alloge neic hematopoietic stem cell transplantation from matched sibling and unrelated donors. Bone Marrow Transplant. 2006;38(12):799-805. 4. Saber W, Opie S, Rizzo JD, Zhang M-J, Horowitz M-M, Schriber J. Outcomes after matched unrelated donor versus identical sibling hematopoietic cell transplantation in adults with acute myelogenous leukemia. Blood. 2012;119(17):3908-3916. 5. Gragert L, Eapen M, Williams E, et al. HLA match likelihoods for hematopoi etic stem-cell grafts in the U.S. registry. N Engl J Med. 2014;371(4):339-348. 6. Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplan tation. Blood. 2007;110(13):4576-4583. 7. Gupta V, Tallman MS, He W, et al. Comparable survival after HLA-wellmatched unrelated or matched sibling donor transplantation for acute myeloid leukemia in first remission with unfavorable cytogenetics at diag nosis. Blood. 2010;116(11):1839-1848. 8. Saber W, Cutler CS, Nakamura R, et al. Impact of donor source on hema topoietic cell transplantation outcomes for patients with myelodysplastic syndromes (MDS). Blood. 2013;122(11):1974-1982. 9. Al Malki MM, Gendzekhadze K, Yang D, et al. Long-term outcome of alloge neic hematopoietic stem cell transplantation from unrelated donor using tacrolimus/sirolimus-based GvHD prophylaxis: impact of HLA mismatch. Transplantation. 2020;104(5):1070-1080. 10. Kornblit B, Storer BE, Andersen NS, et al. Sirolimus with CSP and MMF as GVHD prophylaxis for allogeneic transplantation with HLA antigenmismatched donors. Blood. 2020;136(13):1499-1506. 11. Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone Dr Prakash Singh Shekhawat MMUD transplant PTCy | 81 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 The authors thank Steven Devine, MD, for discussion, feedback, and critical reading of the manuscript and Sally Mokhtari, PhD, for reviewing the manuscript. marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104(7):1923-1930. 12. Pidala J, Lee SJ, Ahn KW, et al. Nonpermissive HLA-DPB1 mis match increases mortality after myeloablative unrelated allogeneic hematopoi etic cell transplantation. Blood. 2014;124(16):2596-2606. 13. Fleischhauer K, Shaw BE, Gooley T, et al; International Histocompatibility Working Group in Hematopoietic Cell Transplantation. 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Sci Immunol. 2016;1(3):eaag1672. 18. Braud V, Jones EY, McMichael A. The human major histocompatibility com plex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol. 1997;27(5):11641169. 19. Romagnani C, Pietra G, Falco M, et al. Identification of HLA-E-specific alloreactive T lymphocytes: a cell subset that undergoes preferential expan sion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells. Proc Natl Acad Sci U S A. 2002;99(17):11328-11333. 20. Petersdorf EW, Gooley TA, Malkki M, et al; International Histocompatibility Working Group in Hematopoietic Cell Transplantation. HLA-C expression levels define permissible mismatches in hematopoietic cell transplanta tion. Blood. 2014;124(26):3996-4003. 21. Woolfrey A, Klein JP, Haagenson M, et al. HLA-C antigen mismatch is asso ciated with worse outcome in unrelated donor peripheral blood stem cell transplantation. Biol Blood Marrow Transplant. 2011;17(6):885-892. 22. Fernandez-Viña MA, Wang T, Lee SJ, et al. Identification of a permissi ble HLA mismatch in hematopoietic stem cell transplantation. Blood. 2014;123(8):1270-1278. 23. Powers A, Stowell CP, Dzik WH, Saidman SL, Lee H, Makar RS. Testing only donors with a prior history of pregnancy or transfusion is a logical and cost-effective transfusion-related acute lung injury prevention strategy. Transfusion. 2008;48(12):2549-2558. 24. Terasaki P, Lachmann N, Cai J. Summary of the effect of de novo HLA antibodies on chronic kidney graft failure. Clin Transpl. 2006:455-462. 25. Focosi D, Zucca A, Scatena F. The role of anti-HLA antibodies in hema topoietic stem cell transplantation. Biol Blood Marrow Transplant. 2011;17(11):1585-1588. 26. Ciurea SO, Thall PF, Wang X, et al. Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplanta tion. Blood. 2011;118(22):5957-5964. 27. Ciurea SO, Al Malki MM, Kongtim P, et al. Treatment of allosensitized patients receiving allogeneic transplantation. Blood Adv. 2021;5(20):4031-4043. 28. Ciurea SO, Cao K, Fernandez-Vina M, et al. The European Society for Blood and Marrow Transplantation (EBMT) consensus guidelines for the detec tion and treatment of donor-specific anti-HLA antibodies (DSA) in haploidentical hematopoietic cell transplantation. Bone Marrow Transplant. 2018;53(5):521-534. 29. Al Malki MM, Tsai N-C, Palmer J, et al. Posttransplant cyclophosphamide as GVHD prophylaxis for peripheral blood stem cell HLA-mismatched unre lated donor transplant. Blood Adv. 2021;5(12):2650-2659. 30. Luznik L, O’Donnell PV, Symons HJ, et al. HLA-haploidentical bone mar row transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2008;14(6):641-650. 31. Mayumi H, Tokunaga K. Cyclophosphamide-induced chimera-type toler ance to allografts: an overview of drug-induced immunological tolerance. Fukuoka Igaku Zasshi. 1990;81(1):20-39. 32. Mayumi H, Umesue M, Nomoto K. Cyclophosphamide-induced immuno logical tolerance: an overview. Immunobiology. 1996;195(2):129-139. 82 | Hematology 2022 | ASH Education Program 38. Malmström V, Catrina AI, Klareskog L. The immunopathogenesis of sero positive rheumatoid arthritis: from triggering to targeting. Nat Rev Immunol. 2017;17(1):60-75. 39. Koura DT, Horan JT, Langston AA, et al. In vivo T cell costimulation blockade with abatacept for acute graft-versus-host disease prevention: a first-indisease trial. Biol Blood Marrow Transplant. 2013;19(11):1638-1649. 40. Watkins B, Qayed M, McCracken C, et al. Phase II trial of costimulation block ade with abatacept for pre ven tion of acute GVHD. J Clin Oncol. 2021;39(17):1865-1877. 41. Kean LS, Burns LJ, Kou TD, et al. Improved overall survival of patients treated with abatacept in combination with a calcineurin inhibitor and methotrexate following 7/8 HLA-matched unrelated allogeneic hemato poietic stem cell transplantation: analysis of the Center for International Blood and Marrow Transplant Research Database. Blood. 2021;138(suppl 1): 3912. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000398 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/74/2021682/74arslan.pdf by guest on 09 December 2022 33. Kanakry CG, Ganguly S, Zahurak M, et al. Aldehyde dehydrogenase expres sion drives human regulatory T cell resistance to posttransplantation cyclo phosphamide. Sci Transl Med. 2013;5(211):211ra157. 34. Wachsmuth LP, Patterson MT, Eckhaus MA, Venzon DJ, Gress RE, Kanakry CG. Post-transplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression. J Clin Invest. 2019;129(6):2357-2373. 35. Shaw BE, Jimenez-Jimenez AM, Burns LJ, et al. National Marrow Donor Program-sponsored multicenter, phase II trial of HLA-mismatched unre lated donor bone mar row trans plan ta tion using post-trans plant cyclo phosphamide. J Clin Oncol. 2021;39(18):1971-1982. 36. Battipaglia G, Galimard J-E, Labopin M, et al. Post-transplant cyclophos phamide in one-antigen mismatched unrelated donor transplantation ver sus haploidentical transplantation in acute myeloid leukemia: a study from the Acute Leukemia Working Party of the EBMT. Bone Marrow Transplant. 2022;57(4):562-571. 37. Battipaglia G, Labopin M, Kröger N, et al. Posttransplant cyclophospha mide vs antithymocyte globulin in HLA-mismatched unrelated donor trans plantation. Blood. 2019;134(11):892-899. ARE ALTERNATIVE DONORS NOW MAINSTREAM IN ALLOGENEIC TRANSPLANT ? Hematology 2022—what is complete HLA match in 2022? Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/Be The Match, Minneapolis, MN Allogeneic hematopoietic cell transplantation (alloHCT) often represents the only curative treatment for various malignant and nonmalignant disorders. Initially, the only suitable donors were considered human leukocyte antigen (HLA)– matched or partially matched relatives. The founding of international unrelated donor and umbilical cord blood registries expanded unrelated donor options and access for patients. In the absence of a matched sibling donor (MSD) with 13% to 51% availability, the current consensus recommends use of a matched unrelated donor (MUD) at HLA-A, B, C, and DRB1 with consideration of matching at HLA-DPB1 and -DQB1. MUD donor availability (donor willing and available to donate) ranges from 29% to 78% with African American patients on the lower end and white non-Hispanic patients with the highest likelihood of a match. Recent studies comparing donor to no-donor treatment options in malignant disease consistently point to substantially better outcomes following alloHCT. In the absence of an MSD or MUD, alternative donor choices turn to haploidentical related (Haplo), mismatched unrelated donor (MMUD), and umbilical cord blood (UCB). Novel strategies for alloHCT, including the use of posttransplant cyclophosphamide-based graft vs host disease prophylaxis, have expanded the safety and effectiveness of transplant procedures across HLA barriers using Haplo and MMUD. The less restrictive matching requirements for UCB transplant are well documented and allow for transplant across multiply mismatched HLA alleles. When all donor options are considered, nearly all patients have an available donor. Here we discuss the likelihood of donor availability, complete HLA match by available donor type, and current controversies warranting future research. LEARNING OBJECTIVES • Describe the degree of HLA matching and association of matching/mismatching on outcomes for available allogeneic donor options • Understand the likelihood of availability and hierarchy of donor type selection for allogeneic hematopoietic cell transplantation Introduction CLINICAL CASE A 60-year-old Hispanic man presents with de novo acute myeloid leukemia (M2;inv 3/t(3;3)). Induction and consolidation therapy achieved complete remission with no measurable residual disease based on next-generation sequencing. Family studies identifed 1 sibling as a full human leukocyte antigen (HLA) match. However, they have a history of malignant melanoma and were deemed ineligible to donate. One additional (65-year-old) sibling is haploidentical to the patient and is willing and eligible to donate. An unrelated donor search identifes multiple young (<30 years old) well-matched (8/8-considering HLA-A, B, C, and DRB1) and single mismatched (7/8) unrelated donors and multiple cord blood units available. Allogeneic hematopoietic cell transplantation (alloHCT) often represents the only curative treatment for various malignant and nonmalignant disorders. Over the approximately 50 years since the frst alloHCTs were attempted and demonstrated engraftment and curative potential, the defnition of optimal matching has continued to evolve. Initially, the only suitable donors were considered HLA matched or partially matched relatives. The founding of the National Marrow Donor Program (NMDP), Anthony Nolan Registry, and other international donor registries expanded the potential to identify suitable donors outside of the patient’s immediate family. Unrelated donor registries have grown to include over 40 million volunteer donors and cord blood registries to over 800 000 banked umbilical cord blood units worldwide (World Marrow Donor Association stats). HLA testing Dr Prakash Singh Shekhawat HLA and donor selection | 83 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 Stephen R. Spellman Table 1. HLA typing techniques and resulting resolution Approach Interpretation Resolution Application Results Serology Cellular assay based on complement fixation by HLA-specific antibodies Cell death—yes/no Low Family screening Null allele confirmation A2, A24 Sequence-specific primers (PCR-SSP) HLA sequence-specific PCR primers Amplification—yes/no Low to high, dependent on DNA sequence coverage Family screening Verification typing Low—A*02:XX, A*24:XX or A*02AB, A*24:BC High—A*0201g, A*24:02g Sequence-specific oligonucleotide probes (PCR-SSOP) HLA sequence-specific oligonucleotide probes that bind to polymorphic sequences of amplified DNA Probe binding—yes/no Low to high depending on DNA sequence coverage Family screening Verification typing Low—A*02:XX, A*24:XX or A*02AB, A*24:BC High—A*02:01G, A*24:02G Sanger sequencebased typing (SBT) HLA amplicon sequencing using base termination Base pair reads and consensus alignment High to allele level depending on coverage All High—A*02:01G, A*24:02G Allele—A*02:01:01:03, A*24:02:01:01 Next-generation sequencing (NGS) Multiple platforms, based on massive parallel sequencing reactions Base pair calling and consensus alignment High to allele-level depending on coverage All High—A*02:01G, A*24:02G Allele—A*02:01:01:03, A*24:02:01:01 technologies and matching strategies have evolved significantly as well, making the characterization of match between unrelated individuals more routine.1 The advent of DNA-based typing tech nologies and enhanced databases of well-characterized HLA allele sequences have increased the precision and accuracy of typing, leading to improved matching (Table 1). Yet, the question remains, what is the optimal match? The answer is not straightforward, as the optimal HLA match for a given patient will vary based on the donor options available at the time of need and clinical fac tors (eg, timing of transplant, patient size/weight, and prior HLA sensitization). HLA typing for search and match determination To facil i tate HLA match assess ment, patients and poten tial donors (related and unrelated) should be high-resolution HLA typed using DNA-based methods for HLA-A, B, C, DRB1, and DPB1.2 Extended typing can include HLA-DRB3/4/5, DQB1, DQA1, and DPA1, especially in the case of highly sensitized patients to avoid mismatches that may be a target of anti-HLA antibodies (eg, donor-specific antibodies) to minimize the risk of graft fail ure.3 Traditional DNA-based techniques for HLA typing focused on the anti gen rec og ni tion domain (ARD).2 Technological advances now make the routine sequencing of the entire HLA gene feasible. A recent study found that mismatching outside of the ARD was associated with increased risk of acute graftvs-host disease (GVHD) grades II to IV but with no increased risk of transplant-related mortality or decreased overall survival (OS),4 suggesting that matching for allelic variation outside the ARD has limited impact in the matched unrelated donor (MUD) setting. HLA typing should be verified via confirmatory testing prior to finalizing donor selection for alloHCT through an American Society of Histocompatibility and Immunogenetics or European Federation for Immunogenetics accredited laboratory, prefer ably with guidance from an accredited histocompatibility and immunogenetics laboratory director to assist the clinical team with interpretation of the typing results and match assessment between the prospective donor(s) and patient. 84 | Hematology 2022 | ASH Education Program What are the available donor options and likelihood of availability? The conventional optimal donor choice is an HLA-matched fam ily member, most often a matched sibling. However, the rate of matched sibling donor (MSD) availability can vary substantially based on ethnicity and age of the patient with rates ranging from 13% to 51%.5 For those patients without a matched sibling, the first-choice alternative donor option is an MUD. This recom mendation is supported by the findings of the annual Center for International Blood and Marrow Transplant Research (CIBMTR) Center Specific Analysis. The 2021 report included >25 000 unre lated and related alloHCTs performed between 2017 and 2019 in the United States with follow-up through 1 year after alloHCT. A comparison of alternative donor types can be assessed using the odds ratio of OS at 1 year compared with MSD as the baseline with an odds ratio (OR) for MUD of 0.87 (95% confidence inter val [CI], 0.78-0.97, P=.010), haploidentical related (Haplo) OR of 0.76 (0.68-0.84, P<.001), mismatched unrelated donor (MMUD) (7/8) OR of 0.69 (0.59-0.81, P<.001), and umbilical cord blood (UCB) (multiple UCB ≥4/6) OR of 0.50 (0.39-0.64, P<.001) (see the visual abstract). This very large, contemporary, multicenter analysis of real-world data clearly demonstrates the hierarchy for donor selection prioritizing matched siblings, MUDs, and then mismatched graft sources. Despite the large international pool of >40 million volunteer unrelated donors, MUD availability varies and is most substan tially affected by the ethnic background of the patient. MUD donor availability (donor willing and available to donate) ranges from 29% to 78%, with African American patients on the lower end and white non-Hispanic patients with the highest likelihood of a match (NMDP internal analysis). In the absence of an MUD, alternative donor choices turn to Haplo, MMUD, and UCB. Novel strategies for alloHCT, including the use of posttransplant cyclo phosphamide-based graft-vs-host disease prophylaxis, have expanded the safety and effectiveness of transplant procedures across HLA barriers using Haplo6 and MMUD.7,8 The less restrictive matching requirements for UCB transplant are well documented and allow for transplant across multiply mismatched HLA alleles.9 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 Methodology Matched sibling donors As noted above, an MSD is the benchmark against which allalter native donor options are evaluated. Donor choice is generally lim ited in the MSD setting and with donor age tightly correlated to patient age. Given that multiple studies suggest that lower donor age is associated with better OS in both the matched unrelated and haploidentical alloHCT setting,12-14 questions have been raised about whether an older sibling donor is still the best option when multiple donor choices are available. A CIBMTR study of older MSD (≥50 years old) vs younger MUD (<50 years old) alloHCT for lymphoma and leukemia reported superior outcomes in MSD.15 A recent analysis comparing older MSD (≥50 years old) to youn ger MUD (≤35 years old) alloHCT for myelodysplastic syndromes found that MUD was associated with lower relapse and improved dis ease-free sur vival (DFS), suggesting that in cer tain cir cum stances (eg, high risk of relapse), MUD may be advantageous.16 In addition, availability of younger Haplo or MMUD may warrant investigation as an alternative to an older matched sibling as out comes for alldonor types continue to improve. One potential concern in older donors is the higher rates of clonal hematopoiesis of indeterminant potential (CHIP) associ ated with increasing age.17 Prior studies differ on the findings regarding risk/benefit of CHIP in the alloHCT setting. Frick et al18 reported increased risk of chronic GVHD following alloHCT with a related donor harboring CHIP, while Gibson et al19 reported potential benefits of DNMT3A variants in faster engraftment and better disease control. While these data raise potential concerns about the suitability of older donors for optimal transplant out comes, there are scarce data to support prioritizing alternative donors over an available MSD at present. HLA matching in MUD These data were reviewed and sum ma rized in the NMDP/ CIBMTR donor selection guidelines published in Blood in 2019 and provided recommendations for optimal MUD matching in the context of conventional calcineurin inhibitor-based GVHD prophylaxis.20 The optimal level of match in the MUD setting was established in the seminal publication by Lee et al.21 This piv otal study dem on strated that matching for HLA-A, B, C, and DRB1 was associated with superior OS and lower rates of acute GVHD21 (Figure 1). This level of match is often referred to as 8/8 matching. HLA-DQB1 match was not associated with any outcomes as an isolated mismatch and did not confer any addi tional risk when paired with HLA-A, B, C, or DRB1. Inclusion of DQB1 in matching is often referred to as 10/10 matching. Most (>95%) cases matched at 8/8 will also be matched at 10/10, so generally bring along a match at HLA-DQB1. These findings were replicated in subsequent analyses in a more contemporary pop ulation,22 as well as by graft type,23 nonmalignant disease,24 and international cohorts.25 HLA-DPB1 mismatching in the context of an 8/8 or a 10/10 match is associated with increased risk of nonrelapse mortal ity driven by higher GVHD and lower risk of relapse with no association with OS.21,22 A model pioneered by Fleischhauer et al26 based on the immunogenicity of HLA-DPB1 in the context of 3 T-cell epitope (TCE) groups provides a methodology for mitigating the risks associated with HLA-DPB1 mismatching. In multiple analyses, the selection of HLA-DPB1 TCE permissive mismatches was associated with improved OS and reduced risk of GVHD, further adding to the strategy for selecting an optimal 8/8 MUD.22,26,27 In addition, a recent analysis found that Figure 1. Survival of patients with early-stage disease depending on degree of HLA matching (8/8, 7/8, and 6/8) for HLA-A, B, C, and DRB1. (Figure courtesy of Lee et al.21) Dr Prakash Singh Shekhawat HLA and donor selection | 85 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 When alldonor options are considered, there is a high likelihood that allpatients will have an available donor.10 How should alternative donor options be prioritized? Varying reports suggest situations where 1 donor type may be preferred over others (eg, Haplo vs double UCB in adult patients receiv ing alloHCT for lymphoma or acute leukemia based on BMT CTN 1101,11 younger alternative donors in the context of older MSD, MMUD vs Haplo), but much controversy remains. The BMT CTN 1702 protocol, “Clinical Transplant-Related Long-Term Outcomes of Alternative Donor Allogeneic Transplantation (CTRL-ALT-D)” (NCT03904134), is inves ti gat ing the out comes of alloHCT fol lowing biological assignment to an MUD or alternative donor (Haplo, UCB, or MMUD) based on likelihood of MUD availability. This trial reflects current consensus that an MUD, if available in a timely manner, is the preferred alternative to an MSD and will shed further light on alternative donor choice and outcomes. This includes an evaluation of the urgency of need and potential to obtain various donor types. Table 2. HLA matching in matched unrelated donor allogeneic hematopoietic cell transplantation Reference Consensus guidelines Dehn et al20 (2019) 1. 8/8 match at ARD level for HLA-A, B, C, DRB1 2. Younger donor age 3. Match or permissive HLA-DPB1 TCE 4. Minimize mismatches at HLA-DRB3/4/5 and DQB1 5. Avoid DSA targets including DQA1 and DPA1 New research published, since release of guidelines Reference Study type Comparison Key findings Mayor et al (2021), ultra-high resolution N=5140 10/10 MUD, first alloHCT for ALL, AML, or MDS. Myeloablative or RIC Observational—multicenter 12/12 Ultra-high resolution vs ≤11/12 UHR matching associated with lower risk of acute GVHD II-IV, no associations with survival outcomes Arrieta-Bolaños et al28 (2022), TCE core N=5140 10/10 MUD, first alloHCT for ALL, AML, or MDS. Myeloablative or RIC Observational—multicenter 10/10 MUD HLA-DPB1 permissive core alleles vs noncore alleles vs nonpermissive 10/10 MUD HLA-DPB1 TCE3 permissive core associ ated with reduced risk of acute GVHD II-IV and TRM compared to nonpermissive mismatches ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; DSA, donor-specific anti-HLA antibodies; MDS, myelodysplastic syndromes; RIC, reduced intensity conditioning; TRM, transplant-related mortality; UHR, ultrahigh resolution; 8/8, high-resolution match at HLA-A, B, C, and DRB1; 10/10, high-resolution match at HLA-A, B, C, DRB1, and DQB1; 12/12, HLA match at HLA-A, B, C, DRB1, DQB1, and DPB1. the TCE3 group could be further categorized into core and noncore alleles to reduce the risk of GVHD and transplantrelated mortality compared with nonpermissive mismatches.28 The potential to find an MUD with an HLA-DPB1 allele match may be limited, but the potential to identify a permissive mis match is highly likely and imputable to support identification of donors with missing DPB1 typing29,30 (Table 2). The 1 non-HLA donor characteristic that consistently associ ates with improved survival after alloHCT is younger donor age and is prioritized above extended matching at HLA-DPB1 and other loci in the current donor selection guidelines.12,13,20 International donor registries now prioritize recruitment of younger donors (≤40 years old), and NMDP has established donor age as a metric of product quality emphasizing the use of donors ≤35 years old. Further stud ies are required to val i date the effects of extended HLA matching and younger donor age in MUD in the context of novel GVHD prophylaxis strategies, such as posttransplant cyclophosphamide (ptCy), abatacept, and advanced graft engineering. HLA matching in mismatched graft sources Related haploidentical donors The selection and prioritization of Haplo donors is limited by fam ily size. The Euro pean Blood and Marrow Transplantation group recently published consensus recommendations for donor selection in Haplo alloHCT focused mainly on non-HLA donor characteristics.31 Novel HLA match and mismatch asso ci a tions with Haplo alloHCT out comes were published after these recommendations. Solomon et al. reported an association between mismatching at HLA class II loci and decreased relapse and improved OS in a single-center study of T-replete Haplo alloHCT using ptCy.32 A recent large multicenter study reported HLA locus-specific associations with various outcomes and proposed a model for Haplo donor selection to optimize DFS. 86 | Hematology 2022 | ASH Education Program The study evaluated overall degree of high-resolution matching, impact of individual loci, HLA-DPB1 TCE matching, and matching for the conserved exon 1 leader sequence of HLA-B (B leader) previously found to associate with acute GVHD in the MMUD set ting.33 The model recommends prioritizing HLA-B leader match, HLA-DPB1 TCE nonpermissive mismatch (opposite of MUD), DRB1 mismatch, and DQB1 match for optimal DFS and was codified in an online tool (Table 3).34 Mismatched unrelated donors Recommendations for the pri or i ti za tion of HLA match and mismatch in MMUD are predominately based on past experi ence in the setting of calcineurin inhibitor–based GVHD pro phylaxis strategies.20 While multiple studies investigated the potential to apply algorithms that prioritize mismatches based on structural similarity or peptide binding affinity, most have failed validation in large multicenter studies.35 FernandezViña et al36 described a permissive mismatch at HLA-C where the alleles (C*03:03 and C*03:04) only differ outside the anti gen recognition domain, but the extension of these findings to other mismatches is limited. Hurley et al37 reported that mis matches at HLA-A, B, C, and DRB1 limited to the host-vs-graft direction were well tolerated and similar to a full match but only applicable in the setting of mismatching at a homozygous locus in the patient. HLA-B leader matches associated with lower risk of acute GVHD.33 Prioritizing HLA-DPB1 TCE permis sive mismatches and minimizing overall level of mismatch at extended HLA class II loci can also contribute to better out comes, including OS in the MMUD setting26,38 (Table 4). The recently described asso ci a tions between HLA match and mismatch in the multiply mismatched Haplo ptCy setting warrant investigation in MMUD.34 The expanded donor pool in the MMUD setting can support additional selection criteria (eg, donor-specific anti-HLA antibodies in highly sensitized patients and potential to prioritize favorable match/mismatches). Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 Population 4 Table 3. HLA matching in haploidentical related donor allogeneic hematopoietic cell transplantation Reference Consensus guidelines Ciurea et al31 (2020) T-replete Haplo using ptCy-based GVHD prophylaxis: 1. Avoid DSA (MFI <1000) 2. Younger donor over older 3. Male donor for male recipient 4. Sibling or offspring over parent 5. Father preferred over mother 6. ABO match over minor over major mismatch New research published, since release of guidelines Population Trial/study design Comparison(s) Key findings N=208 consecutive first alloHCT for hematologic malignancy receiving T-cell replete Haplo with ptCy GVHD prophylaxis Observational—single center 1. Total number of mismatches 2. Individual locus match/mismatch effects on outcomes 1. HLA-DRB1 and HLA-DPB1 nonpermissive mismatch independently associated with improved OS 2. HLA-A mismatch increased chronic GVHD Fuchs et al34 (2022) N=1484, first alloHCT for ALL, AML, or MDS, adult patients receiving T-cell replete Haplo with ptCybased GVHD prophylaxis Observational—multicenter 1. Total number of mismatches 2. Individual locus match/mismatch effects on outcomes 1. No association with number of mismatches 2. HLA-B leader match improves OS and DFS; HLA-DRB1 GVH vector mismatch decreases relapse and improves DFS; HLA-DPB1 nonpermissive mismatch improves OS and DFS; HLA-C match decreases chronic GVHD MFI, mean fluorescence intensity. Table 4. HLA matching in mismatched unrelated donor allogeneic hematopoietic cell transplantation Reference Consensus guidelines Dehn et al (2019) 1. 7/8 match at ARD level for HLA-A, B, C, DRB1 2. Younger donor age 3. Match or permissive HLA-DPB1 TCE 4. Minimize mismatches at HLA-DRB3/4/5 and DQB1 5. Avoid DSA targets including DQA1 and DPA1 20 New research published, since release of guidelines Study Population Trial/study design Comparison Results Petersdorf et al33 (2020) N=33 982 total cohort with N=1457 HLA-B mismatched, first alloHCT for multiple diseases, CNI-based GVHD prophylaxis Observational study Exon 1 B leader match vs mismatch in 9/10 MMUD B leader match associated with decreased acute GVHD, no association between match status and survival Shaw et al7 (2021) N=80 7/8-4/8 MMUD alloHCT for hematologic malignancies, ptCy-based GVHD prophylaxis with bone marrow grafts Prospective trial 7/8 matched vs <7/8 matched No differences in any outcomes CNI, calcineurin inhibitor; 7/8, single high-resolution mismatch at HLA-A, B, C, and DRB1; 9/10, single high-resolution mismatch at HLA-A, B, C, DRB1, and DQB1. Umbilical cord blood Traditionally, UCB match is considered at HLA-A, B at the antigen level and HLA-DRB1 at high resolution to achieve a minimum of a 4/6 match to the patient. In the single UCB alloHCT setting, matching at HLA-C and at high resolution for HLA-A, B, C, and DRB1 is associated with improved outcomes.39 This is not always practical in the setting of UCB HCT where there is a stronger emphasis placed on achieving a minimum total nucleated and/or CD34 cell dose, limiting UCB choice. Expert recommendations from the NMDP/CIBMTR20 and American Society of Transplant and Cellular Therapy9 suggest selecting the best match consid ering HLA-A, B, C, and DRB1 and avoiding UCB <4/8 matched at high resolution or <4/6 using the traditional match standards (Table 5). Dr Prakash Singh Shekhawat HLA and donor selection | 87 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 Study Solomon et al32 (2020) Table 5. HLA matching in umbilical cord blood allogeneic hematopoietic cell transplantation Reference Consensus guidelines Dehn et al20 (2019) ≥4/6 HLA-A and -B antigen match, HLA-DRB1 high-resolution match, ≥4/8 high-resolution match meeting minimum cryopreserved cell dose for single unit alloHCT of TNC ≥2.5×107/kg and CD34+ cells ≥1.5×105/kg and double-unit alloHCT TNC ≥1.5×107/kg and CD34+ cells ≥1.0×105/kg per unit Politikos et al9 (2020) ≥4/6 HLA-A and -B antigen match, HLA-DRB1 high-resolution match, ≥4/8 high-resolution match meeting minimum cryopreserved cell dose for single-unit alloHCT of TNC ≥3.0×107/kg and CD34+ cells ≥2.0×105/kg and double-unit alloHCT CD34+ cells ≥1.5×105/kg per unit TNC, total nucleated cell count. As transplant continues to evolve and growth inevitably occurs in the HLA mismatched setting thanks to safer, more effective approaches to minimize acute and chronic GVHD without risking increased relapse and infections, we will gain more insights into the matching/mismatching to optimize outcomes. Prioritization of alternative donor types (Haplo, MMUD, and UCB) in the absence of an MSD or MUD remains controversial, with varying programs preferring one approach over others. Until these data mature, the optimal match will vary based on the donor type available to a given patient and the choice of donors within that selection pool. CLINICAL CASE (Cont inu ed) The patient was enrolled on BMT CTN 1702 and bio log i cally assigned to the MUD donor arm due to their good search progno sis score.40 The MUD donor options included multiple 8/8 donors either matched or permissively mismatched for HLA-DPB1 TCE and matched for HLA-DQB1. The transplant team selected the youn gest 10/10 HLA-DPB1 TCE per mis sive mismatched donor available and successfully proceeded to transplant. Acknowledgments The author thanks Bronwen Shaw, Jeffery Auletta, and Steven Devine for helpful discussions and critical review of the manuscript. Conflict-of-interest disclosure Stephen R. Spellman: no competing financial interests to declare. Off-label drug use Stephen R. Spellman: nothing to disclose. Correspondence Stephen R. Spellman, Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/Be The Match, 500 5th N Street, Minneapolis, MN 55401-1206; e-mail:sspellma@nmdp.org. References 1. Spellman S, Setterholm M, Maiers M, et al. Advances in the selection of HLA-compatible donors: refinements in HLA typing and matching over the first 20 years of the National Marrow Donor Program Registry. Biol Blood Marrow Transplant. 2008;14(9 suppl):37-44. 88 | Hematology 2022 | ASH Education Program 2. Nunes E, Heslop H, Fernandez-Vina M, et al. Definitions of histocompatibil ity typing terms. Blood. 2011;118(23):e180-e183. 3. Ciurea SO, Cao K, Fernandez-Vina M, et al. The Euro pean Society for Blood and Marrow Transplantation (EBMT) consensus guidelines for the detection and treatment of donor-specific anti-HLA antibodies (DSA) in haploidentical hematopoietic cell transplantation. Bone Marrow Transplant. 2018;53(5):521-534. 4. Mayor NP, Wang T, Lee SJ, et al. Impact of previously unrecognized HLA mismatches using ultrahigh resolution typing in unrelated donor hemato poietic cell transplantation. J Clin Oncol. 2021;39(21):2397-2409. 5. Besse K, Maiers M, Confer D, Albrecht M. On modeling human leukocyte antigen-identical sibling match probability for allogeneic hematopoietic cell transplantation: estimating the need for an unrelated donor source. Biol Blood Marrow Transplant. 2016;22(3):410-417. 6. Luznik L, O’Donnell PV, Symons HJ, et al. 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HLA match likelihoods for hematopoi etic stem-cell grafts in the U.S. registry. N Engl J Med. 2014;371(4):339-348. 11. Fuchs EJ, O’Donnell PV, Eapen M, et al. Double unrelated umbilical cord blood vs HLA-haploidentical bone marrow transplantation: the BMT CTN 1101 trial. Blood. 2021;137(3):420-428. 12. Kollman C, Spellman SR, Zhang M-J, et al. The effect of donor character istics on survival after unrelated donor transplantation for hematologic malignancy. Blood. 2016;127(2):260-267. 13. Shaw BE, Logan BR, Spellman SR, et al. Development of an unrelated donor selection score predictive of survival after HCT: donor age matters most. Biol Blood Marrow Transplant. 2018;24(5):1049-1056. 14. DeZern AE, Franklin C, Tsai H-L, et al. Relationship of donor age and rela tionship to outcomes of haploidentical transplantation with posttransplant cyclophosphamide. Blood Adv. 2021;5(5):1360-1368. 15. Alousi AM, Le-Rademacher J, Saliba RM, et al. Who is the better donor for older hematopoietic transplant recipients: an older-aged sibling or a young, matched unrelated volunteer? Blood. 2013;121(13):2567-2573. 16. Guru Murthy GS, Kim S, Hu Z-H, et al. Relapse and disease-free survival in patients with myelodysplastic syndrome undergoing allogeneic hemato poietic cell transplantation using older matched sibling donors vs younger matched unrelated donors. JAMA Oncol. 2022;8(3):404-411. 17. Jaiswal S, Natarajan P, Silver AJ, et al. Clonal hematopoiesis and risk of ath erosclerotic cardiovascular disease. N Engl J Med. 2017;377(2):111-121. 18. Frick M, Chan W, Arends CM, et al. Role of donor clonal hematopoie sis in allogeneic hematopoietic stem-cell transplantation. J Clin Oncol. 2019;37(5):375-385. 19. Gibson CJ, Kim HT, Zhao L, et al. Donor clonal hematopoiesis and recipient outcomes after transplantation. J Clin Oncol. 2022;40(2):189-201. 20. Dehn J, Spellman S, Hurley CK, et al. Selection of unrelated donors and cord blood units for hematopoietic cell transplantation: guidelines from the NMDP/CIBMTR. Blood. 2019;134(12):924-934. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 Changing landscape in HCT and implications for optimal alternative donor selection 32. Solomon SR, Aubrey MT, Zhang X, et al. Class II HLA mismatch improves outcomes following haploidentical transplantation with posttransplant cyclophosphamide. Blood Adv. 2020;4(20):5311-5321. 33. Petersdorf EW, Carrington M, O’hUigin C, et al; International Histocompatibility Working Group in Hematopoietic Cell Transplantation. Role of HLA-B exon 1 in graft-versus-host disease after unrelated haemopoietic cell transplantation: a retrospective cohort study. Lancet Haematol. 2020;7(1): e50-e60. 34. Fuchs EJ, McCurdy SR, Solomon SR, et al. HLA informs risk predictions after haploidentical stem cell transplantation with posttransplantation cyclo phosphamide. Blood. 2022;139(10):1452-1468. 35. Spellman S, Klein J, Haagenson M, et al. Scoring HLA class I mismatches by HistoCheck does not predict clinical outcome in unrelated hematopoi etic stem cell transplantation. Biol Blood Marrow Transplant. 2012;18(5): 739-746. 36. Fernandez-Viña MA, Wang T, Lee SJ, et al. Identification of a permissi ble HLA mismatch in hematopoietic stem cell transplantation. Blood. 2014;123(8):1270-1278. 37. Hurley CK, Woolfrey A, Wang T, et al. The impact of HLA unidirectional mis matches on the outcome of myeloablative hematopoietic stem cell trans plantation with unrelated donors. Blood. 2013;121(23):4800-4806. 38. Fernández-Viña MA, Klein JP, Haagenson M, et al. Multiple mis matches at the low expression HLA loci DP, DQ , and DRB3/4/5 associate with adverse outcomes in hematopoietic stem cell transplantation. Blood. 2013;121(22):4603-4610. 39. Eapen M, Klein JP, Ruggeri A, et al; Center for International Blood and Marrow Transplant Research, Netcord, Eurocord, and the European Group for Blood and Marrow Transplantation. Impact of allele-level HLA matching on outcomes after myeloablative single unit umbilical cord blood transplanta tion for hematologic malignancy. Blood. 2014;123(1):133-140. 40. Wadsworth K, Albrecht M, Fonstad R, Spellman S, Maiers M, Dehn J. Unrelated donor search prognostic score to support early HLA consultation and clinical decisions. Bone Marrow Transplant. 2016;51(11):1476-1481. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000326 Dr Prakash Singh Shekhawat HLA and donor selection | 89 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/83/2021755/83spellman.pdf by guest on 09 December 2022 21. Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplan tation. Blood. 2007;110(13):4576-4583. 22. Pidala J, Lee SJ, Ahn KW, et al. Nonpermissive HLA-DPB1 mis match increases mortality after myeloablative unrelated allogeneic hematopoi etic cell transplantation. Blood. 2014;124(16):2596-2606. 23. Woolfrey A, Klein JP, Haagenson M, et al. HLA-C antigen mismatch is asso ciated with worse outcome in unrelated donor peripheral blood stem cell transplantation. Biol Blood Marrow Transplant. 2011;17(6):885-892. 24. Horan J, Wang T, Haagenson M, et al. Evaluation of HLA matching in unre lated hematopoietic stem cell transplantation for nonmalignant disorders. Blood. 2012;120(14):2918-2924. 25. Fürst D, Müller C, Vucinic V, et al. High-resolution HLA matching in hema topoietic stem cell transplantation: a retrospective collaborative analysis. Blood. 2013;122(18):3220-3229. 26. Fleischhauer K, Shaw BE, Gooley T, et al; International Histocompatibility Working Group in Hematopoietic Cell Transplantation. Effect of T-cell-epi tope matching at HLA-DPB1 in recip i ents of unre lated-donor haemopoietic-cell transplantation: a retrospective study. Lancet Oncol. 2012;13(4):366-374. 27. Malki MMA, Gendzekhadze K, Stiller T, et al. Protective effect of HLADPB1 mis match remains valid in reduced-inten sity con di tion ing unre lated donor hematopoietic cell transplantation. Bone Marrow Transplant. 2020;55(2):409-418. 28. Arrieta-Bolaños E, Crivello P, He M, et al. A core group of structurally similar HLA-DPB1 alleles drives permissiveness after hematopoietic cell transplan tation. Blood. 2022;140(6):659-663. 29. Tram K, Stritesky G, Wadsworth K, Ng J, Anasetti C, Dehn J. Identification of DPB1 permissive unrelated donors is highly likely. Biol Blood Marrow Transplant. 2017;23(1):81-86. 30. Sajulga R, Madbouly A, Fingerson S, et al. Predicting HLA-DPB1 permissive probabilities through a DPB1 prediction service towards the optimization of HCT donor selection. Hum Immunol. 2021;82(12):903-911. 31. Ciurea SO, Al Malki MM, Kongtim P, et al. The European Society for Blood and Marrow Transplantation (EBMT) consensus recommendations for donor selection in haploidentical hematopoietic cell transplantation. Bone Marrow Transplant. 2020;55(1):12-24. AUTOIMMUNE HEMOLYTIC ANEMIAS Cold AIHA and the best treatment strategies Department of Pediatrics, Division of Hematology/Oncology, Baylor College of Medicine, Houston, TX Cold-reactive autoimmune hemolytic anemia (AIHA) is rare among the hemolytic anemias. It results when 1 of a variety of processes causes the generation of immunoglobulin M (IgM) autoantibodies against endogenous erythrocytes, resulting in complement activation and predominantly intravascular hemolysis. Cold AIHA is typically a primary lymphoproliferative disorder with marrow B-cell clones producing pathogenic IgM. More rarely, secondary cold AIHA (cAIHA) can develop from malignancy, infection, or other autoimmune disorders. However, in children cAIHA is typically post infection, mild, and self-limited. Symptoms include a sequelae of anemia, fatigue, and acrocyanosis. The severity of disease is variable and highly dependent on the thermal binding range of the autoantibody. In adults, treatment has most commonly focused on reducing antibody production with rituximab-based regimens. The addition of cytotoxic agents to rituximab improves response rates, but at the expense of tolerability. Recent insights into the cause of cold agglutinin disease as a clonal disorder driven by complement form the basis of newer therapeutic options. While rituximab-based regimens are still the mainstay of therapy, options have now expanded to include complement-directed treatments and other B-cell-directed or plasma-cell-directed therapies. LEARNING OBJECTIVES • Understand the role of novel therapeutics in the treatment of primary cold agglutinin disease • Compare the management of adult and pediatric cold autoimmune hemolytic anemia CLINICAL CASE 1 A 65yearold woman is admitted for anemia identified dur ing a workup for fatigue. Her hemoglobin level is 6.5 g/dL, and she is sent to the hospital for admission. She reports purple discoloration of her fingers during the winter. Her exam demonstrates pallor, scleral icterus, and jaundice. She has a positive polyspecific direct antiglobulin test (DAT), negative immunoglobulin G (IgG) and positive C3d, undetectable haptoglobin, lactate dehydrogenase (LDH) of 620 U/L, unconjugated hyperbilirubinemia at 4.8 mol/L, and an elevated absolute reticulocyte count (ARC) at 240 × 109/L. Her C3 and C4 are undetectable, and cold agglutinin titer is 1024 at 4 °C. Bone marrow (BM) biopsy does not identify malignancy but is consistent with cold agglutininassociated lymphoproliferative BM disease, with nodular Bcell aggregates, the absence of paratra becular growth, and the fibrosis and lymphoplasmacyt oid cells seen in lymphoplasmacytic lymphoma. Serum protein electrophoresis demonstrates monoclonal IgMκ. The sample is negative for MYD88 L265P mutation. Flow cytometry on her BM sample demonstrates a ratio of κ/λ positive B cells of 6. She is diagnosed with primary cold 90 | Hematology 2022 | ASH Education Program agglutinin disease (CAD). She receives a warmed erythro cyte transfusion and is started on rituximab and benda mustine. As a bridge pending Bcell depletion, she starts weekly sutimlimab infusions, which are spaced to every 2 weeks after the first 2 doses. Her hemolysis resolves and she remains in remission at 3 months. Her sutimlimab is discontinued without any further hemolytic anemia. CLINICAL CASE 2 An 11yearold boy is admitted for severe anemia and hyperbilirubinemia. Ten days prior, he had nasal conges tion, rhinorrhea, cough, and lowgrade fever. His symp toms progressed to include headache, dizziness, pallor, worsening fatigue, dark urine, and yellowing of his eyes. His pediatrician referred him to the emergency room. On exam, he is tachycardic with a systolic ejection murmur. He has diffuse pallor, jaundice, and scleral icterus, as well as mild hepatosplenomegaly. His labs show a white blood cell count of 344 × 109/L, a hemoglobin level of 5.4 g/dL, a mean corpuscular volume of 104.3 fL, and a platelet count of 344 × 109/L. He has an inappropriately low reticulocyte Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 Jenny McDade Despotovic and Taylor Olmsted Kim Table 1. Suggested diagnostic evaluation for CAIHA Suggested adult CAIHA workup Hematologic testing Required for pediatrics CBC Yes Peripheral smeara Yes DAT Yes Hemolysis screena Yes a a LDH, haptoglobin, bilirubin, reticulocyte count, urinalysis Cold agglutinin titera Thermal amplitude Introduction C3 and C4 levels Based on clinical scenario Immunoglobulin quanitification Based on clinical scenario Serum protein electrophoresis + immunofixationa Cold autoimmune hemolytic anemia (cAIHA) is caused by IgM autoantibodies whose κ light chains bind erythrocyte I (or i) anti gens at temperatures below 37 °C.1 IgM-bound red blood cells (RBCs) agglutinate and activate complement. C3b on the RBC surface triggers phagocytosis via the hepatic reticuloendothe lial system. Terminal complement is activated in the vasculature, leading to intravascular hemolysis.2 Cold AIHA is commonly primary but can be secondary to another disorder. In adults, CAD is a clonal disorder driven by low-grade B-cell proliferation in the absence of an overt malig nancy. Proliferating B cells produce IgM, which drives hemolysis through complement activation (Visual Abstract).2 CAD is more common than cold agglutinin syndrome (CAS), which is defined as cold hemolytic anemia arising secondary to another disorder such as autoimmune disease, infection, or malignancy.3 Bone marrow studiesa Histologic examination Flow cytometry Infectious workup Mycoplasma Recommended Viral testing: HIV, HBV. HCV, EBV Recommended Autoimmune screening Antinuclear antibodiesa Based on clinical scenario dsDNA Based on clinical scenario Malignancy screeninga CT chest abdomen and pelvis Minimum required for diagnosis of CAD vs CAS; other studies based on clinical scenario. EBV, Epstein-Barr virus; HBV, hepatitis B virus; HCV, hepatitis C virus. a Diagnostic workup The recommended workup for cAIHA is outlined in Table 1. The patient his tory and phys i cal exam require atten tion to signs of malignancy or infectious etiology, including evaluation for lymphadenopathy and hepatosplenomegaly. Evidence of acro cyanosis is important in both diagnosis and for guiding treat ment choices. Patients may report worse symptoms in colder temperatures or exacerbations during illnesses.4 Direct antibody testing The initial laboratory workup demonstrates hemolysis with an elevated reticulocyte count and positive hemolytic markers. A positive DAT confirms immune-mediated hemolysis. The DAT is typically strongly C3d positive but may also be weakly IgG pos itive for reasons not fully understood. IgM readily activates the classical complement pathway on the erythrocyte surface. Upon warming, the antibody detaches from the RBC surface before it can be detected in the DAT assay, but bound C3b remains. C3b-bound erythrocytes are phagocytosed by the hepatic retic uloendothelial system (extravascular hemolysis). For cells that survive phagocytosis, surface C3b is degraded into less active complement components, including C3d, which is reported in clinical DAT results. Intravascular hemolysis occurs via terminal complement activation with lysis from the action of the C5b-C9 membrane attack complex on the RBC surface (Visual Abstract).5 The clas sic teach ing is that cAIHA is pri mar ily intra vas cu lar; however, extravascular hemolysis in the liver predominates, especially in stable disease. Baseline activity of the regulatory proteins CD55 and CD59 impedes complement activation on the RBC surface, and intravascular hemolysis plays a larger role in disease exacerbation or severe disease.5 Performing a DAT and antibody identification can be techni cally challenging. With a full understanding of the clinical sce nario, trans fu sion med i cine may be able to opti mize test ing and also provide hematology insights into DAT strength or the behavior of antibodies in the sample. Collaboration with trans fusion medicine in the management of cAIHA is also critical for treatments such as transfusion or plasmapheresis. Other laboratory features The mean corpuscular volume is elevated, reflecting reticulocy tosis, or falsely elevated due to RBC agglutination.6 RBC agglu tination is apparent on peripheral smear (Figure 1). As with the patient in Clinical Case 1, complement levels are typically low due to consumption.7 The cold agglutinin titer is at least 64 in CAD and often sub stan tially higher. Cold agglu ti nin ther mal prop er ties have impli ca tions for the clin i cal course: a higher thermal amplitude, which is the highest temperature at which Dr Prakash Singh Shekhawat Cold AIHA treatment | 91 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 count (2.8%; ARC, 38 000). The peripheral smear shows normo cytic anemia with spherocytes, clumped cells, and an absence of schistocytes. DAT IgG is negative and DAT C3 2 is positive, with cold autoantibody identified. He has a mild transamini tis, unconjugated hyperbilirubinemia, undetectable haptoglo bin, and elevated LDH. He is transfused at presentation using a blood warmer and is started empirically on a 5-day course of azithromycin. He is found to be mycoplasma IgM and IgG pos itive. He requires a second transfusion 2 days later for a hemo globin level of 6.4 g/dL. He remains hospitalized for 5 days with his room kept warm and is advised against cold showers and drinking cold beverages. At the time of discharge, his hemoglo bin level is stable at 8.1 g/dL, his ARC is 152 000 (5.7%), and his hemoglobinuria and jaundice have resolved. His hemoglobin normalizes within 2 weeks during outpatient follow-up. and deep venous thrombosis, which should be considered in making therapy decisions.1 Supportive management the cold agglutinin binds, equates to antibodies binding at a wider range of temperatures and more severe disease. This test ing is technically difficult, requiring the sample to be maintained at 37 °C to 40 °C until the serum has been removed.4 Patients should be evaluated for infection, the presence of an overt malignancy, or systemic autoimmunity. CAS refers to cold antibody–mediated hemolytic anemia that has devel oped secondary to another condition. CAD refers to primary cAIHA driven by the low-grade lymphoproliferation of clonal B cells producing IgM. Serum protein electrophoresis con firms a CAD diagnosis and typically shows monoclonal IgMκ. Required for adult patients in order to distinguish CAS and CAD, BM assess ment dem on strates clonal lymphoprolifera tive infiltration, and immunophenotyping shows an abnormal B-cell clone with a B-lymphocyte κ/λ ratio greater than 3.5.8 A computed tomographic scan of the chest, abdomen, and pelvis is necessary to rule out underlying malignancy, partic ularly lymphoma. Considerations for treatment CAD management is based on the degree of anemia and symp tomatology. Those with mild or compensated anemia may not need treatment. Disease exacerbations occur in infection, sur gery, or cold exposure and may necessitate intervention.1,9 In a study of 232 CAD patients, 36% had mild (hemoglobin level >10) or fully compensated anemia.1 Another 37% had moderate ane mia (hemoglobin 8-10 g/dL), and 27% had severe anemia with hemoglobin levels lower than 8.1 Some patients are transfusion depen dent, with an esti mated 40% of patients hav ing been transfused.1,7,10 Those living in colder climates experience coldinduced ischemic symptoms, including cyanosis of areas farther from the body core such as the tips of the ears, the nose, and the digits. While treatment for moderate to severe anemia or both ersome acral symptoms is accepted, fatigue as an indication for therapy is less clear.11 Fatigue is a prominent complaint for many patients and thought to correlate with complement activity.5 Independent of anemia severity, CAD carries an increased risk for thromboembolic events, including stroke, myocardial infarction, 92 | Hematology 2022 | ASH Education Program Therapy to avoid In warm AIHA, opsonized RBCs are destroyed by splenic macro phages. In cAIHA, C3b-coated RBCs are primarily phagocytosed by the hepatic reticuloendothelial cells; therefore, splenectomy is not indicated in most CAD cases.14 Corticosteroids, the mainstay of treatment in warm AIHA, are ineffective in cAIHA and should be avoided.17 Despite a lack of supporting data and an unfavorable side effect profile, cortico steroids are widely employed.1,7,18 Intravenous Ig is ineffective in AIHA, in contrast to its use in other immune cytopenias.19 Other immune suppressive agents are of limited utility.17 Pharmacologic management B-cell directed therapy Therapies for CAD are summarized in Table 2, and a proposed treatment algorithm is presented in Figure 2. Rituximab is the mainstay of treatment for CAD. It is tolerated well and induces responses in 45% to 60% of patients. Complete responses are rare.20,21 The median time to response to rituximab is approxima tely 1.5 to 3 months, with a median remission duration of approx imately 6 months.20,21 Most patients expe ri ence relapse upon B-cell repopulation, typically within 1 year.21 Given its moderate efficacy and high relapse rate with mono therapy, rituximab is used in com bi na tion with the cyto toxic agents fludarabine and bendamustine. Rituximab with fludara bine has superior efficacy over rituximab alone and may induce responses in those refractory to rituximab monotherapy.22 How ever, fludarabine has higher toxicity; in a prospective uncon trolled trial, 57% of patients developed an infection, and 41% had grade 3 to 4 hematologic toxicity (14% with grade 4 neutrope nia).22 Rituximab with bendamustine has an approximately 70% overall response rate with a more favorable side effect profile. For those with comorbidities, rituximab monotherapy is the recommended first-line therapy, while for fit individuals likely to tolerate cytotoxic therapy, rituximab with bendamustine is the treatment of choice.17 For those who fail initial treatment or have relapsing symptoms, second-line therapies include complement inhibitors, bortezomib, or, for those well enough to tolerate it, ibrutinib or rituximab with fludarabine.17 Bortezomib tar gets long-lived plasma cell pop u la tions. In a trial of 21 refrac tory patients, 32% gained trans fu sion Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 Figure 1. Peripheral blood smear from patient with cold agglutinin disease showing red blood cell clumping (100×). Image courtesy of Dr Tarek M. Elghetany. Patients should avoid cold temperatures, with attention to keep ing acral areas warm.10,12 For symptomatic anemia, transfusion can be given using an in-line blood warmer. Without warming, hemolysis is exacerbated, leading to line occlusion during the transfusion.12 Plasmapheresis may be indicated for patients with severe, symptomatic anemia requiring rapid intervention. A 1 to 1.5 times plasma volume exchange decreases IgM and improves anemia.13 For children, plasmapheresis is used as a temporizing mea sure pending spontaneous disease resolution. While in adults with CAD, plasmapheresis should be followed by more durable therapy.14 Other supportive measures used for hemolytic anemia include folate supplementation and, potentially, erythropoietin.15,16 Table 2. Pharmacologic management of CAD Dose Rituximab Rituximab-fludarabine 375 mg/m2 weekly×4 45%-60% 76% Median duration of response Clinical considerations CR: normalized hemoglobin level and absence of hemolysis or CAD symptoms; PR: hemoglobin increase ≥1-2 g/dL (varies by study), transfusion independence and improved CAD symptoms, 50% reduction in IgM concentration 6.5-11 months (observed) Risk of rituximab infusion reactions CR: normalized hemoglobin level, absence of hemolysis, CAD symptoms, undetectable IgM, no evidence of clonal proliferation; PR: hemoglobin increase >/=2 g/dL, transfusion independence and improved CAD symptoms, 50% reduction in IgM concentration >66 months (observed) Definition of response Confirm vaccination status prior to dosing Months for effect Risk of rituximab infusion reactions Not appropriate for frail patients with comorbidities due to risk for infection, neutropenia Months for effect Fludarabine 40 mg/m2 on days 1-5, 29-34, 57-61, and 85-89 Rituximab-bendamustine Rituximab 375 mg/m2 q28 days×4 71% CR: normalized hemoglobin level, absence of hemolysis, CAD symptoms, undetectable IgM, no evidence of clonal proliferation; PR: hemoglobin increase >/=2 g/dL, transfusion independence and improved CAD symptoms, 50% reduction in IgM concentration >32 months (observed) Risk of rituximab infusion reactions Not appropriate for frail patients with comorbidities due to risk for infection, neutropenia Months for effect Bendamustine 90 mg/m2 on days 1, 2 q28 days×4 Eculizumab 600 mg weekly×4, then 900 mg every other week through week 26 54% Decrease in LDH level pre and post therapy >/=250 U/L Must be used continuously to maintain effect Rapid onset; may be used as a temporizing measure Vaccinate against encapsulated bacteria; meningococcal prophylaxis until vaccinated Will not improve acrocyanosis Sutimlimab 6500 mg weekly×2, then every other week though 26 weeks 54% Hemoglobin increase >/=1.5 g/dL, transfusion independence, no use of CAD treatments Must be used continuously to maintain effect Rapid onset; may be used as a temporizing measure Vaccinate against encapsulated bacteria; meningococcal prophylaxis until vaccinated Will not improve acrocyanosis Ibrutinib Bortezomib 420 mg/d 1.3 mg/m2 on days 1, 4, 8, 11 100% 32% CR: hemoglobin >12 g/dL; PR 10-12 g/dL or ≥2 g/dL Rise in hemoglobin >/=2 g/dL, transfusion independence Studies reported on ~ median of 12 months daily use Requires acyclovir prophylaxis 16 months (observed) Requires acyclovir prophylaxis Useful for acrocyanosis 7500 mg for patients weighing >/=75 kg. CR, complete response; PR, partial response. a Dr Prakash Singh Shekhawat Cold AIHA treatment | 93 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 Rituximab 375 mg/m2 on days 1, 29, 57, and 85 Overall response rate Other promising/experimental therapies Figure 2. Treatment algorithm for cold agglutinin disease. Adapted with permission from S. Berentsen.17 independence, and a subset of individuals achieved durable remission.23,24 Pediatric cAIHA Complement-directed therapy The intravascular component of CAD is driven by terminal com plement activation on the RBC surface (Visual Abstract). Eculi zumab, an anti-CD5 mono clo nal anti body inhibiting ter mi nal complement, is used in other complement-driven conditions, including paroxysmal nocturnal hemoglobinuria and atypi cal hemolytic uremic syndrome.25,26 The DECADE trial was an open-label, prospective nonrandomized phase 2 trial of eculi zumab in CAD patients.27 Eculizumab showed an overall response rate of 54%, with response defined as a decrease in LDH greater than or equal to 250 U/L. The median increase in hemoglobin level was modest, at less than 1 g/dL over the 26-week study period.28 Patients with a narrower thermal amplitude (indicating milder disease) had a better response to eculizumab.27 Sutimlimab, a monoclonal antibody–inhibiting serine protease C1s in the classical complement pathway, recently completed testing in a single-arm phase 3 clinical trial of nontransfused CAD patients (CARDINAL).11,28,29 Patients tolerated the drug well and saw an increase in hemoglobin level of approximately 2.6 g/dL from baseline by week 3, with a hemoglobin level greater than or equal to 11 g/dL maintained from week 3 to the end of the 26-week study period. Notably, the hemoglobin level response in this study was earlier and more robust than studies using ecu lizumab. Sutimlimab also improved fatigue.11 These findings were val i dated in a ran dom ized pla cebo-con trolled phase 3 study (CADENZA), with 73% of patients meeting the primary end point of hemoglobin levels rising more than or equal to 1.5 g/dL without the need for transfusion of additional CAD-directed therapies.30 94 | Hematology 2022 | ASH Education Program CAD is driven by clonal B-cell expan sion that pro duces IgM. Bruton’s tyrosine kinase inhibitors, phosphatidylinositol 3-kinase δ inhibitors, or B-cell lymphoma 2 (BCL2) inhibitors are efficacious in other clonal B-cell disorders and have potential in CAD. Bruton’s tyrosine kinase inhibitors such as ibrutinib are effective in both CAD and CAS.35 Daratumumab, an antibody against plasma cell CD38, used in multiple myeloma, has successfully resolved hemo lysis, reduced agglutinin titers. and resolved non–complementmediated symptoms in refractory CAD.36,37 cAIHA is rare in children compared to adults. The majority of cases develop secondary to infection, with Mycoplasma pneumoniae being the most com mon cause. Epstein-Barr virus, influenza, cytomegalovirus, and varicella are also implicated.38 Infectious testing is recommended for allchildren, and autoim mune screening is suggested (eg, antinuclear antibodies titer) in adolescents and teens. An extensive workup to distinguish CAD from CAS such as that described in Clinical Case 1 is not necessary; cAIHA caused by low-grade clonal B-cell lymphop roliferation is not reported in children. Treatment for pediatric cAIHA is focused on supportive care during the acute presenta tion while awaiting spontaneous disease resolution. Measures include avoiding cold exposure and warming blood products or other intravenous infusions. Though in this case the child’s Mycoplasma pneumoniae was treated, most infections driving pediatric cAIHA are self-limited. In an emergency, plasmaphe resis can be utilized. Rituximab and complement-directed ther apies have not been studied in pediatric cAIHA.39 Paroxysmal cold hemoglobinuria Paroxysmal cold hemo glo bin uria (PCH) is uncom mon in chil dren and exceed ingly rare in adults.5 PCH is caused by the Donath-Landsteiner antibody directed against the erythrocyte P antigen.6 IgG fixes complement at cold temperatures. After cooling and rewarming the sample, complement is amplified, and RBCs undergo intravascular hemolysis.5,39 PCH is triggered by viral infections and treatment is supportive. Because it is IgGmediated, PCH patients may be steroid responsive.39 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 Sutimlimab is being studied in an open-label multicenter trial for transfused CAD patients (NCT03347396). Sutimlimab is the only US Food and Drug Administration-approved drug for adults with CAD to decrease the need for transfusion. BIVV020, an anti-C1s antibody, is in a phase 1 trial assessing safety and tolerability in CAD patients (clinicaltrials.gov identifier: NCT4269551).29 Pegcetacoplan, a C3 inhibitor, successfully causes the cessation of hemolysis in in vitro models.31 In paroxysmal noc turnal hemoglobinuria clinical trials, it is well tolerated and has superior clinical outcomes compared to eculizumab.32,33 A phase 3, randomized, double-blind, placebo-controlled multicenter trial evaluating pegcetacoplan in CAD is recruiting (NCT05096403). Unlike rituximab-based protocols, complement-directed treat ments require ongoing dosing. Because complement inhibitors do not stop IgM binding and RBC agglutination, ischemic symp toms are not alleviated with this class of drugs, though h emolysis may improve.34 The most appro pri ate use of com ple mentdirected drugs may be alleviating acute, severe anemia or bridg ing to durable rituximab-based regimens. Conclusion CAD is a rare cause of AIHA. In adults it is typically primary, driven by clonal lymphoproliferation leading to IgM production, thereby triggering complement. The under stand ing of CAD pathophysiology is improving, and treatment options have expanded. Novel therapeutics targeting clonal B cells or com plement have shown success, and there are more new therapies on the horizon. Increasingly, patients are able to receive ther apy to improve anemia, acral symptoms, and fatigue. With more well-tolerated treatment options available, the risk/benefit ratio of treating cAIHA is shifting toward improved symptom control for more patients. Jenny McDade Despotovic: consultancy: Novartis; research fund ing: Novartis; honoraria: Novartis, Dova, Amgen; royalties: Upto date. Taylor Olmsted Kim: consultancy: Novartis. Off-label drug use All drugs are off-label for pediatric patients. For adults, sutim limab is the only FDA-approved therapy for cold agglutinin dis ease. The rest listed in Table 2 are off-label. Correspondence Jenny McDade Despotovic, Baylor College of Medicine, 6701 Fan nin St, Ste 1580, Houston, TX 77030; e-mail: jmdespot@txch.org References 1. Berentsen S, Barcellini W, D’Sa S, et al. Cold agglutinin disease revisited: a multinational, observational study of 232 patients. Blood. 2020;136(4):480488. 2. Randen U, Trøen G, Tierens A, et al. Primary cold agglutinin-associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow dis tinct from lymphoplasmacytic lymphoma. Haematologica. 2014;99(3):497504. 3. Jäger U, Barcellini W, Broome CM, et al. Diagnosis and treatment of auto immune hemolytic anemia in adults: recommendations from the First Inter national Consensus Meeting. Blood Rev. 2020;41(May):100648. 4. Berentsen S. How I manage patients with cold agglutinin disease. Br J Haematol. 2018;181(3):320-330. 5. Berentsen S. New insights in the pathogenesis and therapy of cold agglu tinin-mediated autoimmune hemolytic anemia. Front Immunol. 2020;11(7 April):590. 6. Kim TO, Despotovic JM. Primary and secondary immune cytopenias: eval uation and treatment approach in children. Hematol Oncol Clin North Am. 2019;33(3):489-506. 7. Berentsen S, Ulvestad E, Langholm R, et al. Primary chronic cold agglutinin disease: a population based clinical study of 86 patients. Haematologica. 2006;91(4):460-466. 8. Berentsen S, Tjønnfjord GE. Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia. Blood Rev. 2012;26(3):107-115. 9. Ulvestad E, Berentsen S, Mollnes TE. Acute phase haemolysis in chronic cold agglutinin disease. Scand J Immunol. 2001;54(1-2):239-242. 10. Swiecicki PL, Hegerova LT, Gertz MA. Cold agglu ti nin dis ease. Blood. 2013;122(7):1114-1121. 11. Röth A, Barcellini W, D’Sa S, et al. Sutimlimab in cold agglutinin disease. N Engl J Med. 2021;384(14):1323-1334. 12. Gabbard AP, Booth GS. Cold agglu ti nin dis ease. Clin Hematol Int. 2020;2(3):95-100. 13. Szczepiorkowski ZM, Winters JL, Bandarenko N, et al; Apheresis Applica tions Committee of the American Society for Apheresis. Guidelines on the use of therapeutic apheresis in clinical practice—evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher. 2010;25(3):83-177. 14. Berentsen S. Complement acti va tion and inhi bi tion in auto im mune hemo lytic ane mia: focus on cold agglu ti nin dis ease. Semin Hematol. 2018;55(3):141-149. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000369 Dr Prakash Singh Shekhawat Cold AIHA treatment | 95 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/90/2021751/90despotovic.pdf by guest on 09 December 2022 Conflict-of-interest disclosure 15. Fattizzo B, Michel M, Zaninoni A, et al. Efficacy of recombinant erythropoi etin in autoimmune hemolytic anemia: a multicenter international study. Haematologica. 2021;106(2):622-625. 16. Go RS, Winters JL, Kay NE. How I treat autoimmune hemolytic anemia. Blood. 2017;129(22):2971-2979. 17. Berentsen S. How I treat cold agglutinin disease. Blood. 2021;137(10):12951303. 18. Mullins M, Jiang X, Bylsma LC, et al. Cold agglutinin disease burden: a lon gitudinal analysis of anemia, medications, transfusions, and health care uti lization. Blood Adv. 2017;1(13):839-848. 19. Flores G, Cunningham-Rundles C, Newland AC, Bussel JB. Efficacy of intra venous immunoglobulin in the treatment of autoimmune hemolytic ane mia: results in 73 patients. Am J Hematol. 1993;44(4):237-242. 20. Schöllkopf C, Kjeldsen L, Bjerrum OW, et al. Rituximab in chronic cold agglutinin disease: a prospective study of 20 patients. Leuk Lymphoma. 2006;47(2):253-260. 21. Berentsen S, Ulvestad E, Gjertsen BT, et al. Rituximab for primary chronic cold agglutinin disease: a prospective study of 37 courses of therapy in 27 patients. Blood. 2004;103(8):2925-2928. 22. Berentsen S, Randen U, Vågan AM, et al. High response rate and durable remissions following fludarabine and rituximab combination therapy for chronic cold agglutinin disease. Blood. 2010;116(17):3180-3184. 23. Rossi G, Gramegna D, Paoloni F, et al. Short course of bortezomib in ane mic patients with relapsed cold agglutinin disease: a phase 2 prospective GIMEMA study. Blood. 2018;132(5):547-550. 24. Carson KR, Beckwith LG, Mehta J. Successful treatment of IgM-mediated autoimmune hemolytic anemia with bortezomib. Blood. 2010;115(4):915. 25. Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355(12):12331243. 26. Legendre CM, Licht C, Muus P, et al. Terminal com ple ment inhib i tor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368(23):2169-2181. 27. Röth A, Bommer M, Hüttmann A, et al. Eculizumab in cold agglutinin dis ease (DECADE): an open-label, pro spec tive, bicentric, nonrandomized phase 2 trial. Blood Adv. 2018;2(19):2543-2549. 28. Nikitin PA, Rose EL, Byun TS, Parry GC, Panicker S. C1s inhi bi tion by BIVV009 (sutimlimab) prevents complement-enhanced activation of auto immune human B cells in vitro. J Immunol. 2019;202(4):1200-1209. 29. Jäger U, D’Sa S, Schörgenhofer C, et al. Inhibition of complement C1s improves severe hemolytic anemia in cold agglutinin disease: a first-inhuman trial. Blood. 2019;133(9):893-901. 30. Röth A, Berentsen S, Barcellini W, et al. Sutimlimab in patients with cold agglutinin disease: results of the randomized placebo-controlled phase 3 CADENZA trial. Blood. 2022;140(9):980-991. 31. Risitano AM, Ricklin D, Huang Y, et al. Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria. Blood. 2014;123(13):2094-2101. 32. de Castro C, Grossi F, Weitz IC, et al. C3 inhibition with pegcetacoplan in subjects with paroxysmal nocturnal hemoglobinuria treated with eculi zumab. Am J Hematol. 2020;95(11):1334-1343. 33. Hillmen P, Szer J, Weitz I, et al. Pegcetacoplan versus eculizumab in parox ysmal nocturnal hemoglobinuria. N Engl J Med. 2021;384(11):1028-1037. 34. Berentsen S. Cold agglutinins: fending off the attack. Blood. 2019;133(9): 885-886. 35. Jalink M, Berentsen S, Castillo JJ, et al. Effect of ibrutinib treatment on hemolytic anemia and acrocyanosis in cold agglutinin disease/cold agglu tinin syndrome. Blood. 2021;138(20):2002-2005. 36. Tomkins O, Berentsen S, Arulogun S, et al. Daratumumab for disabling cold agglutinin disease refractory to B-cell directed therapy [published online 11 July 2022]. American J Hematol. 37. Zaninoni A, Giannotta JA, Gallì A, et al. The immunomodulatory effect and clinical efficacy of daratumumab in a patient with cold agglutinin disease. Front Immunol. 2021;12(March):649441. 38. Kim TO, Bussel JB. Autoimmune Hemolytic Anemia in the Adolescent Females. Springer Nature Switzerland AG; 2020. 39. Teachey DT, Lambert MP. Diagnosis and management of autoimmune cyto penias in childhood. Pediatr Clin North Am. 2013;60(6):1489-1511. AUTOIMMUNE HEMOLYTIC ANEMIAS Susan T. Johnson and Kathleen E. Puca Clinical Education and Diagnostic Laboratories Immunohematology Reference Laboratory, Versiti Blood Center of Wisconsin, Milwaukee, WI The serologic evaluation of autoimmune hemolytic anemia (AIHA) confirms the clinical diagnosis, helps distinguish the type of AIHA, and identifies whether any underlying alloantibodies are present that might complicate the selection of the safest blood for any needed transfusion. The spectrum of testing is generally dependent on the amount and class (immunoglobulin G or M) of autoantibody as well as the resources and methodologies where testing is performed. The approach may range from routine pretransfusion testing, including the direct antiglobulin test, to advanced techniques such as adsorptions, elution, and red cell genotyping. When transfusion is needed, the selection of the optimal unit of red blood cells is based on urgency and whether time allows for the completion of sophisticated serologic and molecular testing methods. From the start of when AIHA is suspected until the completion of testing, communication among the clinical team and medical laboratory scientists in the transfusion service and immunohematology reference laboratory is critical as testing can take several hours and the need for transfusion may be urgent. The frequent exchange of information including the patient’s transfusion history and clinical status, the progress of testing, and any available results is invaluable for timely diagnosis, ongoing management of the patient, and the safety of transfusion if required before testing is complete. LEARNING OBJECTIVES • Describe the difficulties in determining ABO group/RhD type and in identifying alloantibodies in patients with autoantibodies • Discuss strategies for the selection of blood for safe transfusion, keeping in mind patient history and completed laboratory testing • Explain the importance of communication among clinicians and laboratory professionals in the approach to transfusion Introduction Autoimmune hemolytic anemia (AIHA) is serologically defined as warm or cold based on the class of immunoglobulin (Ig), either IgG or IgM, causing hemolysis. Warm autoantibodies are IgG and primarily detected in vitro at 37 °C and in the indirect antiglobulin test (IAT), while cold autoantibodies (CAAs) are IgM and bind preferentially to red blood cells (RBCs) at colder temperatures in clinical testing (Table 1). Despite these simplified definitions, the serologic evaluation of patients presenting with these strong autoantibodies is a challenge for any laboratory. The 96 | Hematology 2022 | ASH Education Program extent of testing capabilities varies among hospital transfusion services and immunohematology reference laboratories (IRLs). An overview of the various tests employed and a reference for the clinical cases are provided in Table 2. CLINICAL CASE 1 A 35-year-old woman with a vague history of idiopathic thrombocytopenia presented to the emergency department with a complaint of fatigue, malaise, head- Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies Table 1. Summary of classic serologic findings in AIHA Type of AIHA Antibody class Antibody specificity Optimum test phase of antibody reactivity Warm IgG Broadly reactive Cold IgM Mixed Type IgG and IgM DAT result Eluate result IAT 2-4+ IgG and C3 or IgG only Positive—reactive with all/majority of panel RBCs I/i RT, 37 °Ca 2-3+ C3 only Not usually performed Broadly reactive RT, 37 °C, IAT 3-4+ IgG and C3 Positive—reactive with all/majority of panel RBCs ache, and cough. Vital signs are temperature, 37.8 °C; blood pressure, 114/55 mmHg; heart rate, 98/min; respiratory rate, 20; and oxygen saturation, 100% on room air. Initial laboratory studies show a positive COVID test, a white blood cell count of 10 100/µL (4200-11 000), a hemoglobin (Hgb) level of 4.4 g/dL (12.0-15.5), and a platelet count of 6000/µL (140 000-450 000). Type and screen is ordered to prepare 2 units of RBCs and 1 unit of apheresis platelets for transfusion. The patient’s blood type is O-positive, but routine antibody screening by a column agglutination test is strongly positive (4+) with allcells, includ ing the patient’s own cells. The direct antiglobulin test (DAT) is strongly positive (4+) with polyspecific reagent. Monospecific testing with anti-IgG and anti-C3d is not available at this labo ratory. With further testing, the patient sample is strongly pos itive (3+) with allpanel cells at saline IAT. The clinical team is notified of the results and the need to send out patient samples to the IRL for further testing, with an anticipated delay of at least 6 to 8 hours. Subsequent labs show reticulocytes, 24.4%; absolute reticulocyte count, 278 000/µL (10 000-120 000); lactate dehydrogenase, 692 U/L (82-240); total bilirubin, 1.5 mg/dL (0.2-1.0); haptoglobin less than 8 mg/dL (30200); and microspherocytes on peripheral smear, suggestive of a diagnosis of warm AIHA (WAIHA). Communication between the laboratory staff and clinical team reveals the patient is clinically stable, has a history of one missed abortion, and no prior trans fusions. The safety of releasing RBC units before completion of testing and the low risk of any reactions were discussed with providers. Pretransfusion testing in patients with WAIHA For any RBC trans fu sion, ini tial rou tine tests include an ABO group, an RhD type, and an antibody detection test (also known as an antibody screen). The primary purpose of antibody detec tion testing is to detect and identify an antibody that could poten tially react with trans fused RBCs and cause hemo ly sis. Antibody detection involves the IAT to identify whether clinically significant alloantibodies or autoantibodies are present. For 90% or more of pretransfusion samples, the results are negative and interpretation, straightforward.1 The presence of an antibody that is broadly reactive, dem onstrating similar agglutination with all reagent RBCs including the patient’s own RBCs, is classified as an autoantibody. A salient point in the interpretation of autoantibodies is their correlation with clinical and other laboratory findings. An independent assessment of the presence or absence of hemolytic anemia is essential. “Benign” autoantibodies are fairly common in sero logic workups but generally not associated with strong positive DAT or hemolysis.2 The case demonstrates one of the more perplexing problems for medical laboratory scientists (MLS) working in the hospital laboratory—distinguishing whether an underlying alloantibody is present in a sample containing a strong warm autoantibody (WAA). As seen in Table 3, allRBCs strongly agglutinate in the presence of a WAA at the IAT, easily masking the detection of an underlying alloantibody. The prevalence of RBC alloantibodies is higher in patients with WAIHA, ranging from 12% to 54% with a mean of 38% (Table 4). For this reason, further studies to eval uate for the presence of a clinically significant alloantibody are necessary for the selection of blood. Initially, a method that does not enhance antibody binding (known as the no-enhancement method or saline IAT) is under taken. This step may omit or weaken the reac tiv ity of a low-titer auto an ti body and allow detection of any underlying alloantibodies.15 Direct antiglobulin test The presence of an autoantibody in the plasma is the result of allantigen sites bound on the patient’s RBCs and should trigger the MLS to perform a DAT. The DAT is a critical diagnostic tool for AIHA and is initially performed by testing the patient’s washed RBCs with polyspecific antihuman globulin (AHG), which con tains both anti-IgG and anti-C3d. The use of a conventional test tube continues to be the pri mary method for the DAT, requiring manual observation for agglu tination of the patient RBCs after the addition of AHG. It is reliable and often used as confirmatory testing for other DAT techniques. In addition, to detect C3d, a test tube method using anti-C3d is optimal. With the expanding role of automation in hospital lab oratories, a DAT can be performed using column agglutination testing (known as gel or beads) or solid-phase methods. While these alternative methods for the DAT may be easier to perform and pro vide less sub jec tiv ity when interpreting the reac tion results (mainly due to end point reading by automation), in the setting of AIHA the authors have not observed the other methods to be more sensitive than a well-performed DAT by tube tech nique. Correlation with patient history is paramount to assess the significance of any positive DAT result regardless of method. When the DAT is pos i tive with polyspecific AHG, fur ther testing with monospecific reagents is required to differentiate whether IgG, C3d, or both are coating the patient RBCs and to Dr Prakash Singh Shekhawat Serologic evaluation of AIHA patients | 97 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 a results after 15-to-30 minute incubation at 37 °C. 2-4+, agglutination strength; RT, direct agglutination at room temperature. Table 2. Immunohematology methods used in evaluating patients with warm and cold autoantibodies Method Description/purpose Comments/pitfalls ABO group/RhD type Determine patient’s ABO group and RhD type • Presence of high titer CAA may cause autoagglutination at room temperature. Steps to resolve this may include: o Warm/wash patient RBCs at 37 °C o Perform testing at 37 °C o Treat RBCs with thiol reagent (DTT/2-ME) to remove IgM coating patient RBCs DAT • Polyspecific AHG Detects IgG and/or C3 (indirectly detects IgM) on patient’s RBCs • Positive in >90% of cases of AIHA • Screening assay; when positive must repeat testing with anti-IgG and anti-C3d to define protein coat ing patient RBCs DAT • Monospecific o Anti-IgG o Anti-C3d Individual reagents to detect whether IgG, C3d, or both on patient’s RBCs • Both reagents plus control must be performed • False positives can occur due to autoagglutination (most common with IgM) Antibody detection (screen) Use of 2-3 reagent RBCs of known phenotype to screen for auto and/or alloantibody in patient’s serum • Detects IgM CAA when performed at IS/RT (direct agglutination) using a test tube method • Detects IgG WAA at IAT with any method. • Detection depends on test method used (test tubes, CAT, SP) • May be negative if allautoantibody bound to patient’s RBCs Antibody identification Use of panel reagent RBCs (10-16) of known phenotype to determine specificity of antibody. • Reactivity at IS/RT (direct agglutination) confirms CAA. Some show autoanti-I or -i specificity. • Confirms broad reactivity or specificity of WAA at IAT. • Subsequent testing when antibody is detected to identify antibodies of clinical significance • Identifies specificity of any underlying alloantibody once autoantibody removed from patient serum Crossmatch Confirms ABO compatibility by either serologic (IS) or electronic/computer. • If known clinically significant alloantibody, must perform serologic crossmatch at IAT (also known as AHG or full crossmatch) Phenotype Serologically type patient RBCs using known antisera. • Performed when patient has not been transfused in last 3 months • Strong positive DAT may cause false positive; not allantisera able to give accurate results when DAT is positive • Partial antigens or variant antigens may not be detected Elution • Elution removes IgG bound to the patient’s RBCs using a dilute acid solution. Resulting eluate is then tested against a panel of reagent RBCs to check for specificity of the RBC bound IgG. o Positive with allcells confirms WAA. o If negative, suspect drug-dependent antibody. • Once diagnosis of AIHA, no need to perform with each subsequent pretransfusion test • Also used in suspected delayed transfusion reaction to detect binding of alloantibody to circulating transfused RBCs Adsorption—autologous • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying allo antibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Performed when patient has not been transfused in last 3 months • Patient RBCs are first treated to remove bound autoantibody to allow sites for autoantibody in plasma to bind • Requires sufficient quantity of patient RBCs for removal of autoantibody Adsorption—allogeneic • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using phenotyped donor RBCs; then adsorbed plasma tested to identify under lying alloantibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using donor RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Typically, 3 donor RBCs of known phenotype (eg, R1R1, R2R2, rr) are selected • May miss antibody to high prevalence antigen Routine: 98 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 Advanced: Table 2. Immunohematology methods used in evaluating patients with warm and cold autoantibodies (Continued) Method Description/purpose Comments/pitfalls Genotype • Molecular methods used to determine patient’s prob able phenotype. • More accurate than serologic phenotyping; generally, provides more comprehensive antigen profile • Recommended when patient has been transfused in last 3 months • Unusual, rare genetic variants or partial antigens may not be detected Prewarm technique • Wash patient RBCs with 37 °C saline prior to testing. • Warm patient plasma and reagent RBCs at 37 °C separately prior to mixing and performing testing to avoid CAA reactivity. • Avoids CAA from binding to patient or donor RBCs • Clinically significant, weakly reactive alloantibodies may not be detected in a prewarm IAT Thermal amplitude study • Incubate patient serum with reagent RBCs for 2 hours at 30 °C and 37 °C. • Determines if CAA has ability to bind to reagent RBCs at warm body temperature. • Should be performed with adult RBCs (positive for I) and cord blood RBCs (positive for i) • Typically, only need to perform once to aid in diagnosis Advanced: Table 3. Typical antibody detection (screening) results for autoantibodies and alloantibodies Cold autoantibody Warm autoantibody No antibody detected Alloantibody detected Reagent red cells RT 37C IAT RT 37C IAT RT 37C IAT RT 37C IAT 1 4+ 3+ 1+ 0 0 3+ 0 0 0 0 0 0 2 4+ 3+ 1+ 0 0 3+ 0 0 0 0 0 3+ 3 4+ 3+ 1+ 0 0 3+ 0 0 0 0 0 0 Autocontrol* 4+ 3+ 1+ 0 0 4+ 0 0 0 0 0 0 Results after mixing patient plasma with reagent RBCs. Agglutination is graded 1-4+; no agglutination is indicated as 0. *Autocontrol contains patient plasma plus patient RBCs. Italics indicate typical optimum reactivity of cold autoantibody. Bold indicates typical optimum reactivity of warm autoantibody. Italics and bold indicate typical reactivity of alloantibody. RT, room temperature (direct agglutination/immediate spin at room temperature); 37C, results after incubation at 37 °C for 10-30 minutes; IAT, results after addition of AHG. Table 4. Patients with WAIHA and underlying alloantibodies Number of alloantibodies detected Reference % samples with underlying alloantibodies Number of samples Morel et al3 (1978) 8 20 40 Branch and Petz4 (1982) 5 14 36 Wallhermfechtel et al5 (1984) 19 125 15 Laine and Beattie6 (1985) 41 109 38 James et al (1988) 13 41 38 Issitt et al8 (alloadsorptions) (1996) 13 34 38 Issitt et al8 (autoadsorptions) (1996) 5 41 12 Leger and Garratty9 (1999) 105 263 40 7 Maley et al (2005) 39 126 31 Das and Chaudhary11 (2009) 7 23 30 10 Yürek et al (2015) 7 36 19 Park et al13 (2015) 87 161a 54 Shirey et al (2002) 8 20 40 TOTALS 389 1013 38% 12 14 124 were warm autoantibodies, 21 cold autoantibodies with wide thermal range, and 16 were cold-reactive only. a Dr Prakash Singh Shekhawat Serologic evaluation of AIHA patients | 99 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 CAT, column agglutination technique; IS, immediate spin (direct agglutination at room temperature); 2-ME, 2-mercaptoethanol; RT, room tempera ture, direct testing; SP, solid phase technique. CLINICAL CASE 1 (Cont inu ed) Testing at the IRL confirms a strongly positive DAT, including 4+ reactivity with both anti-IgG and anti-C3d. Strongly reac tive autoantibody is present in the patient serum and eluate. The autoantibody remains strongly positive with allpanel cells despite no enhancement at the IAT, necessitating the perfor mance of specialized adsorption techniques. RH and K phenotyping show that the patient’s RBCs are E− and K− and C+, c+, and e+. RBC genotyping is ordered to determine the extended phenotype. Adsorption—allogeneic and autologous Adsorption studies are the optimal method to remove autoanti body from the patient’s plasma and leave alloantibody behind, if present. When a sample is referred, the first question asked by the IRL MLS is whether the patient has been transfused and when. If transfused within the last 3 months, allogeneic (using donor cells of known phenotypes) adsorption is performed over autologous (using the patient’s own RBCs) adsorption. Even a small amount of transfused RBCs in the patient’s sample can adsorb out alloantibodies.18 Another factor for the use of alloadsorption is the patient’s degree of anemia. For patients with an Hgb level less than 5g/dL or small pediatric patients, it is unlikely that an adequate quantity of patient RBCs can be collected to successfully perform repeated autoadsorptions. Autologous adsorp tions are logis ti cally eas ier to per form and preferred over alloadsorptions for 2 reasons. One, removing antibody using the patient’s own RBCs proves the autoimmune nature of the autoantibody. Second, autoadsorptions will not remove an alloantibody to a high prevalence antigen. Patient RBCs must first be chem i cally treated to remove autoantibody. Chemicals such as ZZAP (dithiothreitol (DTT)/2-mercaptoethanol + ficin/papain) or enzyme treating (ficin/papain) alone can remove autoantibody to free up anti 100 | Hematology 2022 | ASH Education Program gen sites for more autoantibody binding. Repeat adsorptions (1 to 4) based on the strength of the autoantibody present (1+-4+) in the patient’s serum are required, generally taking 3 to 4 hours. Allogeneic adsorptions are arguably one of the most compli cated immunohematology tests to perform and are confusing to interpret. They are labor-intensive, generally requiring 4 to 8 hours to complete. An adequate inventory of phenotyped blood donors, typically only available in IRLs, is required for the proper antigen makeup and volume of donor RBCs to perform the adsorptions. Generally, 3 different donor RBCs are selected, and the antigen makeup of each must be carefully considered to reveal any clin ically significant alloantibody since alldonor RBCs adsorb the autoantibody. If an alloantibody is present, as depicted in Figure 1, it will bind to donor RBCs that express the corresponding antigen but remain behind if donor RBCs lack the antigen. Typically, 2 or 3 rounds of adsorptions deplete the autoantibody. Regardless of which adsorption method is used, the adsorbed plasma must be retested to detect and identify any alloantibodies. If testing is negative, one can be relatively assured that no alloantibodies are present. In our case, 4 autoadsorptions and 4 alloadsorptions both failed to completely remove the WAA. Any further adsorptions would be impractical and likely increase the risk of missing weakly reactive alloantibodies. These rare situa tions illustrate the importance of RBC phenotyping/genotyping in patients with WAIHA. Patient RBC phenotyping and genotyping Knowing the patient’s RBC phe no type can be a pow er ful resource to predict which alloantibodies the patient can make and to guide the selection of blood. At a minimum, the patient should be typed for RH (C, E, c, e) and K. Extended typing for additional blood group antigens (Fya/Fyb, Jka/Jkb, M/N, and S/s) is possible today with the use of monoclonal reagents. However, when the patient has a strong positive DAT, determining the extended phenotype can be problematic due to autoagglutination. The patient’s RBCs can be chemically treated with EDTAglycine hydrochloric acid or chloroquine to remove IgG prior to testing. These pretreatment methods are often tedious, and the treated RBCs cannot be phenotyped for allblood group anti gens as EDTA-glycine hydrochloric acid denatures KEL blood group sys tem anti gens, and chlo ro quine treat ment reduces RH antigen expression.16 More importantly, phenotype by sero logic method should not be performed if the patient has been recently transfused. RBC genotyping (RCG) is becoming an increasingly valuable, cost-effective tool in transfusion practice. As a send-out test for most laboratories, results are unlikely to be available for 48 to 72 hours. RCG allows testing of more blood group systems and avoids the technical difficulties that may occur with serologic testing when the patient’s RBCs are coated with antibody. In addition, recent transfusion does not pose a problem, as DNA for molec u lar anal y sis is iso lated from the patient’s mono nu clear cells. Using the patient’s predicted phenotype from RCG, a strategy of prophylactic antigen-matched RBC units (RH [D, C, E, c, e], K, and where feasible JK, FY) may prevent alloimmunization, simplify subsequent pretransfusion testing, and possi bly reduce the frequency of adsorption studies.14,19 In a survey of academic hospital transfusion services and IRLs (n=54) on the Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 help classify the type of hemolytic anemia (Table 1). In patients with WAIHA, more com monly both IgG and C3d coat the patient’s cells.16 While the DAT alone is not diagnostic of hemo lytic anemia, stronger reactions (2-4+) denote higher antibody den sity on the RBC sur face and are more likely asso ci ated with hemolysis. Similarly, the presence and amount of C3 (C3b and/or C3d) on the RBC membrane is an important predictor of immune hemolysis.2,17 Conversely, a negative DAT does not rule out AIHA and may be seen in 5% to 10% of patients with hemolytic anemia.16,17 To confirm the positive DAT is due to an autoantibody, an elu tion pro ce dure should be performed as part of the ini tial investigation. The predominant method utilized for eluate prep aration removes cell-bound IgG autoantibody by the addition of a dilute acid solution to the patient’s RBCs. The acid/RBC mix is centrifuged, and the supernatant (eluate) is buffered back to a normal pH. Antibody identification is performed on this eluate. The results are key in differentiating WAIHA from drug-induced immune hemolytic anemia. In WAIHA the eluate contains the IgG autoantibody, whereas with a negative eluate, a drug-dependent antibody would be suspected. testing practices for patients with WAA, 68% performed genotyping and 75% provided prophylactic antigen-matched RBC units, although policies varied on indications for use.20 Transfusion support in patients with AIHA Generally, the deci sion to trans fuse AIHA patients should be based on sim i lar indi ca tions as for ane mic patients with out AIHA. In asymptomatic patients, a restrictive transfusion strat egy (ie, Hgb <7 g/dL) should be practiced.21 The use of bed rest and supplemental oxygen in patients with severe anemia (ie, Hgb <6 g/dL) could help defer transfusion until testing is com plete. The patient’s clin i cal sta tus, comorbidities, and symp toms should guide the decision to transfuse regardless of Hgb level and compatibility test results. When symptoms of signifi cant hypoxia, confusion, angina, or hemodynamic instability are present, RBC transfusion should not be withheld, even when testing is incomplete. The presence of reticulocytopenia in an AIHA patient with severe anemia should be regarded as a medi cal emergency. Reticulocytopenia is a poor prognostic indicator representing an inadequate erythropoietic response and higher transfusion requirements.16,22 When transfusion is medically necessary, the patient’s phy sician should be assured that transfused RBCS are unlikely to cause an acute hemolytic transfusion reaction. The risk for a transfusion reaction in patients with AIHA appears to be no higher than in other patients requiring transfusion. Chen et al.23 found that reactions occurred in 1.6% of 450 transfused AIHA patients, with febrile and allergic reactions the most frequent and no reports of hemolytic adverse events. Most patients with AIHA tolerate serologically incompatible RBC transfusions quite well, without a significant increase in their underlying hemolysis.12,13,23 Dr Prakash Singh Shekhawat Serologic evaluation of AIHA patients | 101 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 Figure 1. Adsorption procedure to remove WAA using allogeneic RBCs. Example of the multistep process for allogeneic adsorptions to remove WAA to identify whether an underlying alloantibody is present (eg, anti-Jka). Three donor RBCs of known phenotypes (ie, R1R1, R2R2, and rr) are selected and treated with enzyme or ZZAP. The patient’s serum (or plasma) is added to an aliquot of each of the 3 donor RBCs and incubated for 10 to 60 minutes at 37 °C. Three separate adsorptions are performed, each undergoing 1 to 4 rounds of adsorption in which the patient’s serum is repeatedly incubated with a new set of donor RBCs. Following an adequate number of adsorptions, adsorbed serum samples are tested against reagent RBCs. If 1 or more aliquots are positive, an alloantibody can be identified based on the pattern of reactivity and knowledge of the antigens expressed on each aliquot of donor RBCs used for adsorption. Selection of blood for transfusion Selection of the optimal RBC unit for transfusion depends on the urgency, capabilities and resources of the hospital transfusion service, and the status of the pretransfusion testing. When there is life-threatening anemia and no time for completion of testing, emergency-release, group O RBC units should be given (Figure 2). The avoidance of hemodynamic collapse and clinical deteriora tion from profound anemia far outweighs the risk of hemolytic reaction from an alloantibody. If the patient has no history of transfusions or pregnancy, the probability that underlying alloantibodies are present is quite low, and one can be reassured of the safety of the transfused RBCs. Extended phenotyping of the patient’s RBCs beyond ABO/Rh D can be done relatively quickly and guides the selection of pro phylactic antigen-matched blood for urgent transfusions (Figure 2). Even partial phenotyping, at a minimum RH (C, c, E, e) and K, and selecting the “best-matched” RBC unit from the available inventory may provide some measure of safety and reduce the risk of development of clinically significant alloantibodies.20,24 In patients with WAA and concomitant alloantibodies, at least 50% are directed against the RH and K antigens.25,26 102 | Hematology 2022 | ASH Education Program Providing prophylactic phenotype-matched RBCs beyond RH and K may be particularly advantageous for patients with ongo ing transfusion needs or previously identified alloantibodies.20,25 Delaney et al found that patients with WAA and preexisting alloantibodies had a higher incidence of new alloantibody for mation (51.8% vs 37.0%), suggesting that the use of prophylac tic antigen-matched RBC units in these patients could prevent additional alloimmunization.25 The degree of antigen-matching depends on the extent of phenotyping performed, when RCG results are available, the supply of appropriate phenotyped/genotyped donor RBCs, and the urgency of transfusion. The tech nique used for crossmatching blood for patients with WAA varies based on local policies.20 One critical point that cannot be overemphasized is that allblood is serologically incom pat i ble. Crossmatching RBC units to search for “least incom pat i ble” should be dis cour aged as there is no clin i cal benefit for the patient. Likewise, the use of this term does not properly convey the most important aspect of pretransfusion test ing—the exclu sion of alloantibodies and/or the selec tion of RBCs based on extended phenotype matching. Discussion with the clinical team should include the extent of compatibility Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 Figure 2. Initial selection of blood for patients with WAIHA. The algorithm is a suggested approach for the selection of RBC units in patients with WAIHA who are initially evaluated by the transfusion service. Serologic phenotyping of the patient’s RBCs and provision of prophylactic antigen-matched RBC units are dependent on the available antisera at the institution and the patient’s recent transfusion history. If it is discovered that the patient has a known history of alloantibodies, provide corresponding antigen-negative RBC units unless it would delay transfusion in urgent situations. testing performed, assurance of the safety of the transfusion, and the fact that the survival of transfused RBCs are comparable to the patient’s own RBCs.27 CLINICAL CASE 1 (Cont inu ed) Throughout a case, such as the one described, the need for frequent, effective dialogue between the clinical team and trans fu sion ser vice for shared deci sion-mak ing on any trans fu sion needs cannot be overemphasized. Information from the clinical team should focus on the patient’s clinical status, urgency for transfusion, and any history of previous transfusions, pregnan cies, or prior hospitalizations to assess the likelihood of underly ing alloantibodies. Clear communication to the provider about the extent of required testing, time for completion, and options for blood selection is imperative to enhance understanding of the risks and benefits when transfusion is necessary.28,29 CLINICAL CASE 2 A 73-year-old woman with a his tory of hyper ten sion and cerebrovascular disease presented for further evaluation of chronic anemia, with a current Hgb level of 8.0 g/dL and nor mal iron studies. She denied any significant chest pain, short ness of breath, or headache and had no episodes of bleeding or dark stools. Vital signs were blood pressure, 130/70 mmHg; heart rate, 71/min; temperature, 36.1 °C; oxygen saturation, 100% on room air. Further laboratory studies showed the fol lowing: white blood cell count, 6300/µL (4100-10 000); plate let count, 347 000/µL (150 000-450 000); reticulocytes, 6.6%; abso lute retic u lo cyte count, 156 000/µL (22 000-84 000); haptoglobin lower than 3 mg/dL (50-220); lactate dehydro genase, 270 U/L (88-207); total bilirubin, 5.4 mg/dL (0-1.0); and indi rect bil i ru bin, 4.6 mg/dL. Routine anti body detec tion by solid phase was negative. Blood grouping showed an ABO discrepancy with extra positive reactions in the patient’s reverse grouping. The DAT showed strong autoagglutination in alltubes including the saline control. After multiple washes of the patient’s RBCs with 37 °C saline, the DAT with anti-IgG was neg a tive and anti-C3d was 3+. CAA was iden ti fied at room temperature (RT) and below. Dr Prakash Singh Shekhawat Serologic evaluation of AIHA patients | 103 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 As the patient was stable and did not have rapidly dropping Hgb, her transfer to a tertiary hospital was arranged. She was started on intravenous Ig and methylprednisolone. Repeat labs 20 hours after her initial presentation showed an Hgb level of 3.6 g/dL. Assessment by the team noted that the patient started her men ses and was complaining of mild head ache and dyspnea. The adsorption studies by the IRL were not yet complete. In collaboration with the clinical team, a decision was made by the transfusion team physician to release par tially antigen-matched RBC units (E− and K−) for transfusion. The patient received 4 RBC units over 3 days. RCG results were available at the time of the fourth transfusion and indicated no additional antigen matching was required. Cold AIHA (CAIHA), although serologically presenting with unexpected test results, is not as difficult or time-consuming to work up in the transfusion service or IRL. Automated methods for pretransfusion testing are designed to detect IgG antibodies, and with their growing use in hospital transfusion services, CAA may not be initially detected in the antibody screen. As noted in the case, potent CAAs often cause ABO and Rh typing discrepancies. Patient RBCs that are heavily coated with IgM CAA autoagglutinate at RT, causing unexpected positive results in forward ABO/RhD typing. Washing patient RBCs with warm 37 °C saline prior to testing usually removes the autoagglutination. Rarely, if warming is unsuccessful, RBCs must be chemically treated with DTT, a thiol reagent, to remove IgM auto an ti body. DTT treat ment breaks disulfide bonds of IgM antibodies and therefore dissipates autoagglutination in the ABO forward group. The bigger challenge with CAA is unex pected positive results in the reverse ABO group when the patient’s plasma is mixed with A1 and B reagent RBCs. To elimi nate this interference, the patient’s plasma is warmed to 37 °C before adding to prewarmed A1 and B cells. In rare cases, autol ogous or allogeneic adsorptions are required to remove CAA to obtain a valid reverse group. Classically, in CAIHA the DAT is positive with only C3d, which infers IgM antibody binding. Similar to WAA, the evaluation of other laboratory studies for hemolysis is vital to determine the sig nifi cance of the CAA. Autoagglutination can occur with a strong CAA and lead to false-positive results when performing a DAT, emphasizing the importance of simultaneous testing of controls. Resolution and removal of IgM coating the patient’s cells follow similar steps as for ABO group discrepancy, with first warm washing of the cells and, if needed, DTT treatment. CAAs rarely cause a challenge in ruling out underlying alloantibodies because IgM autoantibody binds to RBCs preferentially at colder temperatures but can carry over to 37 °C and the IAT, while clinically important IgG alloantibodies are reactive only at the IAT (Table 3). Eliminating the use of potentiators in traditional test tube methods and performing testing only at 37 °C (to avoid autoantibody binding at RT) is often sufficient to reduce reactiv ity by the CAA at the IAT. Negative reactions at the IAT indicate that no IgG alloantibodies are present. Rarely, 1 or 2 autologous or allogeneic adsorptions with treated RBCs incubated at 4 °C remove enough CAA to avoid interference in the IAT and allow alloantibodies to be ruled out. Once the under ly ing alloantibodies have been excluded, the selection of RBCs for transfusion typically follows standard practice. The provision of prophylactic RH and K matching is generally recommended if the patient has an ongoing need for transfusion. If ABO grouping results cannot be resolved due to a potent CAA, group O RBCs should be selected. In patients with CAA with severe brisk hemolytic anemia, the use of an in-line blood warmer as well as warming blankets may mitigate any fur ther hemolysis.16 As part of the diagnostic evaluation, a cold agglutinin titer may be ordered; however, the titer result is not reflective of the ability of the CAA to cause immune hemolytic anemia as the test ing is performed at 4°C. In the authors’ opinion, it is an overused test that provides little clinical relevance. A better test to determine the pathological nature of a CAA is a thermal ampli tude study. This testing assesses the ability of the CAA to bind to RBCs at 30 °C or warmer, reflecting body temperature. Some may argue that neither is necessary if standard serologic testing and clinical signs and symptoms support a pathological CAA. CLINICAL CASE 2 (Cont inu ed) Our patient was asymptomatic and did not require transfusion at this time. Neither a cold agglutinin titer nor a thermal ampli tude study was performed as the findings from her serologic test ing, includ ing a DAT, and other lab o ra tory stud ies were consistent with CAIHA. Both WAIHA and CAIHA present unique challenges for pretransfusion testing. The provision of RBCs for transfusion is based on the clinical status of the patient and the extent of testing affected by the class (IgG or IgM) and concentration of autoanti body. Testing for patients with acute WAIHA is the most difficult, requiring complicated, lengthy procedures such as adsorption studies and genotyping that may not be completed prior to needing transfusion. Thus, early and frequent communication is essential for the exchange of information to effectively guide the best approach for immediate and future transfusions. This extra effort provides assurance for the clinical and laboratory teams that units released for transfusion, although incompatible, have a high likelihood of being safe and efficacious for the patient. Conflict-of-interest disclosure Susan T. Johnson: book royalties: AABB Press. Kathleen E. Puca: no competing financial interests to declare. Off-label drug use Susan T. Johnson: nothing to disclose. Kathleen E. Puca: nothing to disclose. Correspondence Susan T. Johnson, Clinical Education and Diagnostic Laboratories Immunohematology Reference Laboratory, Versiti Blood Center of Wisconsin, 638N 18th St, Milwaukee WI 53233; e-mail: stjohnson@ versiti.org. References 1. 2. 3. 4. 5. 6. 7. Hendrickson JE, Tormey CA. Understanding red blood cell alloimmunization triggers. Hematology Am Soc Hematol Educ Program. 2016;2016(1):446451. Wheeler CA, Calhoun L, Blackall DP. Warm reactive autoantibodies: clinical and serologic correlations. Am J Clin Pathol. 2004;122(5):680-685. Morel PA, Bergren MO, Frank BA. A simple method for the detection of allo antibody in the presence of warm autoantibody [abstract]. Transfusion. 1978;18(3):388. Branch DR, Petz LD. A new reagent (ZZAP) having multiple applications in immunohematology. Am J Clin Pathol. 1982;78(2):161-167. Wallhermfechtel MA, Pohl BA, Chaplin H. Alloimmunization in patients with warm autoantibodies. A retrospective study employing three donor alloabsorptions to aid in antibody detection. Transfusion. 1984;24(6):482-485. Laine ML, Beattie KM. Frequency of alloantibodies accompanying autoantibodies. Transfusion. 1985;25(6):545-546. James P, Rowe GP, Tozzo GG. Elucidation of alloantibodies in autoimmune haemolytic anaemia. Vox Sang. 1988;54(3):167-171. 104 | Hematology 2022 | ASH Education Program © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000406 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/96/2021987/96johnson.pdf by guest on 09 December 2022 Conclusion 8. Issitt PD, Combs MR, Bumgarner DJ, Allen J, Kirkland A, Melroy-Carawan H. Studies of antibodies in the sera of patients who have made red cell autoantibodies. Transfusion. 1996;36(6):481-486. 9. Leger RM, Garratty G. Evaluation of methods for detecting alloantibodies underlying warm autoantibodies. Transfusion. 1999;39(1):11-16. 10. Maley M, Bruce DG, Babb RG, Wells AW, Williams M. The incidence of red cell alloantibodies underlying panreactive warm autoantibodies. Immunohematology. 2005;21(3):122-125. 11. Das SS, Chaudhary R. Utility of adsorp tion tech niques in sero log i cal evaluation of warm autoimmune haemolytic anaemia. Blood Transfus. 2009;7(4):300-304. 12. Yürek S, Mayer B, Almahallawi M, Pruss A, Salama A. Precautions surrounding blood transfusion in autoimmune haemolytic anaemias are overestimated. Blood Transfus. 2015;13(4):616-621. 13. Park SH, Choe WH, Kwon SW. Red blood cell transfusion in patients with autoantibodies: is it effective and safe without increasing hemolysis risk? Ann Lab Med. 2015;35(4):436-444. 14. Shirey RS, Boyd JS, Parwani AV, Tanz WS, Ness PM, King KE. Prophylactic antigen-matched donor blood for patients with warm autoantibodies: an algorithm for transfusion management. Transfusion. 2002;42(11):14351441. 15. Cohn CS, Delaney M, Johnson ST, Katz LM. AABB Technical Manual. 20th ed. Association for the Advancement of Blood and Biotherapies; 2020:408409, 434-444. 16. Petz LD, Garratty G. Immune Hemolytic Anemias. 2nd ed. Churchill Livingstone; 2004:69-77,171,215-216,319,395. 17. Meulenbroek EM, Wouters D, Zeerleder SS. Lyse or not to lyse: clinical sig nificance of red blood cell autoantibodies. Blood Rev. 2015;29(6):369-376. 18. Laine EP, Leger RM, Arndt PA, Calhoun L, Garratty G, Petz LD. In vitro stud ies of the impact of transfusion on the detection of alloantibodies after autoadsorption. Transfusion. 2000;40(11):1384-1387. 19. El Kenz H, Efira A, Le PQ , et al. Transfusion support of autoimmune hemo lytic anemia: how could the blood group genotyping help? Transl Res. 2014;163(1):36-42. 20. Ziman A, Cohn C, Carey PM, et al; Biomedical Excellence for Safer Transfusion Collaborative. Warm-reactive (immunoglobulin G) autoantibodies and laboratory testing best practices: review of the literature and survey of current practice. Transfusion. 2017;57(2):463-477. 21. Carson JL, Guyatt G, Heddle NM, et al. Clinical practice guidelines from the AABB: red blood cell trans fu sion thresh olds and stor age. JAMA. 2016;316(19):2025-2035. 22. Barcellini W, Zaninoni A, Giannotta JA, Fattio B. New insights in auto immune hemolytic anemia: from pathogenesis to therapy. J Clin Med. 2020;9(12):3859. 23. Chen C, Wang L, Han B, Qin L, Ying B. Autoimmune hemolytic anemia in hospitalized patients: 450 patients and their red blood cell transfusions. Medicine. 2020;99(2):e18739. 24. Hill QA, Stamps R, Massey E, et al; British Society for Haematology. The diagnosis and management of primary autoimmune haemolytic anaemia. Br J Haematol. 2017;176(3):395-411. 25. Delaney M, Apelseth TO, Bonet Bub C, et al; Biomedical Excellence for Safer Transfusion Collaborative. Red-blood-cell alloimmunization and pro phylactic antigen matching for transfusion in patients with warm autoantibodies. Vox Sang. 2020;115(6):515-524. 26. Garratty G, Petz LD. Approaches to selecting blood for trans fu sion to patients with autoimmune hemolytic anemia. Transfusion. 2002;42(11):13901392. 27. Petz LD. “Least incompatible” units for transfusion in autoimmune hemo lytic ane mia: should we elim i nate this mean ing less term? A com men tary for clinicians and transfusion medicine professionals. Transfusion. 2003;43(11):1503-1507. 28. Petz LD. A physician’s guide to transfusion in autoimmune haemolytic anae mia. Br J Haematol. 2004;124(6):712-716. 29. Ness PM. How do I encourage clinicians to transfuse mismatched blood to patients with autoimmune hemolytic anemia in urgent situations? Transfusion. 2006;46(11):1859-1862. AUTOIMMUNE HEMOLYTIC ANEMIAS Warm autoimmune hemolytic anemia and the best treatment strategies Hematology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA Warm autoimmune hemolytic anemia (wAIHA) is characterized by evidence of red blood cell (RBC) hemolysis and a direct antiglobulin test positive for IgG and sometimes complement. While varying with the extent of the compensatory increase in RBC production, symptoms of anemia predominate, as does jaundice, the latter often exacerbated by concurrent Gilbert’s syndrome. Initial treatment with corticosteroids is highly effective, with over 85% of patients responding but with less than one-third maintaining that response upon weaning. Subsequent rituximab administration in those failing corticosteroids provides complete remission in over 75% of patients and may be long-lasting. Over 50% of patients failing rituximab respond to erythropoiesis-stimulating agents or immunosuppressive agents. Splenectomy is best deferred if possible but does offer long-term remission in over two-thirds of patients. A number of new treatments for wAIHA (fostamatinib, rilzabrutinib, and FcRn inhibitors) show promise. A treatment algorithm for wAIHA is proposed to avoid the excessive use of corticosteroids. LEARNING OBJECTIVES • Recognize the clinical and laboratory features that determine the diagnosis and etiology of wAIHA • Create a pathophysiologybased framework for the treatment of wAIHA with current and future novel therapies CLINICAL CASE A 69yearold woman who was a Jehovah’s Witness pre sented with profound fatigue. Her previous complete blood cell count (CBC) had been normal. Laboratory tests showed the following values: hemoglobin (Hgb), 6 g/dL; reticulocytes, 15%; lactate dehydrogenase (LDH), 560 IU/L; total bilirubin, 2.3 mg/dL; and haptoglobin, less than 10 mg/dL. The smear showed spherocytes, and the direct antiglobulin test (DAT) was strongly positive for immuno globulin G (IgG) and weak for C3. She was treated with folate, 1 mg/d, and prednisone, 60 mg/d, for 3 weeks. Her LDH and bilirubin decreased nearly to normal, and her Hgb level rose to 13 g/dL, so prednisone was weaned over the next 8 weeks to 5 mg/d. Her Hgb level remained stable at 10 g/dL for 2 months on 5 mg of prednisone every other day but then dropped to 7 g/dL. Prednisone was increased to 20 mg/d, and 375 mg/m2 rituximab was administered weekly for 4 weeks, with a return of the Hgb to 12 g/dL. Steroids were again weaned to 5 mg/d, but 3 months later her Hgb level again dropped below 9 g/dL. Mycophenolate (MMF) at 1000 mg twice a day was started and the prednisone was weaned off, with Hgb lev els remaining stable at 10 g/dL for 12 months. The patient developed increasing nausea, and the MMF was discon tinued, with the Hgb level falling slowly to 7.7 g/dL 10 weeks later. The patient refused splenectomy, resumed prednisone at 5 mg/d, and was started on 200 mcg of darbepoetin alpha every 3 weeks, with Hgb rising to 10.5 (the prednisone having been tapered off). After 9 months, her insurance disallowed the darbepoetin alpha, and the patient again resumed prednisone at 5 mg/d. The Hgb level rose to 9.4 g/dL. She entered a clinical trial with fos tamatinib and has been maintained on 150 mg twice a day for 2 years with an Hgb level of 12 g/dL off prednisone. Introduction The normal red blood cell (RBC) survives in the circulation for approximately 120 days (Figure 1A). Hemolytic anemia is considered when RBC survival drops below 100 days.1 In evaluating a patient for hemolytic anemia, it is important to first provide evidence for hemolysis. In patients with hemolysis, a number of laboratory tests become abnormal Warm autoimmune hemolytic anemia: diagnosis and treatment | 105 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 David J. Kuter (Figure 1B). The reticulocyte count and reticulocyte index usually increase as a reflection of the increased egress of young RBCs from the bone marrow as they “shift” into the circulation. The destruction of RBCs results in the release of their LDH, and some times Hgb, into the circulation. Free Hgb may be detectable in the urine and serum and can bind to and reduce levels of hap toglobin; once filtered by the kidney, it may be catabolized by renal tubular cells, which retain iron and produce a positive urine hemosiderin test. The catabolism of RBCs increases bilirubin; the formation of bilirubin and the concurrent production of carbon monoxide in this process are perhaps the most accurate indica tors of RBC turnover.2,3 The above attributes of hemolysis have many exceptions and nuances. As discussed below, the reticulocyte count and index may be low in many patients with autoimmune hemolytic ane mia (AIHA) due to antibodies binding the reticulocyte or nutri tional or bone marrow deficiencies. Given that Gilbert’s disorder affects 5% to 10% of the Caucasian population,2 even mild hemo lysis may result in strikingly elevated bilirubin and symptomatic jaundice. In general, the presence of urine hemosiderin, urine, or serum free Hgb reflects extra vas cu lar hemo ly sis, whereas increased LDH, reduced haptoglobin, and elevated bilirubin may be seen in both intravascular and extravascular hemolysis. 106 | Hematology 2022 | ASH Education Program The documentation of RBC hemolysis is critical for the diagno sis of hemolytic anemia. In a small study of 100 anemic patients, a haptoglobin lower than 25mg/dL was 83% specific and 96% sensitive and had a predictive value of 88% for hemolysis.4 When the haptoglobin level is less than 25mg/dL and the LDH level is greater than the upper limit of normal, sensitivity drops to 50%, but specificity and predictive value are 100%.5 Hemolytic anemia is imprecisely defined as an anemia with shortened RBC survival, an LDH level greater than the upper limit of normal, and a hapto globin level less than the lower limit of normal.1 Many regulatory mechanisms may affect the physiological com pen sa tion for increased RBC destruc tion. These include eryth ro poi e tin and nutri ents such as folic acid, B12, and iron, as well as the general health of the bone marrow. Normal RBC pro duc tion can increase 5-fold to 8-fold and pro vide sig nifi cant compensation for reduced RBC survival. Indeed, for some patients with any type of mild hemolytic anemia, the provision of adequate nutritional resources and erythropoietin may allow adequate compensation with no need for other treatment. There are many causes of hemolytic anemia. As described in Table 1, they include intrinsic defects of the RBC membrane, enzymes, or Hgb. Many disorders extrinsic to the RBC may also cause shortened RBC survival. Nonimmune mechanisms such as Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 Figure 1. RBC production and catabolism in states of normal (A) or accelerated (B) RBC turnover. The RBC half-life of 5 to 10 days is simply illustrative. The changes denoted in RBC production and markers of catabolism vary with the extent of RBC turnover. against mul ti ple RBC types. The con clu sions from the direct antiglobulin test (DAT) and eluate (using the DAT) are as follows: Table 1. Types of hemolytic anemia Intrinsic Membrane/cytoskeletal abnormalities— hereditary spherocytosis Enzyme defects—G6PD, pyruvate kinase deficiency Hgb defects—thalassemia, sickle cell disease Conclusions C3 only Immune complex, cold agglu tinin disease, cold agglutinin syndrome Increased shear—artificial valves DIC, TTP, HUS Infection—malaria, babesiosis Oxidative stress—dapsone, rasburicase Liver failure—spur cell anemia Hypersplenism IgG, negative eluate Drug, absorbed IgG IgG, eluate specific Alloimmune IgG, eluate binds allRBCs wAIHA, drug IgG, +/− C3, eluate binds allRBCs wAIHA AIHA Cold antibody—cold AIHA: cold agglutinin disease, cold agglutinin syndromea Warm antibody—wAIHAb Mixed In summary, AIHA is characterized by evidence of hemolysis and a positive Coombs test. Eighty percent of patients have an IgG (warm antibody) that binds to protein antigens on RBC sur faces at the core body temperature. Twenty percent of patients have an IgM (cold antibody) that binds to polysaccharide antigens on RBC surfaces at temperatures below the core body tempera ture. A small proportion of patients may have both (mixed AIHA). The rest of this review focuses on warm AIHA (wAIHA). Extrinsic Nonimmune Immune Intravascular hemolysis. Extravascular hemolysis. DIC, disseminated intravascular coagulation; G6PD, glucose-6-phos phate dehydrogenase deficiency; HUS, hemolytic uremic syndrome; TTP, thrombotic thrombocytopenic purpura. a b Pathophysiology of wAIHA disseminated intravascular coagulation, infection, and oxidative stress are common. The immune mechanisms causing AIHA are much less common but are characterized by the demonstration of specific antibodies and/or complement binding to RBCs. Critical to the diagnosis of AIHA is the identification of anti bodies against RBC antigens. This is routinely done using many variations of the test originally designed by Robin Coombs.6,7 RBCs are initially assessed for the presence of both immuno globulin and C3 using polyspecific antisera.8 If positive, further assessments with antibodies specific for IgG and C3 are per formed. A com plete assess ment of RBCs test ing pos i tive for IgG includes determining whether eluate autoantibodies react More than 95% of patients with wAIHA have an IgG antibody that binds to RBC antigens independently of temperature. Rarely, antibodies may be IgA and even less com monly, IgM; these may account for the approximately 5% of patients who have “Coombs-negative” wAIHA. These antibodies are polyclonal even in clonal disorders such as chronic lymphocytic leukemia (CLL).9 About one-third fix complement; IgG1 and IgG3 fix com plement more efficiently than the others. The antibodies bind two major RBC anti gens (Figure 2). About 40 000 Rh proteins are fixed to the cytoskeleton in each RBC and do not have a high enough density to activate comple ment. In contrast, there are about 800 000 glycophorin proteins Figure 2. RBC surface antigen targets in wAIHA. Warm autoimmune hemolytic anemia: diagnosis and treatment | 107 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 DAT and eluate results with mild hemolysis, in patients who have concurrent Gilbert’s syndrome.2 Some wAIHA patients sim ply have a mild ane mia and increased mean corpuscular volume and are referred to hematol ogy for assessment for possible myelodysplastic syndrome. The increased mean corpuscular volume is related to the increased numbers of reticulocytes (which are generally larger than more mature RBCs) and is not due to a primary bone marrow disorder. Incidence of wAIHA Figure 3. Peripheral blood smear in patient with wAIHA failing splenectomy. Increased numbers of spherocytes (red arrows) and polychromatophilic reticulocytes (black arrows), as well as one RBC with a Howell-Jolly body (green arrow), are indicated. A, B, C, and D fixed to the membrane of each RBC, a high enough density to activate complement. Once bound to an antibody, the RBC can undergo multiple fates. Most will be bound to splenic macrophages via the FcγRIII receptor, resulting in either phagocytosis of the entire RBC or, more likely, removal of a significant portion of the RBC mem brane-pro duc ing spherocytes (also called microspherocytes) (Figure 3). Unlike the normal RBC, spherocytes are not deform able and, upon entering the splenic Cords of Billroth, undergo destruction. This may account for some of the increased LDH and depressed haptoglobin seen in a disorder commonly described as “extravascular.” About one-third of patients with wAIHA addi tionally have complement bound to RBCs, and these C3b-coated RBCs are then cleared by the C3b receptors on hepatic Kupffer cells. In some of these RBCs, C3b is inactivated to C3d, thereby preventing their subsequent destruction and allowing a popula tion of antibody/complement-coated RBCs to persist. In summary, wAIHA is defined by the following: (1) evidence of hemolysis; (2) an IgG antibody binding to protein antigens on the RBC surface at the core temperature (warm antibody), dem onstrated by a positive Coombs test for IgG in 95% of patients; (3) spherocytes on the peripheral blood smear. RBCs from 30% of patients bind both IgG and C3, and less than 5% are Coombs negative.1,10 Presentation of wAIHA The presenting characteristics are variable. The typical symp toms of ane mia are often pres ent, includ ing short ness of breath with exercise, generalized fatigue, dizziness, syncope, malaise, chest pressure/pain, and cognitive dysfunction. Most patients have a reduced health-related quality of life.11 Some patients with mild anemia and significant compensation have no symptoms. Symptoms spe cific to hemo ly sis include jaun dice, spleno megaly, and gallstones. Significant icterus may be present, even 108 | Hematology 2022 | ASH Education Program Causes of wAIHA Table 2 lists the many causes for wAIHA.14,16 Infections and a wide variety of drugs/toxins are commonly associated with wAIHA, but these hemolytic events are mostly transient, usually iden ti fied upon ini tial eval u a tion, and resolve with treat ment of the underlying infection or discontinuation of the associated drug/toxin. What is of concern then in this discussion are those patients in whom wAIHA is chronic and not obviously associ ated with transient infection or drug/toxin exposure. As listed in Table 2, some patients nonetheless have some infection or some drug/toxin expo sure not ini tially iden ti fied. More com monly, about half of patients have no apparent underlying cause. About a quarter have an identifiable lymphoproliferative disease (LPD); as high as 11% of patients with CLL develop wAIHA, with this number increasing after exposure to fludarabine.17 Another quarter of all patients have an identifiable immune/autoimmune disorder, such as common variable immune deficiency, systemic lupus erythematosus, Sjogren’s syndrome, or autoimmune hep atitis. Although 29% of immune thrombocytopenia (ITP) patients Table 2. Causes of chronic wAIHA Idiopathic: ~45% Malignancies: LPDs: ~24% 11% of CLL patients Even higher after fludarabine treatment Immune disorders: ~25% Immune deficiency disorders (common variable immune deficiency) Autoimmune disorders (systemic lupus, ulcerative colitis) ~10% of patients with systemic lupus ITP patients (Evans syndrome) Others: ~5% Viral infection (usually kids) Drugs (huge number) Data reproduced with permission from Hansen et al12 and Tranekær, Hansen, and Frederiksen.14 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 As with many rare diseases, estimates of the epidemiol ogy of wAIHA in adults are inexact. The incidence in adults ranges from 1.77 per 100 000 in Denmark to 2.4 per 100 000 in France, with a prevalence in Denmark of 17 per 100 000.12,13 The median age of presentation in adults is 53 years and 60% are female.14 The disease incidence increases with age, with 0.35 per 100 000 patient-years in those more than 20 years old; 0.51 per 100 000 patient-years in those 20 to 50 years old, and 3.34 per 100 000 patient-years in patients over 50.15 have a pos i tive DAT, few have active hemo ly sis.18 Evans syn drome has an incidence of 1.8 per 1 000 000 patient-years, with the wAIHA and ITP often not concurrent.19 Evaluating the patient for wAIHA History Treatment of wAIHA Who should be treated? Signs of sig nifi cant ane mia such as pal lor, tachy car dia, heart failure, pulmonary edema, and peripheral edema may be pres ent. Asymmetric lower-extremity edema should raise concern for thrombosis. Icterus, particularly of the sclera, is commonly found. While mild splenomegaly is common, the spleen is usually not enlarged enough to be appreciated by palpation. The pres ence of significant splenomegaly on palpation associated with adenopathy should raise the concern of an LPD. Some patients with minimal anemia are sufficiently compen sated for their hemolysis and may not require any therapy. Such asymptomatic patients usually have Hgb level higher than or equal to 10g/dL and might experience a reduced quality of life with therapy. These patients are followed closely, supplemented with folate, and advised to contact their hematologist should they experience signs of worsening hemolysis (eg, darker urine, more icterus, worsening fatigue), especially when stressed by infection or other medical problems. Generally, patients who need treatment are symptomatic, have Hgb levels lower than 10g/dL, or require RBC transfusion. In patients with wAIHA secondary to symptomatic LPDs or immune/autoimmune disorders, therapy directed at the under lying disorder may mitigate the hemolysis. Laboratory examination What are the goals of therapy for wAIHA? Physical examination Table 3 lists the laboratory assessments of patients with wAIHA. A CBC and a reticulocyte count and tests to measure LDH, bilirubin, and haptoglobin identify those with hemolytic anemia. The DAT and eluate assessment confirm whether this is a wAIHA. A review of the peripheral blood smear is mandatory to affirm the pres ence of spherocytes and assess for LPDs. Subsequent tests for underlying causes include antinuclear antibody and rheumatoid factor to assess for autoimmune causation and a serum protein electrophoresis to assess for chronic variable immune defi ciency, as well as flow cytometry and often a chest/abdomen/ pel vic com puted tomo graphic scan looking for LPD. Only in the presence of features (lymphadenopathy, other cytopenias, other abnormalities on the peripheral blood smear) suggestive of other hematologic disorders or in cases of Coombs-negative While desirable, normalization of Hgb is rarely required. Suc cessful therapy strives for a stable Hgb level over 10g/dL, res olution of symptoms, and transfusion independence. Treatment should minimize the adverse effects of immunosuppression. In particular, it is important to avoid the side effects of corticoste roids, with chronic prednisone dosage never exceeding 5mg/d and avoided entirely if possible. Although splenectomy may be a definitive treatment for many, most patients prefer to avoid it; joint decision-making in wAIHA is mandatory. For clin i cal tri als, the First International Consensus Meet ing defined a “response” as an increase in Hgb by more than 2g/dL or normalization of Hgb without a biochemical resolution of hemolysis, along with an absence of transfusion for the last 7 days.1 A complete response (CR) was defined as normalization Table 3. Recommended laboratory tests for chronic wAIHA CBC with differential Folate level Reticulocyte count Erythropoietin levela Review blood smear Serum protein electrophoresis Haptoglobin ANA, rheumatoid factor Bilirubin (direct/total) Flow cytometry for clonal B cellsa LDH level Chest/abdomen/pelvic CT scan Coombs test and eluate assessment (Bone marrow examination)b Fe/total iron-binding capacity/ferritin Prothrombin fragment 1.2a B12 level D-dimer If testing available. b In certain situations (see text). ANA, antinuclear antibody; CT, computed tomographic. a Warm autoimmune hemolytic anemia: diagnosis and treatment | 109 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 The initial evaluation of the patient should include a review of the signs and symptoms of anemia, the prior bouts of such, and the presence of jaundice or dark-colored urine. A personal or family history of other autoimmune conditions is not uncommon. Symp toms of LPD such as adenopathy, night sweats, and splenomegaly should be sought. At this point it is also of paramount importance to assess for recent infections (eg, babesiosis) and exposure to medications or toxins. Evidence of blood loss ought to be consid ered. Assess for symptoms of venous thromboembolism. wAIHA is bone mar row exam i na tion indi cated. In those with Coombs-negative AIHA, a bone marrow examination helps doc ument accelerated erythropoiesis and may exclude other pri mary bone marrow disorders. Measurement of levels of nutrients and erythropoietin assess for adequacy of bone marrow com pensation. Although their role in predicting thrombotic risk has not been demonstrated, this reviewer routinely checks D-dimer and a prothrombin fragment. of Hgb, no evidence of hemolysis (normal bilirubin, LDH, hapto globin, and reticulocytes), and the absence of transfusions. What is the initial therapy for wAIHA? Corticosteroids are the ini tial ther apy for almost allpatients (Table 4). In patients with significant symptomatic anemia, RBC transfusion needs to be considered. All patients should be sup plemented with folic acid of at least 0.4mg/d and repleted with B12 and iron if so deficient. Corticosteroids Prednisone at 1mg/kg/d for 2 to 4 weeks until the Hgb level is over 10 is a common starting dose.20,21 Although dexametha sone is sometimes used, this reviewer finds its effects to be tran sient and cannot recommend its use. Over 85% of patients have a rise in Hgb.22,23 Slow steroid tapering over 5 to 12 weeks to the lowest effective dose is then undertaken, with close monitoring (every 1-2 weeks) of Hgb, reticulocyte count, LDH, and hapto globin. Pneumocystis carinii pneumonia prophylaxis should be considered in patients who are maintained at prednisone doses over 20mg/d for over 1 month. Unfortunately, with steroid taper Table 4. Treatments for chronic wAIHA RBC transfusion Adequate nutritional support Folate, B12, iron Reduce antibody production Corticosteroids 1mg/kg/d Rituximab MMF Azathioprine Cyclophosphamide Cyclosporine Danazol Alemtuzumab Daratumumab Reduce macrophage function Corticosteroids Splenectomy Intravenous immunoglobulin Vincristine Increase RBC production ESAs 110 | Hematology 2022 | ASH Education Program Subsequent treatments for wAIHA Although most patients have a significant response to cortico steroids, with tapering most patients lose their response and require additional therapy. The many current therapy options are listed in Table 4, and the most common are discussed sep arately next. Rituximab Rituximab is highly effective in patients resistant to or depen dent upon corticosteroids. Standard doses of 375mg/m2 weekly for 4 weeks or 2 doses of 1000mg 2 weeks apart have been stud ied. When added to corticosteroids, the response rate is much higher than with corticosteroids alone. In one study, response rates of 75% to 80% at 12 months with mostly CRs (defined as a normal Hgb level on no treatment) were reported; the CR rate with corticosteroids was only 36%.20 The response duration was more than 36 months in 70% of patients. In a second smaller phase 3 study, overall responses at 1 year were 31% (95% CI, 11.058.7; 5 CR) for prednisone alone versus 75% (95% CI, 47.6-92.7; 11 CR, 1 partial remission [PR]) for prednisone plus rituximab.21 After 2 years, 10 in 16 rituximab patients but only 3 in 16 placebo patients maintained a CR (P = 0.011).21 Eighteen wAIHA patients in a third study were treated with a brief course of corticoste roids plus 100mg of rituximab weekly for 4 weeks. All patients responded by 6 and 12 months with approximately 70% CR24; relapse-free survival at 36 months was 76%.25 Splenectomy Although often declined by patients, splenectomy is an effec tive treatment for wAIHA.26,27 Laparoscopic approaches are pre ferred. Presplenectomy vaccination is recommended if there is adequate time. Deep vein thrombosis (DVT) prophylaxis for up to a month following treatment should be considered. CR rates of 75% have been reported in one study and of 81% at 3 years in another. 26,27 Long-term complications of infection and throm bosis need to be considered. Shared decision-making is encour aged. Intravenous immunoglobulin As with most treatments for wAIHA, there are little data. Intra venous immunoglobulin doses of 0.4g/kg/d for 5 days have been studied. In 73 patients the overall response rate was 29 in 73 (40%).28 A 2-g/dL Hgb rise in 2 weeks was seen in 25 of 62 (40%), and a Hgb rise over 2g/dL in 2 weeks with a Hgb higher than 10g/dL was seen in 9 of 61 (15%) patients. The treatment response was short-lived. Mycophenolate Doses of 500 to 1000mg of MMF twice a day have been stud ied. This drug may take 4 to 8 weeks before reaching its full effect. Pooled data from 2 short series show that all 8 patients responded with a significant rise in Hgb.29,30 MMF is well toler Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 RBC transfusions In medically unstable, symptomatic patients, particularly those with Hgb levels lower than 6g/dL, RBC transfusion is indicated. Appropriately crossmatched blood may not be imme di ately available because of the panagglutinin, and therefore type-spe cific blood is used. In general, worsening of hemolysis is more likely to occur with alloimmune antibodies than with autoanti bodies. ing, the initial high response is not maintained: 50% maintained response at 3 months but only 31% to 38% at 12 months. Overall, nearly two-thirds of corticosteroid responders require additional therapy.22 In patients failing to respond rapidly or for those presenting with severe disease, the addition of rituximab should be consid ered (see below). ated except for mild nausea and remains the favorite immuno suppressive drug for this reviewer. Cyclophosphamide A 50-mg/m2 dose of cyclophosphamide daily for 4 days pro duced a CR rate in 6 of 9 patients and a PR rate (absence of trans fusions) in 3 of 9 patients.31 Another study reported a response rate of 59% of 17 patients.32 Single intravenous doses of 600 to 750mg/m2 have also been reported as effective. Erythropoiesis-stimulating agents Approximately 20% to 40% of patients with AIHA have a reduced retic u lo cyte response due to antibodies against reticulocytes or reduced erythropoiesis; this correlates with a poor response. In a recent multicenter prospective study, 51 patients with wAIHA had severely reduced erythropoietin levels compared with other forms of anemia; they were treated with an erythropoiesis-stimulating agent (ESA) beginning at a median (range) of 24 (0.03-187) months from diag no sis.35 The overall response rate (Hgb >10g/dL or ≥2g/dL increase in the absence of transfusions) was 55% at 15 days, 71% at 30 days, 73% at 3 months, 76% at 6 months, and 78% at 12 months. The median (range) Hgb rise at 6 months was 2.1 (0-2.18) g/dL. Other agents Individual case reports and small retrospective analyses have reported therapeutic responses to danazol, cyclosporine, borte zomib, daratumumab, vincristine, eculizumab, and alemtuzumab. Putting it alltogether A proposed treatment algorithm can be found in (Figure 4). As in this case, the initial therapy for most patients should be cor ticosteroids, usually in the form of prednisone at 1mg/kg/d. If the patient is severely affected or shows no response within 2 weeks, rituximab should be given at a dose of either 375mg/m2 for 4 weeks or 2 sep a rate weekly doses of 1000mg each. Corticosteroids are weaned slowly over 8 to 12 weeks, and if an adequate therapeutic response (eg, Hgb over 9-10 g/dL) can not be maintained at a dose of prednisone less than or equal to 5mg/d, rituximab (if not already given) is administered. If an ade quate therapeutic response is not obtained after 2 or 3 months, our preferred approach is 1000mg of MMF twice a day in those who have demonstrated a strong corticosteroid response. In those having never had an adequate corticosteroid response or failing MMF, an ESA, such as 200 mcg of darbepoetin alfa every 2 weeks, is administered for several months in those with an endogenous erythropoietin level under 500 mIU/mL. Through out this treatment algorithm, the lowest effective dose of pred nisone is administered to maintain an adequate Hgb response; prednisone tapering is undertaken whenever another thera peutic modality demonstrates a beneficial effect. In patients refractory to low-dose corticosteroids, rituximab, immunosup pressive agents, and ESAs, we reassess the diagnosis and indi Complications of wAIHA and their treatment Aside from the well-known complications of long-term corticoste roid treatment, 3 other aspects of wAIHA warrant brief comment: • • • Patients face an increased risk of developing LPDs. An LPD developed in 7 of 71 (10%) of patients with wAIHA after a median (range) of 26.5 (9-76) months, suggesting that wAIHA might be the first manifestation of a nascent LPD.36 Patients’ risk of thromboembolism (venous more than arterial) is increased. In a recent study, 15 of 126 (12%) patients devel oped thrombosis.37 Increased LDH and trans fu sions were associated with increased risk. Thrombosis is of particular concern following splenectomy38; appropriate postoperative DVT prophylaxis should be provided. Hospitalized patients should receive routine DVT prophylaxis. Monitoring for diabetes is impaired. Given the reduced red cell survival, hemoglobin A1c levels do not provide accurate glucose monitoring.39 Newer therapies in development for wAIHA wAIHA is a frustrating disorder, and many patients remain on inappropriately high doses of corticosteroids for years. Newer therapies are needed.40 Figure 4. Proposed treatment algorithm for wAIHA. Warm autoimmune hemolytic anemia: diagnosis and treatment | 111 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 Azathioprine wAIHA is not uncommon in dogs; 87 of 93 dogs responded to azathioprine and survived.33 In humans, doses of 100 to 200mg daily have been used, with reported response rates of 26 of 33 (79%) in 2.2 months in one study and 7 of 8 (1 CR, 6 PR) in another.23,32 With discontinuation of the drug, 42% relapsed.32 To avoid severe hema to log i cal tox ic ity, patients need to be screened for deficiencies of thiopurine methyltransferase.34 Immunoproteasome inhibitors such as bortezomib and KZR– 616 may reduce autoantibody formation, as might CD38 inhib itors such as daratumumab. Drugs that block the neonatal Fc receptor shorten IgG half-life and reduce anti-RBC antibody.40 Inhibitors of mediators of phagocytosis such as syk kinase (fos tamatinib) and Bruton kinase (rilzabrutinib) are currently being studied.41,42 Fostamatinib increased Hgb to a level above 10g/dL, with an increase of 2g/dL in 11 of 24 (46%) refractory wAIHA patients; Hgb improvement was often seen after 2 weeks and was maintained with few adverse events.43 Clinical trials are also underway to assess the efficacy of inhibitors of complement fac tors C1 and C3. cations for treatment. Our subsequent preference is for clinical trials, such as those with fostamatinib, rilzabrutinib, or a neonatal Fc receptor inhibitor. While not averse to splenectomy, this is usually reserved for those highly refractory to the therapeutic modalities described above and for whom clinical trials are not appropriate. Acknowledgment I am pleased to thank Dr Drew Provan for the artistry in Figure 2 and the Visual Abstract. Conflict-of-interest disclosure Off-label drug use David J. Kuter: none of the pharmacologic therapies listed have received regulatory approval for the treatment of wAIHA. Correspondence David J. Kuter, Hematology Division, Massachusetts General Hos pital, Ste 118, Rm 110, Zero Emerson Pl, Boston, MA 02114; e-mail: dkuter@MGH.harvard.edu. References 1. Jäger U, Barcellini W, Broome CM, et al. Diagnosis and treatment of auto immune hemolytic anemia in adults: recommendations from the First Inter national Consensus Meeting. Blood Rev. 2020;41:100648. 2. Wagner KH, Shiels RG, Lang CA, Seyed Khoei N, Bulmer AC. Diagnostic criteria and contributors to Gilbert’s syndrome. Crit Rev Clin Lab Sci. 2018;55(2):129-139. 3. Trudel G, Shahin N, Ramsay T, Laneuville O, Louati H. Hemolysis contributes to anemia during long-duration space flight. Nat Med. 2022;28(1):59-62. 4. Marchand A, Galen RS, Van Lente F. The predictive value of serum hapto globin in hemolytic disease. JAMA. 1980;243(19):1909-1911. 5. Galen RS. Application of the predictive value model in the analysis of test effectiveness. Clin Lab Med. 1982;2(4):685-699. 6. Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br J Exp Pathol. 1945;26:255-266. https://www .ncbi.nlm.nih.gov/pmc/articles/PMC2065689/pdf/brjexppathol00160 -0040.pdf. Accessed 26 August 2022. 7. Coombs RR, Edin B, Mourant AE, Race RR. Detection of weak and “incom plete” Rh agglutinins: a new test. Lancet. 1945;246(6358):15-16. 8. Zarandona JM, Yazer MH. The role of the Coombs test in evaluating hemo lysis in adults. CMAJ. 2006;174(3):305-307. 9. Mauro FR, Foa R, Cerretti R, et al. Autoimmune hemolytic anemia in chronic lymphocytic leukemia: clinical, therapeutic, and prognostic features. Blood. 2000;95(9):2786-2792. 10. Barcellini W, Fattizzo B. The changing landscape of autoimmune hemolytic anemia. Front Immunol. 2020;11:946. 11. Hill QA, Horan M, Charlton A, et al. Developing the evidence base for the management of autoimmune haemolytic anaemia (AIHA): the UK experi ence. Br J Haematol. 2021;192(2):e54-e57. 12. Hansen DL, Möller S, Andersen K, Gaist D, Frederiksen H. Increasing inci dence and prevalence of acquired hemolytic anemias in Denmark, 19802016. Clin Epidemiol. 2020;12:497-508. 13. Maquet J, Lafaurie M, Walter O, et al. Epidemiology of autoimmune hemo lytic anemia: a nationwide population-based study in France. Am J Hematol. 2021;96(8):E291-E293. 112 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 David J. Kuter: research funding: Argenx, Biocryst, Immunovant, Rigel, Sanofi (Principia), Takeda (Bioverativ), UCB; consultancy: Alexion (Syntimmune), Amgen, Argenx, BioCryst, Bristol Myers Squibb, Caremark, Cellphire, Cellularity, CRICO, Daiichi Sankyo, Hengrui, Immunovant, Incyte, Kyowa-Kirin, Merck Sharp Dohme, Momenta, Novartis, Pfizer, Platelet Biogenesis, Platelet Disorder Support Association, Rigel, Sanofi (Bioveratif), Sanofi (Genzyme), Sanofi (Principia), Sobi (Dova), Takeda, UCB, Up-To-Date; stock ownership: Rubius. 14. Tranekær S, Hansen DL, Frederiksen H. Epidemiology of secondary warm autoimmune haemolytic anaemia—a systematic review and meta-analysis. J Clin Med. 2021;10(6). 15. Sokol RJ, Hewitt S, Stamps BK. Autoimmune haemolysis: an 18-year study of 865 cases referred to a regional transfusion centre. Br Med J (Clin Res Ed) 1981;282(6281):2023-2027. 16. Engelfriet CP, Overbeeke MA, von dem Borne AE. Autoimmune hemolytic anemia. Semin Hematol. 1992;29(1):3-12. 17. Barcellini W, Fattizzo B, Zaninoni A. Management of refractory autoimmune hemolytic anemia after allogeneic hematopoietic stem cell transplanta tion: current perspectives. J Blood Med. 2019;10(August):265-278. 18. Hollenhorst MA, Al-Samkari H, Kuter DJ. Markers of autoimmunity in immune thrombocytopenia: prevalence and prognostic significance. Blood Adv. 2019;3(22):3515-3521. 19. Hansen DL, Möller S, Andersen K, Gaist D, Frederiksen H. Evans syndrome in adults—incidence, prevalence, and survival in a nationwide cohort. Am J Hematol. 2019;94(10):1081-1090. 20. Birgens H, Frederiksen H, Hasselbalch HC, et al. A phase III randomized trial comparing glucocorticoid monotherapy versus glucocorticoid and rit uximab in patients with autoimmune haemolytic anaemia. Br J Haematol. 2013;163(3):393-399. 21. Michel M, Terriou L, Roudot-Thoraval F, et al. A randomized and doubleblind controlled trial evaluating the safety and efficacy of rituximab for warm auto-immune hemolytic anemia in adults (the RAIHA study). Am J Hematol. 2017;92(1):23-27. 22. Roumier M, Loustau V, Guillaud C, et al. Characteristics and out come of warm auto im mune hemo lytic ane mia in adults: new insights based on a sin gle-cen ter expe ri ence with 60 patients. Am J Hematol. 2014;89(9):E150-E155. 23. Prabhu R, Bhaskaran R, Shenoy V, Reema G, Sidharthan N. Clinical charac teristics and treatment outcomes of primary autoimmune hemolytic ane mia: a single center study from South India. Blood Res. 2016;51(2):88-94. 24. Barcellini W, Zaja F, Zaninoni A, et al. Low-dose rituximab in adult patients with idiopathic autoimmune hemolytic anemia: clinical efficacy and bio logic studies. Blood. 2012;119(16):3691-3697. 25. Barcellini W, Zaja F, Zaninoni A, et al. Sustained response to low-dose rituximab in idiopathic autoimmune hemolytic anemia. Eur J Haematol. 2013;91(6):546-551. 26. Barcellini W, Fattizzo B, Zaninoni A, et al. Clinical heterogeneity and pre dictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood. 2014;124(19):2930-2936. 27. Giudice V, Rosamilio R, Ferrara I, Seneca E, Serio B, Selleri C. Efficacy and safety of splenectomy in adult autoimmune hemolytic anemia. Open Med (Wars). 2016;11(1):374-380. 28. Flores G, Cunningham-Rundles C, Newland AC, Bussel JB. Efficacy of intra venous immunoglobulin in the treatment of autoimmune hemolytic ane mia: results in 73 patients. Am J Hematol. 1993;44(4):237-242. 29. Howard J, Hoffbrand AV, Prentice HG, Mehta A. Mycophenolate mofetil for the treatment of refractory auto-immune haemolytic anaemia and autoimmune thrombocytopenia purpura. Br J Haematol. 2002;117(3):712-715. 30. Kotb R, Pinganaud C, Trichet C, et al. Efficacy of mycophenolate mofetil in adult refractory auto-immune cytopenias: a single center preliminary study. Eur J Haematol. 2005;75(1):60-64. 31. Moyo VM, Smith D, Brodsky I, Crilley P, Jones RJ, Brodsky RA. High-dose cyclophosphamide for refractory autoimmune hemolytic anemia. Blood. 2002;100(2):704-706. 32. Hantaweepant C, Pairattanakorn P, Karaketklang K, Owattanapanich W, Chinthammitr Y. Efficacy and safety of second-line treatment in Thai patients with primary warm-type autoimmune hemolytic anemia. Hematology. 2019;24(1):720-726. 33. Piek CJ, Junius G, Dekker A, Schrauwen E, Slappendel RJ, Teske E. Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs. J Vet Intern Med. 2008;22(2):366-373. 34. Lennard L. Implementation of TPMT test ing. Br J Clin Pharmacol. 2014;77(4):704-714. 35. Fattizzo B, Michel M, Zaninoni A, et al. Efficacy of recombinant erythropoi etin in autoimmune hemolytic anemia: a multicenter international study. Haematologica. 2021;106(2):622-625. 36. Sallah S, Wan JY, Hanrahan LR. Future development of lymphoproliferative disorders in patients with autoimmune hemolytic anemia. Clin Cancer Res. 2001;7(4):791-794. 37. Fattizzo B, Barcellini W. Autoimmune hemolytic anemia: causes and conse quences. Expert Rev Clin Immunol. 2022;18(7):731-745. 38. Crary SE, Buchanan GR. Vascular com pli ca tions after sple nec tomy for hematologic disorders. Blood. 2009;114(14):2861-2868. 39. Lum G. Artefactually low hemoglobin A1C in a patient with hemolytic ane mia. Lab Med. 2010;41(5):267-270. 40. Kuter DJ. Novel therapies for immune thrombocytopenia. Br J Haematol. 2022;196(6):1311-1328. 41. Podolanczuk A, Lazarus AH, Crow AR, Grossbard E, Bussel JB. Of mice and men: an open-label pilot study for treatment of immune thrombocytope nic purpura by an inhibitor of Syk. Blood. 2009;113(14):3154-3160. 42. Langrish CL, Bradshaw JM, Francesco MR, et al. Preclinical efficacy and antiinflammatory mechanisms of action of the Bruton tyrosine kinase inhibitor rilzabrutinib for immune-mediated disease. J Immunol. 2021;206(7):14541468. 43. Kuter DJ, Rogers KA, Boxer MA, et al. Fostamatinib for the treatment of warm antibody autoimmune hemolytic anemia: phase 2, multicenter, open-label study. Am J Hematol. 2022;97(6):691-699. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000405 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/105/2021916/105kuter.pdf by guest on 09 December 2022 Warm autoimmune hemolytic anemia: diagnosis and treatment | 113 Dr Prakash Singh Shekhawat BEYOND ROUTINE FRONTLINE THERAPY OF CML Christian Niederwieser and Nicolaus Kröger Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Germany Molecular therapy with tyrosine kinase inhibitors (TKIs) has significantly reduced the indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in chronic myeloid leukemia (CML). Treatment-free remission can be obtained in about 50% of patients with an optimal response. However, cure rates up to 90% are restricted to patients receiving HSCT. Timing is essential since HSCT in the early stages of the disease has the best outcome. Patients in a more advanced phase (AdP) than chronic-phase (chP) CML undergo HSCT with suboptimal outcomes, and the gap between chP and AdP disease is widening. First-line therapy should start with first- or second-generation (G) TKIs. Patients failing treatment (BCR-ABL1 transcripts of greater than 10% at 3 and 6 months and greater than 1% at 12 months) should be switched to second-line TKIs, and HSCT should be considered. Patients not responding to 2G-TKI therapy as well as patients in an accelerated phase (AP) or blast crisis (BC) are candidates for HSCT. Therapy resistant BCR-ABL1 mutations, high-risk additional cytogenetic abnormalities, and molecular signs of leukemia progression should trigger the indication for HSCT. Patients who, despite dose adjustments, do not tolerate or develop severe adverse events, including vascular events, to multiple TKIs are also candidates for HSCT. In AdP CML, TKIs do not show long-lasting results, and the outcome of HSCT is less optimal without pretransplant therapy. In these patients the induction of chP2 with TKIs, either alone (AP) or in combination with intensive chemotherapy (BC), followed by HSCT should be pursued. LEARNING OBJECTIVES • Understand the criteria for considering allogeneic stem cell transplantation for CML patients • Define the risk factors for outcomes after allogeneic stem cell transplantation for CML patients • Acquire the knowledge of how to use TKIs before and after transplantation in CML patients Introduction Chronic myeloid leukemia (CML), a disease of predominantly older adult (age >60 years) and male patients, is in many aspects a model for malignant diseases. Described by Virchow and Bennett in 1845 as leucocythemia, it was the first malignancy with a common chromosomal alteration.1,2 After molecular identification of the breakpoints, the main molecular pathomechanisms were unraveled.3,4 Hematopoietic stem cell transplantation (HSCT) was shown to be the only curative option.5 Almost at the same time, treatment with interferon alfa was shown to induce cytogenetic remissions (CyR) in a small proportion of patients.6 The use of BCR-ABL1 transcripts for disease monitoring and donor lymphocyte infusion (DLI) to treat relapse after HSCT were the next hallmarks in the treatment of this disease.7,8 Finally, in 2001 a specific inhibitor of BCR-ABL tyrosine kinase revolutionized the treatment of CML.9 In 2010, the discontinuation of TKIs in patients with optimal 114 | Hematology 2022 | ASH Education Program response was described and the term “operational cure” used.10 After decades of development, HSCT was increasingly used worldwide, and more than 1.5 million transplants (now 90 000 annually) were reported up to 2019.11 Patients with early chronic-phase (chP) CML were considered an ideal indication for HSCT.12 Interval-diagnosis HSCT (<12 months and >12 months), disease stage (chP, accelerated phase [AP], and blast crisis [BC]), donor (related and unrelated), and gender differences were recognized as major prognostic factors influencing transplant-related mortality (TRM; nonrelapse mortality caused by graft-versus-host disease [GVHD], infections, or toxicities), and relapse incidence (European Society for Blood and Marrow Transplantation [EBMT] risk score).12 Alternative donors became available in 1989 using cord blood and haploidentical donors by ex vivo or in vivo T-cell depletion.13,14 In 1998 reducedintensity preparative regimens for older adults and patients Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 Transplantation in CML in the TKI era: who, when, and how? with comorbidities were developed.15 Today, TRM of 5% to 10% in chP CML and 20% to 25% in advanced phase (AdP) CML remain, and GVHD represents a major complication after HSCT. After the availability of TKIs, HSCT activity in the EBMT registry continuously decreased for chP but remained stable for AP/BC (Figure 1). Nowadays, more patients in non-chP than in chP are transplanted worldwide,11 which underlines the importance of optimizing outcome by improving the timing of HSCT. Oral TKI therapy in patients with chP CML resulted in an over all survival (OS) of 80% at 10 years, but only approximately 50% remained on first-generation (1G) treatment.9,16 After first, sec ond, and third G-TKIs became available, major molecular remis sions (MMR) and even treatment-free remissions were observed.17 Despite this success, a significant proportion of patients become refractory or progress, become intolerant, or develop cytopenia. Approximately 31% of patients switch from first-line to sec ond-line therapy after 11 months.18 Reasons for switching include failure in 32%, intolerance in 57%, and other issues in 12% of patients. Recently, asciminib has demonstrated a higher MMR rate in combination with a more favorable safety profile as bosutinib.19 Despite being well tolerated overall, side effects on TKIs, especially in higher G-TKIs, occur (eg, vasculopathy up to 53% of patients on nilotinib, vascular events in 37% on ponatinib, and pleural effusion in approximately 20% on dasatinib).20,21 Other studies showed an intolerance of 16% or 7% on treatment with dasatinib (5 years) and/or imatinib (5 and 10 years).16,22 Despite HSCT being the only cura tive treat ment, tim ing assumes an essential role in this process. Decisions should not be taken too early and incur unnecessary risks or too late, which could jeopardize the outcome after HSCT. Results in AdP CML (especially in BC) are poor with single TKI therapy and single HSCT (see below). Recommendations from experts in the field and published results are of fundamental importance in this process. CLINICAL CASE 1 A 38-year-old con struc tion worker presented with sud den fatigue, nosebleeds, and pneumonia. He had leukocytosis (112 × 109/L), thrombocytopenia (15 × 109/L), and anemia (hemo glo bin level of 8.4 g/dL). Initially, an acute mye loid leu ke mia (AML) was suspected after the bone marrow aspiration revealed 97% blasts with a myeloid phenotype. In addition, a Philadelphia chromosome (Ph) in 20 out of 20 metaphases and a complex aberrant karyotype (including monosomy 7) were detected. A T315I mutation was described in the molecular ana lyses and a blast crisis (BC) of CML diagnosed. CLINICAL CASE 2 At a routine checkup, a 60-year-old woman had 30×109/L leu kocytes, 400×109/L platelets, and a hemoglobin level within a normal range. She currently experienced shortness of breath but otherwise felt healthy. After referral to a hematologist, a left shift was found in the differential but no enlargement of the lymph nodes or spleen. Since the Ph chromosome was detected in all metaphases upon bone marrow aspiration, chP CML and a lowrisk EUTOS long-term-survival-score was diagnosed. Who should undergo transplantation Outcomes of HSCT have improved substantially during the last decades. In a real-world analysis, the Swedish registry reported an OS of 96% at 5 years in chP1 in patients with a median age spec tive of 43 years using matched donors.23 In another pro study, the German CML study group reported 3-year OS rates Dr Prakash Singh Shekhawat HSCT in CML | 115 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 Figure 1. Development of allogeneic stem cell transplantation for CML in Europe from 1990 to 2020 (EBMT registry) showing chP and AdP CML (AP and BC). considered for HSCT, while de novo AP CML might become eligi ble for HSCT if the response to TKIs is not optimal. Recommendations according to the ELN and to the NCCN regarding allogeneic stem cell transplantation are outlined in Table 1. Although AdP CML occurs in a minority of patients (de novo 10%; 5% on dasatinib and 7% on imatinib develop AdP after 5 years), out comes are infe rior to chP CML after HSCT and TKIs alone. Outcomes of BC HSCT in the pre-imatinib era were reported to be only 21% OS at 2 years.28 1G-TKIs in patients with BC resulted in a median OS of 7 to 10 months, while treatment with 2G-TKIs (nilotinib or dasatinib) resulted in an OS of 32% and 30% at 2 years, respectively. In a retrospective study with 104 patients, 1-3G-TKIs plus intensive chemotherapy (IC) and TKIs plus hypomethylating agents led to a higher rate of CRi (57.5% vs 33%), a higher complete CyR rate (45% vs 10.7%), and more patients proceeding to HSCT (32.5% vs 10.7%) than TKIs or IC alone. Long-term results were similar in the combinations and clearly inferior to TKIs or IC alone (OS, 30%-28% vs 13%-0% at 5 years).31 HSCT resulted in long-term OS in patients with advanced CML (34% CI, 23-46, at 15 years). OS was improved in non-BC patients at HSCT with donors 36 years of age or youn ger and with a higher CD34+ cell dose in the graft.32 The ELN and NCCN provide information on induction chemotherapy according to AML-based morphology and acute lymphocytic leukemia treatment. The ELN emphasizes the need to attempt to return to chP CML with subsequent HSCT without delay and that patients with untreated BC should not undergo HSCT. A study found that in patients in remission for BC, conventional risk factors such as advanced age, poor performance status, a longer interval from diagnosis to HSCT, myeloablative con di tion ing (MAC), and unre lated donors remained the major deter mi nants of out come, whereas in those with active BC at transplant, unrelated donor transplantation was associated with prolonged leukemia-free survival (LFS).33 Similar results for advanced CML in the AP or BC or pretreated with TKIs beyond Table 1. Recommendations for allogeneic stem cell transplantation in CML according to the ELN and the NCCN Chronic phase (chP) Accelerated phase (AP) Blast phase (BC) ELN 2020 - Disease resistant or intolerant (suboptimal response to 2 or more TKIs) - For the very rare patient with inadequate recovery of normal hematopoiesis - Resistance to 2G-TKIs (first or second line) ponatinib or experimental agent - Failure to respond to ponatinib after 3 months’ treatment - Emergence of high-risk cytogenetics: observe closely, consider intensification of treatment (ponatinib, early allo-SCT) ELN 2020 - A patient presenting in AP should be treated as a high-risk patient, becoming eligible for HSCT if the response is not optimal - A patient progressing to AP during treatment should immediately be considered for HSCT ELN 202025 - Attempt at return to chP2 - Addition of chemotherapy based on AML regimens for myeloid BP (such as dasatinib or ponatinib + FLAG-IDA) or ALL regimens for lymphoid BP (such as imatinib or dasatinib + hyperfractionated CVAD) recommended - After CP2 is achieved proceed to allo-SCT without delay - Transplantation in active BP is not recommended NCCN guidelines26 - If TKI-resistant disease BCR-ABL1 (IS) >10% at >3 months, switch to alternate TKI and evaluate for HSCT NCCN guidelines26 - Disease progression to AP while on TKI therapy should be considered for HSCT - Patients who present with AP at diagnosis should be treated with a TKI, followed by evaluation for allogeneic HSCT based on response to therapy after 3, 6, or 12 months NCCN guidelines26 - Recommendation does not depend on response - After therapy with morphology-based induction chemotherapy + TKI in lymphoid and myeloid blast crisis or TKI plus steroids in lymphoid blast crisis and sole TKI in myeloid blast crisis 25 25 CVAD, chemotherapy combination used to treat some types of acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Hyper-CVAD includes the drugs cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride (Adriamycin), and dexamethasone; FLAG-IDA, fludarabine, highdose cytosine arabinoside (AraC), idarubicin, and granulocyte colony-stimulating factor (G-CSF); IS, international scale. 116 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 of 88% and 94% after elective HSCT in high-risk chP (but a 0-1 EBMT score) and in imatinib-failure chP CML patients, respec tively. TRM of only 8% was described in this multicenter study, which compares favorably to TRM of 26% in an earlier random ized study after IFN-based treatment in chP CML.24 TKI therapy with only imatinib led approximately 96% OS at 3 years in the same study.24 The European LeukemiaNet (ELN) criteria and the US National Comprehensive Cancer Network (NCCN) guide lines are updated on a regular basis and guide the treatment not only in chP CML. The difference between the 2 involves the definition of response to therapy. Transcripts higher than 10% at 3 months define patients with possible TKI-resistant disease and after 3 months with definite TKI-resistant disease (NCCN guidelines). In the ELN guidelines, transcripts higher than 10% are defined as a warning at 3 months and if confirmed within the next 3 months as failure. Failure means changing to another TKI if therapy started with 1G-TKI but to assessment for HSCT only if therapy started with 2G-TKI. In NCCN guidelines, possible TKI resistance automatically leads to evaluation for HSCT.25,26 On the other hand, the ELN recommendations specify resistance to 2GTKI as an indication for HSCT. HSCT results in patients with AP CML are clearly inferior to those in chP1. Before the TKI era, outcomes of HSCT in AP CML were 35% at 2 years and in the imatinib era, 59% at 2 years (com pared to 88% and 94% in early chP CML).27,28 In a prospective study, Jiang et al demonstrated an advantage for HSCT over TKI in AP CML (6-year OS, 83.3% vs 51.4%).29 Similar results of 50% to 60% OS at 5 years after imatinib, nilotinib, and dasatinib (60%70% OS and 10% MMR at 2 years) and bosutinib (60% OS and 11% MMR at 4 years) were reported with TKI monotherapy.30 Ponatinib had slightly higher response rates (84% OS and 34% MMR at 1 year), but randomized comparisons are lacking. Patients with de novo AP CML treated with nilotinib or dasatinib (70% MMR and 90% OS at 3 years, respectively) have been reported to have superior results than AP CML that develops while on therapy.30 Patients with AP CML under treatment should immediately be The patient was treated with an AML-like induction chemother apy in combination with ponatinib, a 3G-TKI to overcome the resistance to the T315I mutation. After reaching chP, the patient was referred to an HSCT program. at 3 and 6 months of treatment and higher than 1% at 12 months are considered treatment failures and should lead to second-line TKI treatment (either 2G- or 3G-TKI or a TKI responsive to the detected mutation). A donor search should be initiated upon resistance to 2G-TKI treatment.25 Approximately 65% respond to second-line treatment with BCR-ABL1 transcripts of less than or equal to 10%. Nonresponding patients (>10% transcripts at 3 months) have durable CyR of only 50% at 4 years.51 HSCT in early chP CML has been shown to have the best out come, even after first-line TKI resistance.12 In addition, TKI ther apy before HSCT has been associated with better posttransplant outcomes.32 A Center for International Blood and marrow Transplant Research (CIBMTR) study confirmed the beneficial effect of pretransplant TKIs in chP CML for posttransplant survival.53 Delaying HSCT to late chP CML incurs the risk of progression to AdP disease, with unfavorable results. Patients not eligible for HSCT (>80 years of age) should continue on 3G-TKIs or new inhibitors in the testing phase. In pediatric patients, lifelong TKI therapy, including side effects, needs to be balanced with an increased HSCT TRM at the beginning and a possible increased morbidity with chronic GVHD.54 The goal in advanced CML is a return to a chP followed by HSCT. The choice of pretransplant TKIs for AdP CML is not well stan dard ized, but dasatinib and nilotinib have at least safely been admin is tered before HSCT with out increased TRM.50,55 Thus, it is appropriate to use TKIs to reduce the disease burden before HSCT for AP CML. The search for a donor should be initi ated at diagnosis and HSCT planned after a response (Table 2). In BC, a downgrade to chP2 should be considered a clinical condi tion requiring, whenever possible, treatment with a combination of TKIs with IC or TKIs with hypomethylating agents followed by HSCT.31 The combination of TKIs plus IC with HSCT has been shown to result in 54% OS at 2 years in AdP CML.27 HSCT remains a unique therapeutic option for patients in chP CML after the fail ure of 2 TKIs or in those potentially harboring the T315I mutation (after a trial of ponatinib therapy).56 CLINICAL CASE 2 (Cont inu ed) CLINICAL CASE 1 (Continued) CLINICAL CASE 1 (Cont inu ed) After therapy with hydroxyurea, the patient was started on the 1G-TKI imatinib. Her response according to ELN criteria was optimal after 3 months. After she discussed the possibility of HSCT with her hematologist, it was decided to continue TKI therapy under continuous BCR-ABL1 monitoring. Timing: when to perform a transplant It is generally acceptable to start first-line therapy with TKIs (first or second generation) in chP CML without affecting the out come of HSCT.25 The response and tol er ance to the TKI determine further treatment. The response to the TKI depends on the EUTOS long-term survival score and the presence of high-risk cytogenetics and TKI mutations.51 The presence of the T315I mutation, which is responsive to ponatinib, is a trigger for HSCT. The ELN provides regular recommendations on how to man age TKI treatment according to BCR-ABL1 transcript reduction at defined time points.52 BCR-ABL1 transcripts higher than 10% After weighing the pros and cons, the patient decided to undergo HSCT from the HLA-matched brother at a JACIE-certified transplant center. Three months after diagnosis, he underwent peripheral blood HSCT. CLINICAL CASE 2 (Continued) After 2 years of optimal response, the patient showed increased BCR-ABL1 transcripts. Molecular analyses detected V299L resis tant to dasatinib and Y253H resistant to nilotinib, but both were sensitive only to ponatinib. Treatment with ponatinib was started. Since her 70-year-old brother, her only sibling, had CLL, an unrelated donor search was initiated. Following intoler ance to ponatinib (causing headaches and vomiting) but after achieving BCR-ABL1 negativity and lacking access to asciminib, an HSCT was considered. Dr Prakash Singh Shekhawat HSCT in CML | 117 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 frontline therapy have been reported by other investigators and are listed in Table 2.34-41 The emergence of high-risk addi tional chromosomal aberrations (ACAs; high-risk ACAs include +8, a second Ph chromosome, i(17q), +19, −7/7q-, 11q23, 3q26.2, and complex aberrant karyotypes) predict a poorer response to TKIs and a higher risk of progression. According to ELN cri te ria, patients with high-risk ACAs are con sid ered high-risk patients,25 and they should be observed and considered for intensification of treatment, including early HSCT. Gene muta tions (RUNX1, ASXL1, IKZF1, WT1, TET2, IDH1/2, CBFB/MYH11, TP53) are found in CML and might be associated with progres sion to BC.42,43 Such mutations may lead to a genetically based risk classification in the future with the potential for combina tion with non–BCR-ABL1 targeted therapy. The molecular land scape of muta tions, espe cially concerning epi ge net ics, has been featured in recent publications.44 Optimal preconditions for HSCT are a low CI Sorror score and good performance status, in addition to the requirement for a suit able donor, pref er a bly a human leu ko cyte antigen (HLA)– compatible sibling followed by an unrelated donor (10 out of 10 matches).45 If no matched donor is available, alternative donors, such as haploidentical HSCT with post cyclophosphamide or mismatched donors or cord blood, should be taken into con sideration.46-49 In addition, HSCT is feasible in patients previously treated with 2G-TKIs with a posttransplant complication rate compara ble to that of TKI-naive or imatinib-treated patients.50 Table 2. Selection of CML studies using HSCT including patients beyond frontline therapy Authors Number of patients HSCT y Disease stage (patient %) TKI used before HSCT (patient %) TRM % DFS/LFS/ PFS/RI % OS % Study type chP 449 177 (<18y) 272 (18-29y) 2001-2010 chP1 (100) TKI (60) <18y TKI (48) 18-29y 13 (at 1y), 18 (at 3y), 20 (at 5y) LFS 59% 57 (<18y) 60 (18-29y) n.s. 75% OS (at 5y) 76% (<18y) 74% (18-29y) n.s. Retrospective, multicenter, children and young adults (CIBMTR) Yassine et al35 199 (adults) 97 (children/ adults) Metaanalysis chP (100) Meta-analysis 20 (adults) 28 (children/ adults) Adult DFS 66%, adults/ children DFS 47%/PFS 82% Adults 84%, children 91%, adults/ children 76% Meta-analysis resistant/ intolerant chP to >1 TKI Jiang et al29 132 2001-2008 AP (100) Imatinib (66%) Imatinib + HSCT (34) 11 Low risk: imatinib 85% HSCT 95% (PFS at 6y) High risk: imatinib 19% HSCT 100% (PFS at 5y) Low risk: imatinib 100% HSCT 81% (OS at 6y) High risk: imatinib 18% HSCT 100% (OS at 5y) Prospective single-center study (imatinib vs HSCT) Khoury et al57 449 1999-2004 chP2 (41), AP (41), BC (18) Imatinib (50) chP2 33, AP 34, BC 46 (at 1y) chP2 27%, AP 37%, BC 10% (LFS at 3y) chP2 36%, AP 43%, BC 14% (OS at 3y) Retrospective multicenter study (CIBMTR) Zheng et al36 32 2002-2011 AP (59), BC (41) Imatinib (53) Cord 38, sib 12 (at 0.5y) Cord 50% sib 40% (LFS at 5y) Cord 62%, Sib 49% (OS at 5y) Retrospective single-center study (cord vs sib) Radujkovic et al33 170 2004-2016 BC in 2 chP (56) BC active (44) 1G-TKI (59), 2G-TKI (33), 3G-TKI (8) 19.7 (at 1y) 23.3 (at 3y) Active BC: 27.1% 3y BC in remission: 20.2% 3y LFS 34.6, RI 45.7 (at 1y). LFS 26.1, RI 50.7 (at 3y) LFS: active BC: 11.6% 3y BC in remission 33.8% 3y RI: active BC: 56.4.6% 3y BC in remission 45.9% 3y 57.5%(at 1y) 38.5 (at 3y) Active BC: 23.8% (3y) BC in remission 51% (3y) Retrospective EBMT study HSCT in treated vs untreated BC Yang et al41 278 2002-2021 de novo AP (100) Imatinib (67) or 2G-TKI (33; nilotinib 24, dasatanib 7, flumatinib 1) Censored at HSCT TFS 89% (at 6y) Low risk 95 (5y) Interm 76 (5y) High-risk 19 (5y) No significance between 1G-TKI versus 2G for TFS OS 90% (at 6y) Low risk 54 (5y) Interm. 36 (5y) High-risk 10 (5y) No significance between 1G-TKI vs 2G for OS Comparison between imatinib and 2G-TKI before HSCT AP and BC 118 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 Chaudhury et al34 Table 2. Selection of CML studies using HSCT including patients beyond frontline therapy (Continued) Authors Number of patients HSCT y Disease stage (patient %) TKI used before HSCT (patient %) TRM % DFS/LFS/ PFS/RI % OS % Study type Different disease phase 84 2003-2008 chP1 (TKI) (23), chP1 (TKI failure) (44), AP (4), BC (30) Imatinib 8 in chP (at 3y) 18 in AdP (at 3y) CMR at last PCR 88% chP1 (elective) 88%, chP1 (imatinib failure) 94%, AP 59% (OS at 3y) Prospective multicenter study (CML IV study) Jabbour et al55 47 2004-2007 chP1 (34), chP2 (21), AP (25), BC (19) Imatinib failure (100) + 2G-TKI (62) 13% (at 2y) 49% (EFS at 2 y) (muta tion 36% vs no mutation 58%) 63% (OS at 2y) (mutation 44% vs no mutation 76%) Retrospective single-center study Topcuoglu et al37 84 1989-2007 chP1 (79), chP2 (6), AP (15) NA 7% RIC 14% MAC 48% (LFS at 5y) No difference RIC vs MAC 56% (OS at 5y) No difference RIC vs MAC Retrospective single-center study (RIC vs MAC) Oyekunle et al38 68 2002 – 2009 chP1 (40), >chP1 (60) Pre-HSCT TKI (71), post-HSCT TKI (29) NA 54% (LFS at 2y) 63% (OS at 2y) Single institute HSCT in TKI era Milojkovic et al51 5732 2000-2011 Prior TKI: chP (51), chP >1 2(59), AP (14), BC (10), no-TKI: NR Prior TKI (22), no TKI (78) NA Non-TKI 46% Prior TKI 42% (PFS at 5y) Non-TKI 61% Prior TKI 59% (OS at 5y) Retrospective multicenter study (EBMT) Piekarska et al39 25 2008-2013 chP1 (50), chP2/AP (29), BC (21) Dasatinib (53), nilotinib (18), or both (29) 7.1 (chP1), 12.5 (chP2/AP); 50 (BC) RI: 29.6% RI: chP1 21.4% RI: chP2/AP 12.5% RI: BP 50% chP1 92.9%, chP2/AP 85.7% BP 0% (at 1 or 3y) Prospective Lubking et al23 118 2002-2017 chP1 (47.5), chP >1 (40.7), AP/BC (11.9) Imatinib (39.8), imatinib +2G-TKI (33.1), imatinib +2GTKI +3G-TKI (5.1), 2G-TKI (13.6), 2G-TKI +3G-TKI (5.1), no TKI (3.4) 11.6 in chP ≥1 (at 5y) 23.1 in AP/BC (at 5y) chP: 66% molec relapse (at 2y) AP/BC: 71.4% progress to AP/BC 50% to AdP in chP >1 chP 96,3% 70.1% > chP1/AP 36.9% BP AP/BP to chP 70.1% chP TKI resist. 96.8% (allat 5y) Swedish registry study Hu et al40 1223 2001-2013 chP1 (60), chP2 (21), AP (12), BC (7), progression to AP/BC (19) TKI 1 (median, range 0–3) 10–20 (at 1y) NA chP 1 HSCT (vs non-HSCT inferior OS, HR 2.4) No difference chP2 and AP; BC trend favoring HSCT Life expectancy calculation in comparison to no HSCT (CIBMTR) Niederwieser et al27 147 1990-2018 Non-BC (75) BC (25) Prior TKI: 1G (27.2); 1G + 2G (13.6); 1G + 3G (0.7); 2G±3G (38.1); 3G (0.7) 28 (at 15y) 24 in BC (at 5y) 24 non-BC (at 5y) 30% at 10y and 26% at 15y BP 24% 5y Non-BP 31% 5y OS 15y 34% BP 30% 10y; 30% 5y non-BP 41% 10y; 44% 5y Bicentric retrospective study Long-term follow-up Masouridi-Levrat et al50 383 2009-2013 chP1 (38) AP > chP1 (45) BC (16) Prior TKI: dasatinib (40) or nilotinib (17) or sequential ± bosutinib/ ponatinib (43) 18 (at 1y) 24% (at 5y) No difference between 2G-TKIs RFS 40% (at 5y) RI 29% (at 2y) RI 36% (at 5y) 65% (at 2y) 56% (at 5y) chP1 67% AP/chP >1 57% BC 37% EBMT study, retrospective study CMR, complete molecular response; cord, cord blood; NA, not available; n.s., not significantly different; PCR, polymerase chain reaction; PFS, progression-free survival; RI, relapse incidence; sib, sibling; TFS, transformation-free survival. Dr Prakash Singh Shekhawat HSCT in CML | 119 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 Saussele et al24 How to manage transplant therapies detected 6 months after HSCT. Because of a high risk of relapse, ponatinib was restarted, and the BCR-ABL1 transcripts became negative in the blood. After consultation with his physician fol lowing continuous negative BCR-ABL1 tran scripts, the patient opted to terminate TKI treatment 5 years post transplant. CLINICAL CASE 2 (Continued) A matched unre lated donor was found, and after RIC with busulfan/fludarabine, HSCT was performed without complica tions. BCR-ABL1 transcripts remained undetectable post HSCT. No TKI prophylaxis was given, and the patient remained BCRABL1-negative at 5 years after HSCT. Summary Rapid developments in the treatment of CML require continual adaptation regarding indications for curative stem cell trans plantation. Patients with chP CML should be started with 1G- or 2G-TKIs and monitored according to ELN or NCCN guidelines. The emergence of high-risk ACAs predict a poorer response to TKIs and a higher risk of progression and are triggers for HSCT. Patients with resistant disease (NCCN) or failure (ELN) should be regarded as candidates for HSCT. Patients with resistance to G2TKIs should be considered for HSCT if they are not responding to G3 after 3 months. An HLA-identical related or fully matched unrelated donor should be selected, and the intensity of the conditioning regimen should be adjusted according to age and comorbidities. Patients in AP and BC CML should be started on IC with the possible addition of TKIs and are candidates for HSCT. Posttransplant monitoring with quantitative polymerase chain reaction testing is indicated to guide prophylactic or pre emptive TKI treatment with or without DLIs. Conflict-of-interest disclosure Chris tian Niederwieser: no com pet ing finan cial inter ests to declare. Nicolaus Kröger: research funding: Bristol Myers Squibb, Jazz, Neovii, Novartis, Riemser; honoraria: Bristol Myers Squibb, Gilead, Jazz, Neovii, Novartis, Riemser, Sanofi. Off-label drug use Christian Niederwieser: nothing to disclose. Nicolaus Kröger: nothing to disclose. Correspondence Nicolaus Kröger, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Martinistr 52, 20246 Hamburg, Germany; e-mail: nkroeger@uke.uni-hamburg.de. References 1. CLINICAL CASE 1 (Cont inu ed) The patient underwent a transplant, after conditioning with fludarabine and myeloablative busulfan, from an HLA-identical brother and, except for neutropenic fever, had an uneventful posttransplant period. During immunosuppression tapering, he developed mild chronic skin GVHD. However, BCR-ABL1 transcripts were still 120 | Hematology 2022 | ASH Education Program Nowell P, Hungerford D. A minute chromosome in human chronic granulo cytic leukemia. Blut. 1962;8(April):65-66. 2. Rowley JD. Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243(5405):290-293. 3. Groffen J, Stephenson J, Heisterkamp N, Deklein A, Bartram C, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984;36(1):93-99. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 In a CIBMTR study, the prognostic favorable factors for LFS and OS after HSCT in advanced CML were the time from diagnosis to transplantation, good performance status, and access to an HLA fully matched donor.57 In an EBMT study, risk factors for poorer survival were active BC at transplant, advanced age (>45 years), low performance status, a longer interval from diagnosis to transplant, MAC, and the use of an unrelated donor.33 Other studies reported that a low CD34+ cell count in the graft and a higher donor age were adverse risk factors for OS.27 Although no pro spec tive ran dom ized study exists, reduced intensity conditioning (RIC) seems to be a feasible approach that increases accessibility to the transplant procedure.58 Whether younger patients and patients without comorbidities should be transplanted after RIC instead of MAC is still a matter of debate. Similar OS but lower chronic GVHD rates were observed in RIC when compared to MAC.59 RIC may be a reasonable alternative to MAC in the TKI era, but results should be confirmed in a ran domized study. However, due to the high risk of relapse in AdP CML, patients who can tolerate MAC should receive it. Ex vivo T-cell depletion is associated with a high relapse risk, but it has been observed that using anti–T-lymphocyte globulin within the conditioning regimen reduces the incidence of GVHD without increasing the risk of relapse.60 The detec tion of BCR-ABL1 tran scripts early after HSCT has no adverse prognostic significance. However, BCR-ABL1 tran scripts (persistently negative, fluctuating, or persistently positive) 6 months post trans plant pre dict a risk of relapse.61 Several reports sug gest that early posttransplant TKIs (includ ing 2GTKIs) are safe to administer effectively in chP CML but are less effec tive in AdP CML.62,63 Maintenance therapy with TKIs with or without DLIs appears to be safe and has been associated with a lower inci dence of extensive chronic GVHD.27,61 Continued regular long-term monitoring of BCR-ABL1 transcripts is required to anticipate the occasional late relapsing patient; however, both the optimal fre quency of monitoring and the threshold of BCR-ABL1 transcripts for preemptive therapy with TKIs or DLIs need to be established in the contexts of conditioning regimen and graft manipulation. Although the use of posttransplant TKI therapy is widespread, prospective studies are needed to explore the best TKI dose, the treatment duration, and possible coadministration with DLIs. A relapse of CML can occur as late as the second decade after HSCT and involves molecular, cytogenetic, and hematological relapse.5 Relapsed CML has been treated with DLIs, TKIs, che motherapy, and second HSCTs.27,64-67 Combinations of TKI ther apy and DLIs can achieve a 5-year postrelapse survival rate of 62%, and some patients became TKI-free, suggesting a persis tent graft-versus-leukemia effect.67 Molecular remission without GVHD can be achieved, especially when DLIs were given beyond 1 year from HSCT for molecular or cytogenetic relapse.68 26. Deininger MW, Shah NP, Altman JK, et al. Chronic myeloid leukemia, version 2.2021, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2020;18(10):1385-1415. 27. Niederwieser C, Morozova E, Zubarovskaya L, et al. Risk factors for out come after allogeneic stem cell transplantation in patients with advanced phase CML. Bone Marrow Transplant. 2021;56(11):2834-2841. 28. Gratwohl A, Brand R, Apperley J, et al; Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Allogeneic hema topoietic stem cell transplantation for chronic myeloid leukemia in Europe 2006: transplant activity, long-term data and current results. An analysis by the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Haematologica. 2006;91(4):513-521. 29. Jiang Q , Xu LP, Liu DH, et al. Imatinib mesylate versus allogeneic hemato poietic stem cell transplantation for patients with chronic myelogenous leukemia in the accelerated phase. Blood. 2011;117(11):3032-3040. 30. How J, Venkataraman V, Soriano Hobbs, G. Blast and accelerated phase CML: room for improvement. ASH Educational Program. 2021;2021(1):122128. 31. Saxena K, Jabbour E, Issa G, et al. Impact of frontline treatment approach on outcomes of myeloid blast phase CML. J Hematol Oncol. 2021;14(1): 94. 32. Oehler VG, Gooley T, Snyder DS, et al. The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia. Blood. 2007;109(4):1782-1789. 33. Radujkovic A, Dietrich S, Blok HJ, et al. Allogeneic stem cell transplantation for blast crisis chronic myeloid leukemia in the era of tyrosine kinase inhib itors: a retrospective study by the EBMT Chronic Malignancies Working Party. Biol Blood Marrow Transplant. 2019;25(10):2008-2016. 34. Chaudhury S, Sparapani R, Hu ZH, et al. Outcomes of allogeneic hemato poietic cell transplantation in children and young adults with chronic mye loid leukemia: a CIBMTR cohort analysis. Biol Blood Marrow Transplant. 2016;22(6):1056-1064. 35. Yassine F, Reljic T, Moustafa MA, et al. Efficacy of allogeneic hematopoietic cell transplantation in patients with chronic phase CML resistant or intoler ant to tyrosine kinase inhibitors [published online ahead of print 11 March 2022]. Hematol Oncol Stem Cell Ther. 36. Zheng C, Tang B, Yao W, et al. Comparison of unrelated cord blood trans plantation and HLA-matched sibling hematopoietic stem cell transplanta tion for patients with chronic myeloid leukemia in advanced stage. Biol Blood Marrow Transplant. 2013;19(12):1708-1712. 37. Topcuoglu P, Arat M, Ozcan M, et al. Case-matched comparison with stan dard versus reduced intensity conditioning regimen in chronic myeloid leukemia patients. Ann Hematol. 2012;91(4):577-586. 38. Oyekunle A, Zander AR, Binder M, et al. Outcome of allogeneic SCT in patients with chronic myeloid leukemia in the era of tyrosine kinase inhibi tor therapy. Ann Hematol. 2013;92(4):487-496. 39. Piekarska A, Gil L, Prejzner W, et al. Pretransplantation use of the secondgeneration tyrosine kinase inhibitors has no negative impact on the HCT outcome. Ann Hematol. 2015;94(11):1891-1897. 40. Hu B, Lin X, Lee HC, et al. Timing of allogeneic hematopoietic cell trans plantation (alloHCT) for chronic myeloid leukemia (CML) patients. Leuk Lymphoma. 2020;61(12):2811-2820. 41. Yang S, Zhang XS, Gale RP, et al. Co-variates associated with outcomes of tyrosine kinase-inhibitor therapy in persons with chronic myeloid leukae mia ini tially presenting in accel er ated phase. Leukemia. 2022;36(7):18181824. 42. Hehlmann R. The new ELN recommendations for treating CML. J Clin Med. 2020;9(11):3671. 43. McKenney AS, Levine RL. Isocitrate dehydrogenase mutations in leukemia. J Clin Invest. 2013;123(9):3672-3677. 44. Adnan-Awad S, Kankainen M, Mustjoki S. Mutational landscape of chronic myeloid leukemia: more than a single oncogene leukemia. Leuk Lymphoma. 2021;62(9):2064-2078. 45. Sorror ML, Maris MB, Storer B, et al. Comparing morbidity and mortality of HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative and myeloablative conditioning: influence of pretransplantation comorbidities. Blood. 2004;104(4):961-968. 46. Mielcarek M, Furlong T, O’Donnell PV, et al. Posttransplantation cyclophos phamide for prevention of graft-versus-host disease after HLA-matched mobilized blood cell transplantation. Blood. 2016;127(11):1502-1508. 47. Chen H, Si Y, He X, et al. HLA-mismatched hema to poi etic stem cell transplantation for treatment of chronic myelogenous leukemia. Blood. 2008;112(11):1113-1113. Dr Prakash Singh Shekhawat HSCT in CML | 121 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 4. Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature. 1985;315(6020):550-554. 5. Goldman JM, Majhail NS, Klein JP, et al. Relapse and late mor tal ity in 5-year survivors of myeloablative allogeneic hematopoietic cell transplan tation for chronic myeloid leukemia in first chronic phase. J Clin Oncol. 2010;28(11):1888-1895. 6. Talpaz M, Kantarjian HM, McCredie K, Trujillo JM, Keating MJ, Gutterman JU. Hematologic remission and cytogenetic improvement induced by recom binant human interferon alpha A in chronic myelogenous leukemia. N Engl J Med. 1986;314(17):1065-1069. 7. Lee MS, Chang KS, Freireich EJ, et al. Detection of minimal residual bcr/abl transcripts by a modified polymerase chain reaction. Blood. 1988;72(3):893897. 8. Kolb HJ, Mittermüller J, Clemm C, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow trans plant patients. Blood. 1990;76(12):2462-2465. 9. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344(14):1031-1037. 10. Mahon FX, Réa D, Guilhot J, et al; Intergroupe Français des Leucémies Myéloïdes Chroniques. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11(11):1029-1035. 11. Niederwieser D, Baldomero H, Bazuaye N, et al. One and a half million hematopoietic stem cell transplants: continuous and differential improve ment in worldwide access with the use of non-identical family donors. Haematologica. 2022;107(5):1045-1053. 12. Gratwohl A, Hermans J, Goldman JM, et al. Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow trans plantation. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Lancet. 1998;352(9134):1087-1092. 13. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitu tion in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med. 1989;321(17):1174-1178. 14. Luznik L, O’Donnell PV, Fuchs EJ. Post-transplantation cyclophosphamide for tolerance induction in HLA-haploidentical bone marrow transplanta tion. Semin Oncol. 2012;39(6):683-693. 15. Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stem cell trans plantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood. 1998;91(3):756-763. 16. Hochhaus A, Larson RA, Guilhot F, et al; IRIS Investigators. Long-term out comes of imatinib treatment for chronic myeloid leukemia. N Engl J Med. 2017;376(10):917-927. 17. Mahon FX, Delbrel X, Cony-Makhoul P, et al. Follow-up of complete cytoge netic remission in patients with chronic myeloid leukemia after cessation of interferon alfa. J Clin Oncol. 2002;20(1):214-220. 18. Kohlbrenner K, Fabarius A, Reiser M, et al. Therapy in patients with chronic myeloid leukemia outside of clinical trials: results of the German CMLregistry (CML-VI). Paper presented at: European Society of Hypertension 31st European Meeting; 17-20 June 2022; Athens, Greece. 19. Réa D, Mauro MJ, Boquimpani C, et al. A phase 3, open-label, randomized study of asciminib, a STAMP inhibitor, vs bosutinib in CML after 2 or more prior TKIs. Blood. 2021;138(21):2031-2041. 20. Pasvolsky O, Leader A, Iakobishvili Z, Wasserstrum Y, Kornowski R, Raanani P. Tyrosine kinase inhibitor associated vascular toxicity in chronic myeloid leukemia. Cardiooncology. 2015;1(1):5. 21. Brixey AG, Light RW. Pleural effusions due to dasatinib. Curr Opin Pulm Med. 2010;16(4):351-356. 22. Cortes JE, Saglio G, Kantarjian HM, et al. Final 5-year study results of DASISION: the Dasatinib ver sus Imatinib Study in Treatment-Naïve Chronic Myeloid Leukemia Patients trial. J Clin Oncol. 2016;34(20):2333-2340. 23. Lübking A, Dreimane A, Sandin F, et al. Allogeneic stem cell transplantation for chronic myeloid leukemia in the TKI era: population-based data from the Swedish CML registry. Bone Marrow Transplant. 2019;54(11):1764-1774. 24. Saussele S, Lauseker M, Gratwohl A, et al; German CML Study Group. Allogeneic hematopoietic stem cell transplantation (allo SCT) for chronic mye loid leukemia in the imatinib era: evaluation of its impact within a subgroup of the randomized German CML Study IV. Blood. 2010;115(10):1880-1885. 25. Hochhaus A, Baccarani M, Silver RT, et al. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 2020;34(4):966-984. 122 | Hematology 2022 | ASH Education Program 60. Zander AR, Kröger N, Schleuning M, et al. ATG as part of the conditioning regimen reduces transplant-related mortality (TRM) and improves overall survival after unrelated stem cell transplantation in patients with chronic myelogenous leukemia (CML). Bone Marrow Transplant. 2003;32(4):355361. 61. Kaeda J, O’Shea D, Szydlo RM, et al. Serial measurement of BCR-ABL tran scripts in the peripheral blood after allogeneic stem cell transplantation for chronic myeloid leukemia: an attempt to define patients who may not require further therapy. Blood. 2006;107(10):4171-4176. 62. Carpenter PA, Snyder DS, Flowers ME, et al. Prophylactic administration of imatinib after hematopoietic cell transplantation for high-risk Philadelphia chromosome-positive leukemia. Blood. 2007;109(7):2791-2793. 63. Nakasone H, Kanda Y, Takasaki H, et al; Kanto Study Group for Cell Therapy. Prophylactic impact of imatinib administration after allogeneic stem cell transplantation on the incidence and severity of chronic graft versus host disease in patients with Philadelphia chromosome-positive leukemia. Leukemia. 2010;24(6):1236-1239. 64. Goldman JM, Melo JV. Chronic myeloid leukemia—advances in biology and new approaches to treatment. N Engl J Med. 2003;349(15):1451-1464. 65. Kim YJ, Kim DW, Lee S, et al. Cytogenetic clonal evo lu tion alone in CML relapse post-transplantation does not adversely affect response to imatinib mesylate treat ment. Bone Marrow Transplant. 2004;33(2):237242. 66. DeAngelo DJ, Hochberg EP, Alyea EP, et al. Extended follow-up of patients treated with imatinib mesylate (Gleevec) for chronic myelogenous leuke mia relapse after allogeneic transplantation: durable cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease. Clin Cancer Res. 2004;10(15):5065-5071. 67. Savani BN, Montero A, Kurlander R, Childs R, Hensel N, Barrett AJ. Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant. 2005;36(11):1009-1015. 68. Radujkovic A, Guglielmi C, Bergantini S, et al; Chronic Malignancies Working Party of the Euro pean Society for Blood and Marrow Transplantation. Donor lymphocyte infusions for chronic myeloid leukemia relapsing after allogeneic stem cell transplantation: may we predict graft-versusleukemia without graft-versus-host disease? Biol Blood Marrow Transplant. 2015;21(7):1230-1236. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000329 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/114/2021884/114niederwieser.pdf by guest on 09 December 2022 48. Xiao-Jun H, Lan-Ping X, Kai-Yan L, et al. HLA-mismatched/haploidentical hematopoietic stem cell transplantation without in vitro T cell depletion for chronic myeloid leukemia: improved outcomes in patients in accelerated phase and blast crisis phase. Ann Med. 2008;40(6):444-455. 49. Sanz J, Sanz GF. Umbilical cord blood trans plan ta tion from unre lated donors in adult patients with chronic myeloid leukaemia. Best Pract Res Clin Haematol. 2010;23(2):217-222. 50. Masouridi-Levrat S, Olavarria E, Iacobelli S, et al. Outcomes and toxicity of allogeneic hematopoietic cell transplantation in chronic myeloid leukemia patients previously treated with second-generation tyrosine kinase inhibi tors: a prospective non-interventional study from the Chronic Malignancy Working Party of the EBMT. Bone Marrow Transplant. 2022;57(1):23-30. 51. Milojkovic D, Apperley JF, Gerrard G, et al. Responses to second-line tyro sine kinase inhibitors are durable: an intention-to-treat analysis in chronic myeloid leukemia patients. Blood. 2012;119(8):1838-1843. 52. Baccarani M, Castagnetti F, Gugliotta G, Rosti G. A review of the European LeukemiaNet recommendations for the management of CML. Ann Hematol. 2015;94(suppl 2):S141-S147. 53. Lee SJ, Kukreja M, Wang T, et al. Impact of prior imatinib mesylate on the outcome of hematopoietic cell transplantation for chronic myeloid leuke mia. Blood. 2008;112(8):3500-3507. 54. Hafez HA, Abdallah A, Hammad M, et al. Outcomes of allogenic hemato poietic cell transplantation for childhood chronic myeloid leukemia: singlecenter experience. Pediatr Transplant. 2020;24(4):e13664. 55. Jabbour E, Cortes J, Santos FP, et al. Results of allogeneic hematopoietic stem cell transplantation for chronic myelogenous leukemia patients who failed tyrosine kinase inhibitors after developing BCR-ABL1 kinase domain mutations. Blood. 2011;117(13):3641-3647. 56. Nicolini FE, Basak GW, Kim DW, et al. Overall sur vival with ponatinib versus allogeneic stem cell transplantation in Philadelphia chromosomepositive leukemias with the T315I mutation. Cancer. 2017;123(15):28752880. 57. Khoury HJ, Kukreja M, Goldman JM, et al. Prognostic factors for outcomes in allogeneic transplantation for CML in the imatinib era: a CIBMTR analysis. Bone Marrow Transplant. 2012;47(6):810-816. 58. Crawley C, Szydlo R, Lalancette M, et al; Chronic Leukemia Working Party of the EBMT. Outcomes of reduced-intensity transplantation for chronic myeloid leukemia: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood. 2005;106(9):2969-2976. 59. Chhabra S, Ahn KW, Hu ZH, et al. Myeloablative vs reduced-intensity condi tioning allogeneic hematopoietic cell transplantation for chronic myeloid leukemia. Blood Adv. 2018;2(21):2922-2936. BEYOND ROUTINE FRONTLINE THERAPY OF CML Treatment of CML in pregnancy Center for Hematology, Imperial College London, London, UK; and Department of Clinical Hematology, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, UK Since the introduction of tyrosine kinase inhibitors (TKIs) at the beginning of the millennium, the outlook for patients with chronic myeloid leukemia (CML) has improved remarkably. As such, the question of life expectancy and survival has become less problematic while quality of life and family planning have become more so. While TKIs are the cornerstone of CML management, their teratogenicity renders them contraindicated during pregnancy. In recent years, patients who satisfy standardized criteria can stop TKI therapy altogether, and indeed, in eligible patients who wish to become pregnant, these objectives overlap. However, not all patients satisfy these criteria. Some pregnancies are unplanned, and a number of patients are pregnant when diagnosed with CML. In these patients the way forward is less clear, and there remains a paucity of good evidence available to guide treatment. In this article, we summarize the relevant literature and provide a framework for clinicians faced with the challenge of managing CML and pregnancy. LEARNING OBJECTIVES • Manage CML diagnosed in pregnancy • Manage established CML in unplanned pregnancies • Facilitate planned childbirth in established CML CLINICAL CASE A 35-year-old woman was diagnosed with chronic myeloid leukemia (CML) in chronic phase in 2014. There were no additional chromosomal abnormalities, and the European Treatment and Outcome Study (EUTOS) long-term survival score was intermediate. She was started on imatinib and achieved complete cytogenetic remission (CCyR) at 12 months but was not in major molecular remission (MMR) at 2 years. She has no children but indicates that having a child is very important to her. Introduction Targeted tyrosine kinase inhibitors (TKIs) have revolutionized the course of CML. An individual diagnosed with CML, previously a fatal disease, now has an excellent chance of a normal life expectancy.1 Furthermore, for many patients the aim is to achieve deep and sustained molecular responses so that TKI therapy can be discontinued indefinitely. In Western countries the median age at diagnosis is 57 to 60 years, so many patients are women of childbearing age.2 This is even more relevant in emerging nations, where the median age at diagnosis is 30 to 40 years.3 Before the advent of TKIs, pregnancy and CML were largely incompatible, but the ability to induce profound responses and an increasing prevalence in young patients mean that family planning is an increasingly important discussion topic. There are now 6 TKIs in clinical practice and welldefined sets of hematological, cytogenetic, and molecular landmarks to guide management.4 All produce excellent responses in the majority, but their teratogenicity hinders their use in pregnancy. This therefore poses a therapeutic and ethical dilemma for the clinician and patient, and decisions must be made with the disease, mother, and fetus in mind. Poorly controlled CML in pregnancy can lead to adverse outcomes for both parties. Persistently raised white blood cell (WBC) counts can result in leukastasis, which may subsequently result in placental insufficiency, intrauterine growth retardation, or fetal mortality.5 Thus, 2 equally important considerations must be balanced: (1) adequately controlling the disease to avoid progression, and (2) minimizing exposure of the fetus to potentially harmful systemic medications. Despite increasing attention over the last decade, standardized guidance for the management of CML in pregnancy is lacking. Appropriately stratifying patients by Dr Prakash Singh Shekhawat Treatment of CML in pregnancy | 123 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 Harry F. Robertson and Jane F. Apperley disease status at diagnosis and the risk of relapse/progression is helpful when approaching management. They can be generally defined by the following: a) CML diagnosed during pregnancy b) Established CML when the patient is a candidate for treatmentfree remission (TFR) c) Established CML when the patient is not a candidate for TFR CML diagnosed during pregnancy Table 1. Summary of studies describing the management of CML diagnosed in pregnancy Treatment during pregnancy (n, %) Outcome (n, %) 15 HC (5, 33) IFN (3, 20) Leukapheresis (1, 7) Nilotinib (1, 7) Observation (5, 33) Live birth (12, 80) Spontaneous termination (2, 13) Elective termination (1, 7) Imatinib (4, 27) Dasatinib (6, 40) Nilotinib (5, 33) 14 (93) Blast crisis and death (1, 7) 48 Imatinib (10, 30) IFN only (2, 6) IFN + imatinib (3, 10) HC (4, 12) Observation (14, 42)a Live birth (33, 71) Spontaneous termination (1, 2) Elective termination (14, 29) Imatinib (30, 94) Nilotinib (1, 2) Dasatinib (1, 2)a,b 17 (52)a Blast crisis and death (2, 6)a Study N Assi et al9 Chelysheva et al10 TKI after pregnancy end (n, %) Denominator derived from the number of patients who continued pregnancy to term (n=33). One patient had a set of twins, analyzed as 2 pregnancies. a b 124 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat ≥MMR, n (%) Adverse disease outcomes (n, %) Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 A diag no sis of CML dur ing preg nancy is not an uncom mon occurrence; approximately 10% of pregnancy-related leukemias are CML, and 20% of pregnancy-related CML is in patients diag nosed during pregnancy.6,7 Unsurprisingly, patients often meet the delivery of such grave news at this time with profound shock and anxiety surrounding their own health and that of the unborn child. For the clinician, it is important to be empathic, sensitive, and to draw upon the multifaceted skill set of the surrounding health care team, including but not limited to obstetricians, spe cialist nurses, psychologists, and therapists. At diag no sis there are 3 three main aspects to con sider, which guide treatment decisions: disease phase, disease burden and time to delivery. If the patient is presenting in blast crisis or chronic phase with multiple high-risk features, serious consider ation should be given to termination of the pregnancy in order to commence treatment. Blast crisis is now increasingly rare, but the outlook remains bleak, with median survivals around 12 months.8 Fortunately, the vast majority present in the chronic phase. Upon first consultation, a full outline of the treatment options, the associated risks, and an expected pregnancy road map should be discussed with the patient. In addition to isolated case reports, 2 studies provided data on multiple patients presenting with CML in pregnancy (Table 1). Assi et al described 15 patients, the majority of whom achieved at least MMR following the start of TKIs.9 One patient who was man aged with interferon alfa (IFN-α) during pregnancy was resistant to dasatinib, progressed to blast crisis, and died. Chelysheva et al presented 48 patients, of which 33 went to term.10 Imatinib was given in 13 cases at a median of 18 weeks’ gestation (range, 16-35). No congenital abnormalities occurred, but the mothers of 6 of 7 infants born with low birth weights had received imatinib. Two patients demonstrated resistance to multiple TKIs and died secondary to blast crisis. At our center we managed 11 cases of CML diagnosed during pregnancy (Table 2). All patients were alive at last follow-up. Many patients diagnosed in pregnancy present with man ageable cell counts and low-risk disease such that a “watch and wait” strategy is a completely reasonable approach. For patients with rapidly rising WBC counts and/or troublesome symptoms, we discuss below the various options for treatment. Pregnancy itself does not alter the disease course of CML, with the caveat that the true effect of treatment delay on long-term disease con trol is uncertain.11 TKIs should be avoided in pregnancy, particularly during the first trimester, when the majority of organogenesis occurs, and the fetus is most sensitive to maternal systemic medications.12 In both clin i cal and pre clin i cal stud ies, the most com monly observed abnor mal i ties in fetuses exposed to TKIs are bony skull defects and exomphalos.13 Significant “off-tar get” inhi bi tion, most probably of platelet-derived growth factor receptor alpha, is likely responsible for these malformations.14 Pye et al reviewed the largest cohort to date of 125 patients treated with imatinib during pregnancy, with 103 exposed in the first trimes ter. Fifty percent of the pregnancies resulted in live births, and 12 fetal malformations were observed. Moreover, the sim i lar ity of rare malformations in 3 infants suggests that they were drug induced.13 These findings were corroborated by Abruzzese et al, who concluded that although the majority of pregnancies exposed to imatinib result in normal live births, there is a sig nificant risk of fetal abnormality.15 Dasatinib exhibits the highest incidence of fetal abnormalities and should not be used at any point during pregnancy.16 Nilotinib should not be used in the first trimester, and the sparsity of evidence for bosutinib currently supports avoidance throughout pregnancy.17,18 There have been no published reports on the effect of the newest-generation TKIs, ponatinib and asciminib, on human pregnancy outcomes; however; both demonstrated embryo-fetal toxicity in preclinical studies (Ariad and Novartis investigator brochures, respectively). While there is no doubt that TKIs should be avoided in the first trimester, there is growing evidence of their safety when used in later pregnancy. The pregnancy registry of the European LeukemiaNet described 41 women who started on a TKI late in gestation (imatinib=33, nilotinib=8), and while a small number had malformations, these were deemed unrelated to the TKI by Table 2. Summary of patients diagnosed with CML during pregnancy at Hammersmith Hospital, London Characteristics (N=11) Median (range); N [%] Age at diagnosis (years) 33 (25-40) Trimester 1 6 [55] 2 5 [45] CML phase Chronic 11 [100] 80 (14.7-215.4) Sokal score Low 6 [55] Intermediate 2 [18] No data 3 [27] Pregnancy outcome Live birth 10 [91] Miscarriage 1 [9] Management in pregnancy Observation 3 [27] IFNa 2 [18] Leukapheresis 3 [27] IFN + leukapheresis 3 [27] TKI started after pregnancy end Imatinib 8 (73) Dasatinib 3 (27) Duration of TKI delay (months) 7 (1-10) ≥MR1 at 3 mo 11 [100] ≥CCyR at 6 mo 9 [82] ≥MMR at 12 mo 8 [73] One patient was switched from IFN-α to PEG-IFN-α due to an adverse reaction to the drug. a the treating physicians.19 Abruzzese and colleagues confirmed these find ings and reported preg nan cies in 17 patients, 3 of whom started a TKI during pregnancy (imatinib=1, nilotinib=2). All 3 pregnancies resulted in live healthy infants.20 Therefore, when necessary in those with rapidly increasing BCR-ABL1 tran scripts, we recommend consideration of TKIs in later pregnancy (second or third trimester). Patients should be aware of the man ufacturer’s advice to avoid TKIs during pregnancy as well as the potential risks to starting or delaying TKI therapy. A cru cial fac tor in avoiding com pli ca tions is the man age ment of thrombocytosis and leukocytosis. Those with significant thrombocytosis should start low-dose aspirin (75-100mg) and low-molecular-weight heparin, both of which are considered safe in pregnancy, to reduce the risk of thrombosis.21,22 While there is no evidence of a threshold for starting antithrombotic drugs, recent expert recommendations suggest initiation when the platelets exceed 600×109/L.7 Clearly, the benefits of avoiding thrombosis must be carefully balanced against the risk of Established CML: patient is a candidate for TFR Regardless of pregnancy, a number of patients are eligible for trials of treatment discontinuation. Indeed, the numerous tri als of treatment discontinuation in nonpregnant patients can guide management of the pregnant population. Stopping TKIs offers myriad benefits, including a resolution of side effects and a reduction in health care costs and the risk of fetal abnormalities in those attempting pregnancy. Guidelines indicate that patients in the chronic phase who have been on first- or second-line (intolerance only) TKIs for over 5 years and have sustained deep Dr Prakash Singh Shekhawat Treatment of CML in pregnancy | 125 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 WBC (×109/L) pregnancy-related bleeding. There is little evidence to suggest the best time to stop treatment prior to labor, and the decision is ultimately led by the obstetrician based on the expected mode of delivery and associated procedures. There are a number of reports of safe hydroxycarbamide (HC) use during pregnancy, albeit mostly in myeloproliferative neo plasms.23,24 The National Toxicology Program gathered informa tion from 33 patients exposed to HC during pregnancy. They reported major malformations in 8% of those exposed to HC in the first trimester and 14% in the second and/or third trimester. Two stillbirths were noted.25 In the recent expert recommenda tions, HC was not recommended for use during pregnancy.7 Leukapheresis is effective in controlling high WBC counts and can be used safely in pregnancy.26,27 It is, however, not without drawbacks, including availability, cost, and potential problems with repeated intra ve nous access with risks of infec tion and bleeding. Our personal practice is to commence leukapheresis when the WBC count is higher than 100×109/L. IFN-α is the only therapeutic option that exhibits anticancer properties and is safe to use throughout pregnancy.28,29 Polyethylene glycol (PEG)–IFN-α is more commonly used now due to its faster and lon ger-last ing treat ment response. Initially, PEG–IFN-α was contraindicated in pregnancy due to concerns around the preservative. Multiple case reports from patients with myeloproliferative neoplasms and viral hepatitis and expe rience from our own center have not demonstrated any harmful effects.30 The ability of IFN-α to produce meaningful disease control over the remaining period of gestation is variable, and it is associated with unpleasant side effects such as pyrexia and myalgia.31 TKI therapy should begin as soon as possible after pregnancy has concluded. Imatinib and nilotinib are present in breast milk, but the effects of these low drug concentrations on a develop ing infant are unknown.32-36 There is little information on whether the other TKIs are present in breast milk, but it is safer to avoid their use when breastfeeding. Many women wish to breastfeed, but the decision to delay effective therapy in order to facilitate this must be on a case-by-case basis after a thorough discussion of the risks involved. It is clear that a “one-size-fits-all” approach is not possible, and treatment should be tailored based on the patient’s per sonal, cultural, and religious wishes. A multidisciplinary approach is paramount. A treatment-free first trimester is the objective, with management in the second and third trimesters remaining largely the same, with reassurance that any treatment admin istered has a lower risk of causing fetal abnormalities. TKI ther apy should be avoided during breastfeeding, but if the mother is insistent, then it should be limited to 2 to 3 weeks to minimize the risk of disease progression. Established CML: patient is not a candidate for TFR This is the majority of women who represent a spectrum of sce narios ranging from planned pregnancies in those with minimal TKI exposure and a lack of adequate response, to those on longterm treatment who have sustained MMR without satisfying TFR criteria, to those who conceive while on TKI. The evidence for TKI discontinuation in the non-TFR population is growing and shows that conception and pregnancy can be safely navigated. In order to appropriately stratify these patients, the duration of TKI exposure and the molecular response are important consid erations. Women who have received TKI therapy for less than 3 years or who have failed to achieve CCyR or MMR even after 3 years of treatment, such as our patient, have a higher risk of loss of CHR upon TKI discontinuation. The prime consideration in these patients should be the leu ke mic bur den. The most straight forward management is to delay pregnancy and continue TKI therapy in the pursuit of deeper responses. If the patient is on imatinib, this could be switched to a more potent TKI. If this is not feasible due to patient preference, then once pregnancy is confirmed the TKI must be stopped and the patient managed similarly to women diagnosed in pregnancy. A third option is assisted conception, which may be partic u larly rel e vant in older women concerned about age-related fertility loss. Depending on personal circumstances and the con sent of their partner, these women can be considered for ovar ian hyperstimulation, embryo creation, and subsequent embryo implantation in order to reduce the time off TKIs. Women without partners can be offered oocyte or ovarian cryopreservation. Strategies such as TKI holidays, in which highly motivated patients with reg u lar cycles discontinue their TKI after men stru a tion, perform a pregnancy test 5 days post the expected ovulation, and resume their TKI if negative, are theoretically possible but carry a risk of dis ease pro gres sion and can not be rou tinely recommended. For women in MMR but not deep molecular remission, pre vious guidelines recommended that those who had sustained MMR for more than 12 months could safely discontinue their TKI and subsequently regain the same disease control after restarting.7 Table 3 summarizes the studies reporting disease control following TKI discontinuation for pregnancy. These all demon strated that while a significant proportion of patients lose MMR upon TKI discontinuation, their original response can be regained in the majority following TKI resumption.40-43 Furthermore, Lee et al demonstrated, importantly, that almost half of those who lost MMR retained CCyR and could be managed without inter vention throughout pregnancy.44 Table 3. Summary of recent studies reporting disease outcomes in patients who discontinue TKI while in MMR or deeper N (patients, pregnancies) TKI therapy prior to stop, n (%) Time on TKI in months, median (range) ≥MMR at time of stopping TKI, n (%) Loss of MMR, n (%) ≥MMR recovery after TKI restart in those who lost MMR, n (%) 28, 38 ND ND 29 (76) 20 (69) 18 (90)a Chelysheva et al 87, 87 ND ND 87 (100) 6 mo—57% 12 mo—66% 6 mo—50% 12 mo—75% Lee et al44 39, 50 Imatinib 24 (48) Dasatinib 14 (28) Nilotinib 10 (20) Bosutinib 1 (2) Radotinib 1 (2) 77 (6-194) 44 (88) 20 (45.5) ND Lasica et al42 16, 27 Imatinib 13 (81) Dasatinib 2 (13) Nilotinib 1 (6) 32 (3-84) 12 (75) 11 (92) 12 (100) Dou et al43 17, 17 Imatinib 13 (76) Nilotinib 4 (24) 49 (6-102) 17 (100) 10 (59) 10 (100) Study Caldwell et al40 41 The remaining 2 patients were too early for evaluation. ND, no data. a 126 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 molecular remission for more than 2 years are eligible for treat ment discontinuation.4,37 Approximately 50% subsequently lose their MMR, requiring TKI resumption, 80% of which occurs within 4 to 6 months.38,39 Women who wish to become pregnant who are TFR eligible are the most straightforward because they can be managed similarly to their nonpregnant counterparts.4 Assi et al reported planned pregnancies in 7 patients, 6 of whom had a sustained MR4.5 for at least 18 months.9 Five of these patients sustained at least an MMR throughout pregnancy. The 1 remaining patient rapidly lost the response after stopping dasatinib prior to conception but, reassuringly, regained MR4 6 months after restarting. TKIs can be discontinued and the patient mon i tored for increases in transcript levels at 4-to-6-week intervals: a patient remaining in MMR can stay off treatment indefinitely, regardless of pregnancy. In the event of MMR loss, management depends on whether the woman is pregnant. If she is not, then she should resume her original or a more potent TKI and reestablish a deep response, with the possibility of attempting pregnancy in the future. This of course depends on indi vid ual cir cum stances, including, importantly, her age. If this is a consideration, then various possibilities can be considered, including referral for oocyte, ovarian, or embryo storage. If she is pregnant, then man agement depends on the level of tumor burden. Many women reach the end of their pregnancy without losing CCyR or com plete hematological remission (CHR) and do not require treat ment. If intervention is necessary, then the choices are identical to those for women presenting in pregnancy. In any of these scenarios, once conception is confirmed, TKI therapy should be discontinued immediately. There is no need for increased imaging surveillance if no abnormalities are noted at the first routine ultrasound. Those in CCyR should have tran script levels evaluated every 4 to 6 weeks, whereas those with less deep responses should be monitored as appropriate for their individual level of disease control. Management is similar to that of patients diagnosed in pregnancy. A loss of disease control can be managed in a manner similar to those described previously: utilization of IFN-α, therapeutic leukapheresis, and the consideration of TKI use in later preg nancy. Pregnancy and CML can now be successfully managed in unison with minimal risk to both mother and child. A significant pro portion of patients can be managed by observation only, and disease control can be achieved after starting or restarting TKI therapy. If active treatment is required, IFN-α can be used in any trimester with minimal risk to the fetus. More recently, the safe administration of TKIs in the later stages of pregnancy was reported. Most importantly, a multidisciplinary approach should be employed to provide comprehensible and succinct advice to empower both mother and family in the shared decision-making process. Acknowledgment Jane F. Apperley and Harry F. Robertson acknowl edge the sup port of the National Institute of Health Research Imperial Biomedical Research Center. Conflict-of-interest disclosure Harry F. Robertson: no competing financial interests to declare. Jane F. Apperley: advisory board member: Incyte, Novartis; research funding: Incyte, Pfizer; speakers bureau: Incyte, Novartis. Off-label drug use Harry F. Robertson: nothing to disclose. Jane F. Apperley: nothing to disclose. Correspondence Jane F. Apperley, Center for Hematology, Imperial College London, Hammersmith Hospital, Du Cane Rd, London W12 0NN, United Kingdom; e-mail: j.apperley@imperial.ac.uk. References 1. 2. 3. 4. 5. 6. Hehlmann R, Lauseker M, Saußele S, et al. Assessment of imatinib as firstline treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants. Leukemia. 2017;31(11):2398-2406. Höglund M, Sandin F, Simonsson B. Epidemiology of chronic myeloid leu kaemia: an update. Ann Hematol. 2015;94(suppl 2):S241-S247. Malhotra P, Varma S. Chronic mye loid leu kae mia in India. Lancet. 2007;370(9593):1127. Hochhaus A, Baccarani M, Silver RT, et al. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 2020;34(4):966-984. Rohilla M, Rai R, Yanamandra U, et al. Obstetric complications and man agement in chronic myeloid leukemia. Indian J Hematol Blood Transfus. 2016;32(1):62-66. Lichtman M. Acute Myelogenous Leukemia. Williams Hematology. 6th ed. New York: McGraw Hill; 2001:1047. Abruzzese E, Mauro M, Apperley J, Chelysheva E. Tyrosine kinase inhibitors and pregnancy in chronic myeloid leukemia: opinion, evidence, and rec ommendations. Ther Adv Hematol. 2020;11(October):2040620720966120. 8. Jain P, Kantarjian HM, Ghorab A, et al. Prognostic fac tors and sur vival out comes in patients with chronic mye loid leu ke mia in blast phase in the tyrosine kinase inhibitor era: cohort study of 477 patients. Cancer. 2017;123(22):4391-4402. 9. Assi R, Kantarjian H, Keating M, et al. Management of chronic mye loid leukemia during pregnancy among patients treated with a tyrosine kinase inhibitor: a single-center experience. Leuk Lymphoma. 2021;62(4): 909-917. 10. Chelysheva E, Abruzzese E, Rea D, et al. Chronic myeloid leukemia diag nosed dur ing preg nancy: ther apy, out comes and fol low-up. Blood. 2018;132(suppl 1):4255. 11. Lishner M, Avivi I, Apperley JF, et al. Hematologic malignancies in preg nancy: management guidelines from an international consensus meeting. J Clin Oncol. 2016;34(5):501-508. 12. Vandenbroucke T, Verheecke M, Fumagalli M, Lok C, Amant F. Effects of cancer treatment during pregnancy on fetal and child development. Lancet Child Adolesc Health. 2017;1(4):302-310. 13. Pye SM, Cortes J, Ault P, et al. The effects of imatinib on pregnancy out come. Blood. 2008;111(12):5505-5508. 14. Ishida Y, Murai K, Yamaguchi K, et al; Inter-Michinoku Dasatinib Study Group. Pharmacokinetics and phar ma co dy nam ics of dasatinib in the chronic phase of newly diagnosed chronic myeloid leukemia. Eur J Clin Pharmacol. 2016;72(2):185-193. 15. Abruzzese E, Trawinska MM, Perrotti AP, De Fabritiis P. Tyrosine kinase inhib itors and pregnancy. Mediterr J Hematol Infect Dis. 2014;6(1):e2014028. 16. Cortes JE, Abruzzese E, Chelysheva E, Guha M, Wallis N, Apperley JF. The impact of dasatinib on preg nancy out comes. Am J Hematol. 2015;90(12):1111-1115. 17. Barkoulas T, Hall PD. Experience with dasatinib and nilotinib use in preg nancy. J Oncol Pharm Pract. 2018;24(2):121-128. 18. Cortes JE, Gambacorti-Passerini C, Deininger M, Abruzzese E, DeAnnuntis L, Brümmendorf TH. Pregnancy outcomes in patients treated with bosutinib. Int J Hematol Oncol. 2020;9(2):IJH26. 19. Chelysheva E, Turkina A, Rea D, et al. Pregnancy out come in female patients with chronic myeloid leukemia worldwide: analysis of 305 cases of the European LeukemiaNet registry: S881. HemaSphere. 2019;3(suppl 1): 395-396. 20. Abruzzese E, Aureli S, Bondanini F, et al. Chronic myeloid leukemia and pregnancy: when dreams meet reality. State of the art, management and outcome of 41 cases, nilotinib placental transfer. J Clin Med. 2022;11(7): 1801. 21. James AH, Brancazio LR, Price T. Aspirin and repro duc tive out comes. Obstet Gynecol Surv. 2008;63(1):49-57. 22. Deruelle P, Coulon C. The use of low-molec u lar-weight hep a rins in pregnancy—how safe are they? Curr Opin Obstet Gynecol. 2007;19(6):573577. 23. Saleh AJ, Alhejazi A, Ahmed SO, et al. Leukemia during pregnancy: long term follow up of 32 cases from a single institution. Hematol Oncol Stem Cell Ther. 2014;7(2):63-68. 24. Moura AC, Delamain MT, Duarte GBO, Lorand-Metze I, Souza CA, Pagnano KBB. Management of chronic myeloid leukemia during pregnancy: a retrospective analysis at a single center. Hematol Transfus Cell Ther. 2019;41(2):125-128. 25. National Toxicology Program, US Department of Health and Human Services. NTP monograph: developmental effects and pregnancy outcomes associated with cancer chemotherapy use during pregnancy. https://ntp .niehs.nih.gov/ntp/ohat/cancer_chemo_preg/chemopregnancy_monofinal_508.pdf. 26. Galera P, Haynes S, Sulmasy P, et al. Physiological mea surements cor roborate symptomatic improvement after therapeutic leukapheresis in a pregnant woman with chronic myelogenous leukemia. J Clin Apher. 2016;31(4):393-397. 27. Yellu M, Pinkard S, Ghose A, Medlin S. CML in pregnancy: a case report using leukapheresis and literature review. Transfus Apher Sci. 2015;53(3):289292. 28. Balsat M, Etienne M, Elhamri M, Hayette S, Salles G, Thomas X. Successful pregnancies in patients with BCR-ABL-positive leukemias treated with interferon-alpha therapy during the tyrosine kinase inhibitors era. Eur J Haematol. 2018;101(6):774-780. Dr Prakash Singh Shekhawat Treatment of CML in pregnancy | 127 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 Conclusion 7. 128 | Hematology 2022 | ASH Education Program 38. Campiotti L, Suter MB, Guasti L, et al. Imatinib discontinuation in chronic myeloid leukaemia patients with undetectable BCR-ABL transcript level: a systematic review and a meta-analysis. Eur J Cancer. 2017;77(May):48-56. 39. Shah NP, García-Gutiérrez V, Jiménez-Velasco A, et al. Dasatinib discon tinuation in patients with chronic-phase chronic myeloid leukemia and stable deep molecular response: the DASFREE study. Leuk Lymphoma. 2020;61(3):650-659. 40. Caldwell L, Claudiani S, Milojkovic D, et al. Changes in RT-qPCR levels dur ing and after TKI interruptions for attempted conceptions in women with CML. Blood. 2017;130(suppl 1):1612. 41. Chelysheva E, Apperley J, Abruzzese E, et al. Kinetics of the leu ke mic clone in patients with chronic myeloid leukemia during pregnancy. Blood. 2018;132(suppl 1):4254. 42. Lasica M, Willcox A, Burbury K, et al. The effect of tyrosine kinase inhib itor interruption and interferon use on pregnancy outcomes and longterm dis ease con trol in chronic mye loid leu ke mia. Leuk Lymphoma. 2019;60(7):1796-1802. 43. Dou X, Qin Y, Huang X, Jiang Q . Planned pregnancy in female patients with chronic myeloid leukemia receiving tyrosine kinase inhibitor therapy. Oncologist. 2019;24(11):e1141-e1147. 44. Lee JO, Kim DW, Abruzzese E, Apperley J, Caldwell L, Mauro MJ. Kinetics of BCR-ABL after TKI interruption during pregnancy in CML: a multinational retrospective analysis. Blood. 2018;132(suppl 1):4263. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000330 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/123/2021708/123robertson.pdf by guest on 09 December 2022 29. Yazdani Brojeni P, Matok I, Garcia Bournissen F, Koren G. A systematic review of the fetal safety of interferon alpha. Reprod Toxicol. 2012;33(3): 265-268. 30. Beauverd Y, Radia D, Cargo C, et al. Pegylated interferon alpha-2a for essential thrombocythemia during pregnancy: outcome and safety. A case series. Haematologica. 2016;101(5):e182-e184. 31. Talpaz M, Hehlmann R, Quintás-Cardama A, Mercer J, Cortes J. Re-emergence of interferon-α in the treatment of chronic myeloid leukemia. Leukemia. 2013;27(4):803-812. 32. Ali R, Ozkalemkas F, Kimya Y, et al. Imatinib use during pregnancy and breast feeding: a case report and review of the literature. Arch Gynecol Obstet. 2009;280(2):169-175. 33. Russell MA, Carpenter MW, Akhtar MS, Lagattuta TF, Egorin MJ. Imatinib mesylate and metabolite concentrations in maternal blood, umbilical cord blood, placenta and breast milk. J Perinatol. 2007;27(4):241-243. 34. Kronenberger R, Schleyer E, Bornhäuser M, Ehninger G, Gattermann N, Blum S. Imatinib in breast milk. Ann Hematol. 2009;88(12):1265-1266. 35. Burwick RM, Kuo K, Brewer D, Druker BJ. Maternal, fetal, and neonatal imatinib levels with treatment of chronic myeloid leukemia in pregnancy. Obstet Gynecol. 2017;129(5):831-834. 36. Chelysheva E, Aleshin S, Polushkina E, et al. Breastfeeding in patients with chronic myeloid leukaemia: case series with measurements of drug con centrations in maternal milk and literature review. Mediterr J Hematol Infect Dis. 2018;10(1):e2018027. 37. Deininger MW, Shah NP, Altman JK, et al. Chronic myeloid leukemia, version 2.2021, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2020;18(10):1385-1415. BEYOND ROUTINE FRONTLINE THERAPY OF CML Management of TKI-resistant chronic phase CML Precision Cancer Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, Australia; 2Department of Haematology, Royal Adelaide Hospital and SA Pathology, Adelaide, Australia; 3Adelaide Medical School, University of Adelaide, Adelaide, Australia; 4Department of Genetics and Molecular Pathology and Centre for Cancer Biology, SA Pathology, Adelaide, Australia; and 5Clinical and Health Sciences, University of South Australia, Adelaide, Australia 1 Chronic phase CML (CP-CML) patients who are resistant to 2 or more tyrosine kinase inhibitors (TKIs) have limited therapeutic options and are at significant risk for progression to the blast phase. Ponatinib has been the drug of choice in this setting for the past decade, but when given at full dose (45 mg/d), the risk of serious vascular occlusive events is substantial. Lower doses mitigate this risk but also reduce the efficacy. Emerging data suggest that a high dose of ponatinib is important to achieve response, but a lower dose is usually sufficient to maintain response, introducing a safer therapeutic pathway for many patients. The recent development and approval of the novel allosteric ABL1 inhibitor, asciminib, for CP-CML patients with resistant disease provides another potentially safe and effective option in this setting. These recent therapeutic advances mean that for most resistant CP-CML patients who have failed 2 or more TKIs, 2 excellent options are available for consideration—dose modified ponatinib and asciminib. Patients harboring the T315I mutation are also candidates for either ponatinib or asciminib, but in this setting, higher doses are critical to success. Lacking randomized comparisons of ponatinib and asciminib, the best choice for each clinical circumstance is often difficult to determine. Here we review emerging evidence from recent trials and make some tentative suggestions about which drug is preferable and at what dose in different clinical settings using case studies to illustrate the key issues to consider. LEARNING OBJECTIVES • Determine appropriate assessments for CP-CML patients being considered for a change in therapeutic approach because of TKI resistance • Recommend the optimal TKI and the dosing regimen for a CP-CML patient with TKI resistance What disease and patient assessments are indicated for the following 3 patients with resistant chronic phase chronic myeloid leukemia (CP-CML), and what is the best therapeutic approach in each case? CLINICAL CASE 1 A 32-year-old woman with CP-CML and a high-risk EUTOS Long-Term Survival (ELTS) score received frontline dasatinib at 100 mg/d for 4 months, then switched therapy after confirmed early molecular response failure (>10% BCR::ABL1IS). After 4 months of nilotinib at a dose of 400 mg twice daily, there was no improvement in her molecular response. CLINICAL CASE 2 A 55-year-old woman with CP-CML and a low-risk ELTS score was started on nilotinib and achieved a major molecular remission (MMR; <0.1% BCR::ABL1IS) by 12 months. After 18 months, she sequentially switched to bosutinib, then dasatinib, both for intolerance issues, but gradually lost MR2 (>1% BCR::ABL1IS). CLINICAL CASE 3 A 72-year-old man with an intermediate-risk ELTS score failed to achieve MR2 by 12 months on imatinib then received dasatinib at 100 mg/d for 6 months, with an ini- Dr Prakash Singh Shekhawat TKI-resistant CP-CML | 129 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 Timothy P. Hughes1–3 and Naranie Shanmuganathan1–5 tial transient improvement in response (0.5% BCR::ABL1IS). He then rapidly lost molecular response (15% BCR::ABL1IS). Introduction The OPTIC trial Modeling based on the PACE study and other studies suggested the risk of AOEs was significantly lower at lower doses,11,16 lead ing to the approach adopted for the OPTIC trial.13 In OPTIC, patients were randomized to receive either 45 mg/d, 30 mg/d, or 15 mg/d as a starting dose.13 In the case of patients in the higher-dosing cohorts, the dose was reduced to 15 mg/d once MR2 (BCR::ABL1IS < 1%) was achieved.13 As predicted, the dosing strategy of 45 mg/d with dose reduction proved to be signifi cantly less toxic in terms of AOEs compared to the PACE data, where doses were often maintained at 45 mg/d throughout.13 Remarkably, this approach using high doses initially and deescalated doses in maintenance also proved to be highly efficacious and significantly superior to the other 2 arms, with an MR2 rate at 12 months of 52% in the 45-mg cohort vs 36% in the 30-mg cohort and 25% in the 15-mg cohort.13 Furthermore, the OPTIC study provides further insights into the delicate balance of effi cacy and safety that needs to be considered when selecting the initial dose (Figure 1). In resistant cases with the T315I mutation, Figure 1. Overall safety and efficacy of ponatinib based on starting dose. The analysis is a descriptive clinical summary of the data to illustrate the relationship between the efficacy and the AOE rate. TE-AOE, treatment-emergent arterial occlusive events. Reproduced with permission from Cortes et al.13 130 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 For most patients with CP-CML who have achieved and maintained an optimal molecular response, the ongoing manage ment is fairly straight for ward. Ensuring opti mal molec u lar targets are met and maintained, managing drug-related side effects, monitoring drug adherence, and paying careful atten tion to minimize drug-induced organ toxicities are the main priorities.1,2 At the extremes of response, man age ment can be more complex. Patients achieving and maintaining deep molecular responses over several years are considered candi dates for treatment-free remission, although embarking on a treatment-free remission attempt requires extensive discussion and patient education with a careful review of eligibility. Furthermore, once therapy has been ceased, frequent monitoring is essential, as well as a high degree of vigilance looking for molecular relapse.3-6 At the opposite end of the response spec trum, treatment failure to at least 2 tyrosine kinase inhibitors (TKIs) or to just 1 second-generation TKI (2G-TKI) requires expert management while still aiming to maximize optimal responses and also to prevent disease progression and TKI-induced tox ic ity. Treatment fail ure to one or more 2G-TKIs infre quently can be salvaged by switching to another 2G-TKI,7-10 usually in cases in which spe cific ABL1 kinase domain (KD) muta tions have emerged. The clinical development of ponatinib has pro vided an effective option for many of these resistant patients. The PACE study demonstrated its effectiveness in the setting of resistant disease, both in the case of resistance associated with a KD mutation, including the gatekeeper T315I mutation, and in cases without clear identifiable resistance mechanisms.11 The key limitation with ponatinib therapy that emerged in the PACE study and other studies was the very high rate of arterial occlusive events (AOEs) observed, which necessitated the pre mature termination of some ponatinib-based clinical trials and a transient withdrawal from the market.11,12 In the CP-CML arm of the PACE study, the reported rate of AOEs was 31%.11 The therapeutic challenges related to managing these TKIresistant CP-CML patients have become more complex recently because we now have safer and argu ably bet ter ther a peu tic options to consider. The recent demonstration from the OPTIC trial of a less toxic but still efficacious dosing regimen for ponatinib has changed the risk/benefit equation, making ponatinib a more attractive option in this setting.13 Equally promising has been the approval of the allosteric ABL1 inhibitor asciminib, which offers good tolerability and high efficacy in similar high-risk settings.14,15 Clinicians now have several options for patients with 2G-TKI resis tance, but it is often not clear which is the best drug choice and the optimal dose regimen. The prospects of good disease con trol and the risks of toxicity with each option need to be carefully assessed. To provide some clarity about how to make the best decision for a particular patient, we explore the emerging findings from recent trials for resistant patients. We also present and dis cuss several clinical cases to illustrate the value of considering all patient- and drug-specific parameters before selecting the opti mal therapeutic approach for each individual patient. The ASCEMBL trial The ASCEMBL trial randomized CP-CML patients who were intol erant or resistant to 2 or more TKIs to either asciminib or bosutinib in a 2:1 ratio.15 Asciminib dosing was set at 40 mg twice daily based on the tolerability assessments of a number of dosing lev els in the phase 1 study and pharmacokinetic modeling demon strating that either 40 mg twice daily or 80 mg/d of asciminib exceeded the 90% thresh old for mean inhib i tory con cen tra tions.14 It demonstrated a clearly superior rate of MMR (BCR::ABL1IS < 0.1%) by 6 months for asciminib (26% vs 13%).15 However, MR2 may be a more realistic target in this multiresistant setting. The difference was significant here as well—by 12 months, 42% vs 19% had achieved MR2.9 Another major difference between the 2 arms was the rate of discontinuation. In the most recent update,17 43% of patients on the asciminib arm discontinued ther apy, including 24% for lack of efficacy and 6% for adverse events (median follow-up, 19 months). In contrast, 78% were no longer taking bosutinib, with 36% stopping due to lack of efficacy and 24% because of an adverse event.17 There were 7 patients on the asciminib arm who had AOEs at the most recent analysis, which represents a rate of 3.4 per 100 patient-years.17 All 7 patients had received nilotinib pre vi ously, and 3 had received ponatinib.17 Nonetheless, the observed rate of AOEs was higher with asciminib com pared with bosutinib. ASCEMBL dem on strated the clear superiority of asciminib over bosutinib in the resistant and intolerant third-line setting. Comparing ponatinib and asciminib trial data The best way to determine whether ponatinib or asciminib is superior as a third-line option in different clinical settings would be to con duct head-to-head ran dom ized stud ies. However, as these have not been initiated at this stage, we present our opinions based on the data available from the phase 1 asciminib trial, the ASCEMBL trial, and the OPTIC trial to formulate an ini tial assess ment regard ing which drug may be pref er en tial in specific cases (Table 1). OPTIC recruited a higher proportion of patients with KD mutations and a higher proportion of patients who failed to achieve any molecular response to their prior TKI.13 In comparison, ASCEMBL had a higher proportion of intolerant patients compared to OPTIC. Overall response rates look similar when comparing the 45-mg cohort of OPTIC to the asciminib arm of ASCEMBL.13,17 In patients without T315I, the 12-month rates of MR2 were similar; 42% for asciminib and 49% for the ponatinib 45-mg cohort.13,15 However, response rates appear to be more var i able according to the base line response in the asciminib cohort. Notably, only 17% of the patients not in a major cytoge netic response (MCyR) at baseline achieved MMR at 6 months, compared to 40% of patients who were in MCyR at baseline (Figure 2).15 Moreover, the current accepted standard dose of asciminib of 40 mg twice daily may be inadequate to combat highly resistant CML, as evidenced by the higher doses required in T315I-mutated patients.14 Comparable data from OPTIC are not available, but in the 45-mg ponatinib cohort, the MR2 rates were 50% by 12 months regardless of prior response on historical TKI therapy.13 On this (tentative) basis, ponatinib may be a better choice in highly resistant cases, and asciminib, with its superior safety profile, may be preferable in patients with less profound resistance. Another important comparison is the risk of AOEs with each agent. Many variables make this comparison problematic. These include different eligibility criteria regarding cardiac risk factors and previous cardiovascular history, different duration of followup, and different criteria for determining a treatment-emergent AOE. Acknowledging these limitations seems to show an advan tage for asciminib, but this needs careful assessment with ongo ing data from these and other studies. Rates of AOEs per 100 patient-years were 9.6 for the ponatinib 45-mg cohort in OPTIC compared to 3.4 for asciminib in the ASCEMBL trial. Optimal therapy for patients with T315I mutation Both ponatinib and asciminib are active against the T315I muta tion, but in both cases higher doses are important for efficacy. In the OPTIC trial, 15 of 25 (60%) patients with T315I receiving the 45-mg initial dose achieved MR2 by 12 months, compared to 5 of 20 (25%) who started at 30 mg/d.13 Asciminib efficacy against the T315I mutant was assessed in the phase 1 trial, but response rates were low until doses above 150 mg twice daily were given.14 For that reason an expanded cohort of 48 CP-CML and 4 acute-phase CML patients with the T315I mutation were given asciminib at 200 mg twice daily.18 At this dose, molec ular responses were frequent, and toxicity appeared similar to standard -ose asciminib (Table 2). With a median follow-up of 17 months, 35 of 52 (67%) remained on therapy, and 23 of 49 (47%) who were not in MMR at baseline achieved MMR by 24 months.18 This is impressive, especially considering that 28 of 52 (54%) had prior therapy with ponatinib and had developed either intol er ance or resis tance. In ponatinib-naive patients, the rate of MMR was 57% by 6 months, compared to 29% in ponatinib-resistant/intolerant patients.18 Thus, both drugs are highly active when used at the correct dose for patients with the T315I mutation. When patients on ponatinib 45 mg/d deescalated dosing as per the OPTIC pro tocol, the overall rate of loss of response exceeded 30% (the actual rate was not specified in the OPTIC paper).13 Therefore, a risk assessment should be undertaken before dose deescalating in the setting of ponatinib therapy for patients with the T315I mutation. In support of asciminib as the first choice in the T315I setting, tolerance appears superior to ponatinib 45 mg (OPTIC dos ing), while effi cacy appears sim i lar (Table 2).18 In sup port of ponatinib as the first choice, we have more data and longer follow-up to reassure us that responses are stable and durable in T315I patients. Since there is no clear “winner,” the choice should Dr Prakash Singh Shekhawat TKI-resistant CP-CML | 131 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 the advantage of starting at 45 mg/d over 30 mg/d is significant, with MR2 rates by 12 months at 60% and 25%, respectively.13 There was also an advantage with the 45-mg starting dose com pared to the 30-mg cohort in patients with a non-T315I KD muta tion (56% vs 40% MR2 by 12 months).13 In patients with resistance or intolerance without a KD mutation, the advantage was less marked (46% vs 38%).12 The higher efficacy achieved with higher starting doses needs to be weighed against the higher toxicity. In patients with recent AOEs on a prior TKI, even a brief exposure to full-dose ponatinib may prove very hazardous. Treatment-emergent AOEs per 100 patient-years in OPTIC were 3.2 for the 15-mg cohort, 5.3 for the 30-mg cohort, and 9.6 for the 45-mg cohort.13 To distinguish this dosing strategy from the conventional strategy of maintaining the starting dose if it is well tolerated, we refer to this dosing regimen as ponatinib 45 mg (OPTIC dosing) and ponatinib 30 mg (OPTIC dosing) for clarity. Table 1. Comparison of asciminib (ASCEMBL) to ponatinib (OPTIC, 45-mg-dose cohort) Asciminib 40 mg twice daily, arm of ASCEMBL study15,17 Ponatinib 45 mg daily, dose adjusted from OPTIC trial13 Number of patients 157 92 Eligibility ELN201324 treatment failure (61%) or intolerance (38%) Intolerant or resistant to >2 TKIs and not in MR2 Exclusion criteria Within 6 mo prior to starting study treatment of myocardial infarction, angina pectoris, coronary artery bypass graft, or clinically significant cardiac arrhythmias Any history of myocardial infarction, unstable angina, cerebrovascular accident, transient ischemic attack, peripheral vascular disease, NYHA class III or IV congestive heart failure, or reduced left ventricular ejection fraction within 6 mo prior to enrollment, clinically significant cardiac arrhythmias within 6 mo ≥3 prior TKI lines 48% 53% Resistant to last TKI 61% 98% Disparity in favor of ponatinib in highly resistant cases Intolerant patients 38% 2% Disparity in favor of asciminib in intolerant cases MCyR at baseline 29% 35% CCyR at baseline 10% 0% 13% 44% >10% BCR::ABL1 62% NA Cardiac risk factors Not specified 29% had 1, 5% had >1 Follow-up 19 mo 32 months Still on study therapy 57% 47% Discontinuation due to toxicity 6% 17% Discontinuation due to lack of efficacy 24% 19% 42% 44% (49% in non-T315I group) MR2 by 12 mo in patients in CHR or worse on prior TKI NA 27/54 (50%) MR2 by 12 mo in patients who had a better response than CHR to prior TKI NA 14/28 (50%) 29% NA – 34% “overall rate with latest follow-up” MMR by 6 mo in patients in MCyR at baseline 40% NA MMR by 6 mo in patients NOT in MCyR at baseline 17% NA Suggestion from this data that asciminib response may be more dependent on the level of response to prior therapy than is the case for ponatinib AOEs per 100 patient-years 3.4 7.6 (year 1) 5.9 (year 2) In the ponatinib cohort receiving 30 mg initially, AOE rates/100 patient-years were 3 (year 1) and 2 (year 2) Comment KD mutations at baseline (non-T315I) Lower rate of patients with KD mutations in ASCEMBL Results MR2 by 12 mo MMR by 12 mo Consistent with better tolerance to asciminib than ponatinib Note high response rate on ponatinib in highly resistant cohort Data reproduced with permission from Cortes et al,13 Réa et al,15 and Mauro et al.17 CCyR, complete cytogenetic response; CHR, complete hematologic response; NA, not available; NYHA, New York Heart Association. 132 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 Baseline features be individualized by weighing the disease-related factors and the cardiovascular risk. Optimal therapy in the case of other KD mutations Both ponatinib and asciminib are active against most adenosine triphosphate-site KD mutations,19,20 although sensitivity is gen erally lower than it is for unmutated BCR::ABL1. KD mutations of concern for asciminib include F359V/C/I, which either emerged or persisted without evidence of molecular response in several cases in ASCEMBL and the phase 1 study.15,21 This was predicted to be a partially resistant mutation in in vitro studies.21 More data are needed to determine asciminib activity against the F317L mutation, which appears to induce a degree of asciminib resis tance based on in vitro studies (Figure 3).21 However, in allpresented scenarios, higher doses of asciminib may be required for optimal efficacy, analogous to the higher doses necessary to combat the T315I mutation. Any role for 2G-TKIs in this setting? In most cases the opti mal choice of next-line ther apy is between ponatinib and asciminib. There are specific cases in which a 2G-TKI may still have a role. This would include cases in which resistance is driven by a mutation that is well covered by another 2G-TKI. For instance, the development of V299L or F317L mutations is highly sensitive to nilotinib and bosutinib, and F359V/I/C mutations are highly sensitive to both dasatinib and bosutinib (Figure 3).2 Additionally, in some countries eligibility rules prevent access to sequential therapy with either asciminib or ponatinib in the setting of 2G-TKI failure without developing resistance or intol erance to an alternative 2G-TKI. In this setting, a 3-to-6 month trial is generally sufficient to determine if the newly commenced 2G-TKI is likely to be effective and salvage the situation. It is rare for patients who have no improvement in molecular response after 6 months of a salvage therapy to eventually achieve MR2 or better with longer-term exposure. The intolerant patient The focus of this chapter is TKI resistance, but it is worth commenting on the challenging patient who is intolerant to 2 or more TKIs. In this setting the efficacy and tolerability of asciminib look very favorable based on the phase 1 asciminib study,14 and in most cases, ponatinib would not be the primary drug of choice due to its higher rates of intolerance and toxicity. Whether lower doses of ponatinib could achieve comparable response and tol erance to asciminib in these patients, where the predominant challenge is intolerance, remains to be established. Future options Targeting 2 distinct regions on ABL1, the adenosine triphos phate- and myristate-binding sites, with asciminib and conven tional TKIs may also be a future option to combat resistance, with the phase 1 clinical trial yet to formally report outcomes for the relevant treatment arms that investigated combination Dr Prakash Singh Shekhawat TKI-resistant CP-CML | 133 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 Figure 2. Subgroup analysis of the ASCEMBL study: risk difference with 95% CIs for MR3 achievement by 24 weeks of either asciminib or bosutinib. CRF, case report form. Reproduced with permission from Rea et al.15 Table 2. Comparing baseline variables and outcomes for CP-CML patients receiving asciminib at 200 mg twice daily (phase 1 study) or dose-modified ponatinib (OPTIC) Ponatinib 45 mg (OPTIC)13 Ponatinib 30 mg (OPTIC)13 No. of patients 52 94 94 Follow-up 17 mo 32 mo 32 mo Molecular status at baseline BCR::ABL1IS 54%> 10% 25% 1-10% 15% <1% Prior ponatinib therapy 28/52 (54%) NA NA Discontinued therapy 17/52 (33%) 44/94 (47%) For allpatients on 45 mg, not just T315I cohort 53/95 (56%) For allpatients on 30 mg, not just T315I cohort MMR rate at 12 mo 12/21 (57%) in ponatinibnaive cohort NA NA MMR rate at 24 mo 14/21 (66%) in ponatinibnaive cohort NA NA MR2 at 12 mo NA 15/25 (60%) 5/20 (25%) MR2 at 24 mo NA AOEs 2/52 (4%) 9/94 (9.6%) 5/94 (5.3%) Comments Results Asciminib appears to be better tolerated AOE risk with asciminib may be similar to ponatinib 30 mg (OPTIC dosing) Data reproduced with permission from Cortes et al,13 Hughes et al14, and Cortes et al.18 CCyR, complete cytogenetic response; CHR, complete hematologic response; NA, not available. therapy.20 Furthermore, murine models have demonstrated that combining asciminib with ponatinib can inhibit the growth of T315I-containing compound mutations.21 A num ber of novel BCR::ABL1–targeting agents are under investigation for resistant CML. One such drug is vodobatinib, a novel 3G-TKI with limited off-target activity.22 The phase 1 dose escalation study recently reported prom is ing safety and effi cacy results gen er ated in both ponatinib-naive and ponatinib-treated patients.22 Furthermore, cardiovascular toxicity did not appear to be a prominent fea ture at this early stage, which may dis tin guish vodobatinib from the other available agents for resistant CML. Similarly, another 3G-TKI, olverembatinib, has also had early phase 1 results reported in first- and sec ond-gen er a tion-resis tant CP-CML and acute-phase CML.23 Efficacy was observed regard less of the study entry response, pre dom i nantly in T315Imutated patients.23 Responses were less impres sive in the absence of T315I. While longer-term follow-up in larger data sets is required, these agents may offer future options for this resistant patient cohort. CLINICAL CASE 1 (Cont inu ed) This 32-year-old woman with CP-CML and a high-risk ELTS score received frontline dasatinib at 100 mg/d for 4 months, then switched to nilotinib at 400 mg twice daily for 4 months 134 | Hematology 2022 | ASH Education Program after confirmed early molecular response failure. After a total of 8 months, her BCR::ABL1IS remained over 10%, having never fallen below 10%. Cytogenetics at 8 months did not show evidence of clonal evolution, but 18 of 20 metaphases were Ph+. No KD mutation was identified at 4 or 8 months. She had no cardiovascular risk factors or significant comorbidities. Options 1. Continue nilotinib and review at 12 months. 2. Switch to bosutinib. 3. Switch to ponatinib 45 mg/d (OPTIC dosing). 4. Switch to asciminib. 5. Proceed to an allograft. Discussion of options 1. Continue nilotinib: Unless there are significant interruptions to therapy due to toxicity issues or poor adherence that resolve or are overcome, there is very little prospect that the molecular response will improve by continuing the current therapy. In this setting, the risk of disease progression is high. One important consideration is the need to identify those patients who will not achieve response with any TKI as soon as possible so that an allo graft can still be scheduled in the chronic phase, where there is still a good prospect of cure. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 Asciminib 200 mg twice daily18 2. Switch to bosutinib: The likelihood of a response if a third 2G-TKI is given is very low. In ASCEMBL, only 19% achieved MR2 by 12 months on the bosutinib arm, and the response rate was even poorer in patients who were not in an MCyR at baseline, as in this case. 3. Switch to ponatinib 45 mg/d (OPTIC dosing): Given the complete lack of response after 8 months of potent TKI therapy, this may be the best option. In OPTIC, 50% of patients who were not in MCyR at baseline who received 45 mg/d of ponatinib achieved MR2 by 12 months. If a patient is given ponatinib and does not achieve MR2 after a further 6 months, the pros pect of achieving this target with ongoing ponatinib is low, and hence switching to asciminib or proceeding to an allograft would be appropriate. 4. Switch to asciminib: As discussed earlier, responses to asciminib are probably inferior to ponatinib 45 mg/d (OPTIC dos ing) in patients with little or no response to prior therapy. The exception would be in cases in which the risk of a vascular event was high, where asciminib (or lower-dose ponatinib) may be preferred. 5. Proceed to an allograft: While an allograft is not directly recommended at this stage, it would be prudent to tissue type the patient and identify an allogeneic donor in case of failure to respond to third-line therapy. In the event of persistent therapy failure, the patient can proceed to an allogeneic stem cell trans plant promptly without delay. Best option: ponatinib 45 mg daily with a dose reduction to 15 mg daily if MR2 is achieved. CLINICAL CASE 2 (Continued) The 55-year-old woman with CP-CML and a low-risk ELTS score was started on nilotinib and responded well, achieving MMR by 12 months. After 18 months, she developed peripheral vascular disease and switched to bosutinib and then dasatinib due to further toxicity but sequentially lost MMR and MR2 after 3 years. Further assessments: There were no KD mutations or evi dence of clonal evolution on marrow examination. There were substantial interruptions secondary to adverse events as well as periods of poor drug adherence. She had peripheral vascular disease and well-controlled hypertension. Options 1. Switch to imatinib. 2. Switch to ponatinib 45 mg/d with dose reduction once MR2 is achieved. 3. Switch to ponatinib 30 mg/d with dose reduction once MR2 is achieved. 4. Switch to asciminib 40 mg twice daily. Discussion of options 1. Switch to imatinib: It is possible that her loss of response is more related to exten sive inter rup tions to ther apy and poor adherence rather than drug resistance, so imatinib may still be an effective drug in this setting. However, given her intolerance Dr Prakash Singh Shekhawat TKI-resistant CP-CML | 135 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 Figure 3. Heat map summary of IC50 values for TKIs against a panel of Ba/F3 cell lines expressing KD mutations. ATP, adenosine triphosphate. Reproduced with permission from Eide et al.21 of resistance to imatinib that was not driven by a KD mutation. Bosutinib may still be a reasonable choice in this setting, espe cially given its lower risk of AOEs. However, the experience in ASCEMBL would suggest that it may be poorly tolerated. 2. Switch to nilotinib: The F317L mutation is sensitive to nilotinib, but the risk of vascular events is probably too high, espe cially since a higher dose is likely to be needed (400 mg twice daily). 3. Switch to ponatinib 45 mg/d (OPTIC dosing): Patients in the OPTIC study with a non-T315I mutation who received the higher initial dose of ponatinib had a rate of MR2 by 12 months of 56%. The main concern would be the AOE risk given the patient’s history and poorly controlled hypertension. This patient would not have been eligible for the OPTIC trial, so the rate of AOEs would likely be significantly higher than the rate observed on this arm of the OPTIC study. 4. Switch to ponatinib 30 mg/d (OPTIC dosing): Given the concern about the risk of AOEs in this patient, even this modi fied dose represents a significant risk. The rate of AOEs per 100 patient-years was 5.3 for this dose in OPTIC, but this patient would not have been eligible for OPTIC, so the AOE risk is prob ably higher. 5. Switch to asciminib 40 mg twice daily: There are very lim ited data regarding the sensitivity of the F317L mutation to asciminib. In vitro studies suggest a level of resistance greater than that seen with the T315I mutation. This mutation was only found in 2 patients at baseline in the asciminib arm of ASCEMBL; neither had a molecular response.15 Along with the F359C/I/V mutation, we need more clinical data before we can determine whether asciminib is effective in this setting and, if so, at what dose. Best option: bosutinib may be the safest option, with a switch to ponatinib at a modified dose if no molecular response occurs after 3 to 6 months. Acknowledgments CLINICAL CASE 3 (Cont inu ed) A 72-year-old man with inter me di ate-risk ELTS failed to achieve MR2 by 12 months on imatinib. He then received dasatinib at 100 mg/d for 6 months with an initial improve ment in response (best response, 0.5% BCR::ABL1IS) but then lost molecular MR2 (15% BCR::ABL1IS).Further assessments: He had no mutation detected when switching to dasatinib, but when he lost MR2, there was evidence of emergence of the F317L mutation. From a cardiovascular risk perspective, he had a single coronary stent inserted 10 years ago and has poorly controlled hypertension. Conflict-of-interest disclosure Tim o thy P. Hughes: research funding: Novartis, Bristol Myers Squibb; honoraria: Takeda, Novartis, Bristol Myers Squibb. Naranie Shanmuganathan: research funding: Novartis; honoraria: Takeda. Off-label drug use Timothy P. Hughes: nothing to disclose. Naranie Shanmuganathan: nothing to disclose. Options 5. Switch to bosutinib. 6. Switch to nilotinib. 7. Switch to ponatinib 45 mg/d (OPTIC dosing). 8. Switch to ponatinib 30 mg/d (OPTIC dosing). 9. Switch to asciminib 40 mg twice daily. Correspondence Naranie Shanmuganathan, Centre for Cancer Biology and Precision Cancer Medicine Theme, South Australian Health and Medical Research Institute, Royal Adelaide Hospital, Port Rd, 5000 Adelaide, Australia; e-mail: naranie.shanmuganathan@sa.gov.au. Discussion of options 1. Switch to bosutinib: The F317L mutation would appear to be sensitive to bosutinib, but in this patient there was also evidence 136 | Hematology 2022 | ASH Education Program Timothy P. Hughes received support from the National Health and Medical Research Council of Australia (APP1135949) and has the financial support of Cancer Council SA’s Beat Cancer Project on behalf of its donors and the state government of South Australia through the Department of Health. References 1. NCCN Clinical Practice Guidelines in Oncology: Chronic Myelogenous Leukemia. Version 3.2022. Fort Washington, PA; 2022. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 to all 3 2G-TKIs, imatinib may not be well tolerated, and her adherence may be poor once again. 2. Switch to ponatinib 45mg/d (OPTIC dosing) with dose reduction once MR2 is achieved: While she has lost MR2, it is still very pos si ble that she still has TKI-sen si tive dis ease, so she would likely have an excellent response to full-dose ponatinib. However, the risk of AOEs is still significant at this dose, especially given her history, and other options may be equally or more efficacious without the same level of risk. 3. Switch to ponatinib 30 mg/d (OPTIC dosing) with dose reduction once MR2 is achieved: This dose provides a lower risk of AOEs (5.3 events per 100 patient-years compared to 9.6 for the 45-mg ponatinib cohort in OPTIC). However, the rate of molecular response is lower than with the higher dose of ponatinib. In patients with no KD mutations, 46% and 38% achieved MR2 by 12 months in the ponatinib 45-mg and 30-mg dose cohort, respectively. This may not be a major difference as pro gression-free and overall survival were not substantially inferior with the modified dose. This would be an acceptable option except for the fact that the patient has a history of peripheral vascular disease. 4. Switch to asciminib at 40 mg twice daily: This would be a relatively safe and effective option in this setting. Patients in MCyR in ASCEMBL who received asciminib had a 40% rate of MMR by 6 months. The risk of AOEs (per 100 patient-years) in this cohort was 3.4.17 Given her consistent tolerability issues with all 3 2G-TKIs, asciminib would be the option most likely to be well tolerated. Best option: Asciminib at 40 mg twice daily and consider a switch to ponatinib if MR2 is not achieved after 6 to 12 months. Imatinib may be another option, but given the loss of responses on previous therapy, asciminib would be preferentially recommended. 14. Hughes TP, Mauro MJ, Cortes JE, et al. Asciminib in chronic myeloid leuke mia after ABL kinase inhibitor failure. N Engl J Med. 2019;381(24):2315-2326. 15. Réa D, Mauro MJ, Boquimpani C, et al. A phase 3, open-label, randomized study of asciminib, a STAMP inhibitor, vs bosutinib in CML after 2 or more prior TKIs. Blood. 2021;138(21):2031-2041. 16. Dorer DJ, Knickerbocker RK, Baccarani M, et al. Impact of dose intensity of ponatinib on selected adverse events: multivariate analyses from a pooled population of clinical trial patients. Leuk Res. 2016;48(September):84-91. 17. Mauro MJ, Minami Y, Rea D, et al. Efficacy and safety results from ASCEMBL, a multicenter, open-label, phase 3 study of asciminib, a first-in-class STAMP inhibitor, vs bosutinib in patients with chronic myeloid leukemia in chronic phase after ≥2 prior tyro sine kinase inhib i tors: update after 48 weeks [abstract]. Blood. 2021;138(suppl 1). Abstract 310. 18. Cortes JE, Hughes TP, Mauro MJ, et al. Asciminib, a first-in-class STAMP inhibitor, provides durable molecular response in patients (pts) with chronic myeloid leukemia (CML) harboring the T315I mutation: primary effi cacy and safety results from a phase 1 trial. Blood. 2020;136(suppl 1):47-50. 19. Soverini S, Branford S, Nicolini FE, et al. Implications of BCR::ABL1 kinase domain-mediated resistance in chronic myeloid leukemia. Leuk Res. 2014;38(1):10-20. 20. Wylie AA, Schoepfer J, Jahnke W, et al. The allosteric inhibitor ABL001 enables dual targeting of BCR::ABL1. Nature. 2017;543(7647):733-737. 21. Eide CA, Zabriskie MS, Savage Stevens SL, et al. Combining the allo steric inhibitor asciminib with ponatinib suppresses emergence of and restores efficacy against highly resistant BCR::ABL1 mutants. Cancer Cell. 2019;36(4):431-443.e5443e5. 22. Cortes JE, Saikia T, Kim DW, et al. Phase 1 trial of vodobatinib, a novel oral BCR::ABL1 tyrosine kinase inhibitor (TKI): activity in CML chronic phase patients failing TKI therapies including ponatinib. Blood. 2020;136(suppl 1):51-52. 23. Qian J, Shi D, Li Z, et al. Updated safety and efficacy results of phase 1 study of olverembatinib (HQP1351), a novel third-generation BCR-ABL tyro sine kinase inhibitor (TKI), in patients with TKI-resistant chronic myeloid leukemia (CML). Blood. 2021;138(suppl 1):311. 24. Baccarani M, Deininger MW, Rosti G, et al. European LeukemiaNet recom mendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122(6):872-884. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000328 Dr Prakash Singh Shekhawat TKI-resistant CP-CML | 137 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/129/2021810/129hughes.pdf by guest on 09 December 2022 2. Hochhaus A, Baccarani M, Silver RT, et al. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 2020; 34(4):966-984. 3. Mahon FX, Réa D, Guilhot J, et al; Intergroupe Français des Leucémies Myéloïdes Chroniques. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11(11):1029-1035. 4. Ross DM, Branford S, Seymour JF, et al. Safety and efficacy of imatinib ces sation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study. Blood. 2013;122(4):515-522. 5. Saussele S, Richter J, Guilhot J, et al; EURO-SKI Investigators. Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial. Lancet Oncol. 2018;19(6):747-757. 6. Hughes TP, Ross DM. Moving treatment-free remission into mainstream clinical practice in CML. Blood. 2016;128(1):17-23. 7. Garg RJ, Kantarjian H, O’Brien S, et al. The use of nilotinib or dasatinib after failure to 2 prior tyrosine kinase inhibitors: long-term follow-up. Blood. 2009;114(20):4361-4368. 8. Lipton JH, Bryden P, Sidhu MK, et al. Comparative efficacy of tyrosine kinase inhib i tor treat ments in the third-line set ting, for chronic-phase chronic myelogenous leukemia after failure of second-generation tyrosine kinase inhibitors. Leuk Res. 2015;39(1):58-64. 9. Ibrahim AR, Paliompeis C, Bua M, et al. Efficacy of tyrosine kinase inhib itors (TKIs) as third-line therapy in patients with chronic myeloid leuke mia in chronic phase who have failed 2 prior lines of TKI therapy. Blood. 2010;116(25):5497-5500. 10. Hochhaus A, Gambacorti-Passerini C, Abboud C, et al; BYOND Study Investigators. Bosutinib for pretreated patients with chronic phase chronic myeloid leukemia: primary results of the phase 4 BYOND study. Leukemia. 2020;34(8):2125-2137. 11. Cortes JE, Kim DW, Pinilla-Ibarz J, et al. Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5-year results of the phase 2 PACE trial. Blood. 2018;132(4):393-404. 12. Lipton JH, Chuah C, Guerci-Bresler A, et al; EPIC Investigators. Ponatinib versus imatinib for newly diagnosed chronic myeloid leukaemia: an interna tional, randomised, open-label, phase 3 trial. Lancet Oncol. 2016;17(5):612621. 13. Cortes J, Apperley J, Lomaia E, et al. Ponatinib dose-ranging study in chronic-phase chronic myeloid leukemia: a randomized, open-label phase 2 clin ical trial. Blood. 2021;138(21):2042-2050. CONTROVERSIES IN AGGRESSIVE NHL CNS prophylaxis in aggressive B-cell lymphoma Department of Haematology, Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 2Department of Haematology, Vall d’Hebron Institute of Oncology, University Hospital Vall d’Hebron, Barcelona, Spain; and 3Department of Haematology, University College London Hospital, London, United Kingdom 1 The prevention of central nervous system (CNS) relapse in diffuse large B-cell lymphoma (DLBCL) continues to be one of the most contentious areas of lymphoma management. Outcomes for patients with secondary CNS lymphoma (SCNSL) have historically been very poor. However, in recent years improved responses have been reported with intensive immunochemotherapy approaches, and there is a growing interest in potential novel/cellular therapies. Traditional methods for selecting patients for CNS prophylaxis, including the CNS International Prognostic Index, are hampered by a lack of specificity, and there is accumulating evidence to question the efficacy of widely employed prophylactic interventions, including intrathecal and high-dose methotrexate (HD-MTX). Given the potential toxicity of HD-MTX in particular and the ongoing need to prioritize systemic disease control in high-risk patients, there is an urgent need to develop more robust methods for identifying patients at highest risk of CNS relapse, as well as investigating prophylactic interventions with greater efficacy. Here we review new evidence in this field from the last 5 years, focusing on the potential use of molecular diagnostics to improve the identification of high-risk patients, recent large data sets questioning the efficacy of HD-MTX, and the current approach to management of patients with SCNSL. We provide a suggested algorithm for approaching this very challenging clinical scenario. LEARNING OBJECTIVES • Understand the currently available methods for identifying patients at high risk of CNS relapse and the potential for novel molecular diagnostics to improve patient selection in the future • Review recent evidence to question the efficacy of traditional methods for delivering CNS prophylaxis and to evaluate the increasing focus on alternative interventions for this important clinical problem CLINICAL CASE A 62-year-old man with no previous medical history presented in January 2020 with a short history of weight loss, night sweats, hip pain, and bilateral groin lymphadenopathy. His lactate dehydrogenase (LDH) was elevated (>3 times the upper limit of normal). Fluorodeoxyglucosepositron emission tomographic (FDG-PET) imaging revealed widespread hypermetabolic lymphadenopathy (largest lesion, 4 cm diameter) as well as pathological FDG uptake in multiple areas of bone (scapula, L3 vertebra, left hemipelvis) and in the left kidney. A core biopsy from a left inguinal lymph node demonstrated a diagnosis of diffuse large B-cell lymphoma (DLBCL), non-germinal center subtype (Hans algorithm), with overexpression of MYC (>90%) and BCL2 (>60%) by immunohistochemistry— that is, the double-expressor subtype. Fluorescence in situ hybridization (FISH) studies showed no evidence of MYC, BCL2, or BCL6 rearrangements. His Eastern 138 | Hematology 2022 | ASH Education Program Cooperative Oncology Group (ECOG) performance status (PS) was 1, resulting in an international prognostic index (IPI) of 4 (age >60, stage IVB, raised LDH, ≥2 extranodal (EN) sites) and a central nervous system (CNS) IPI of 5 (aforementioned IPI factors plus renal involvement). Six cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) therapy were planned at 21-day intervals. Consideration was given as to whether CNS prophylaxis should be incorporated to reduce risk of CNS relapse. Introduction CNS relapse (otherwise referred to as secondary CNS lymphoma [SCNSL]) is a relatively rare but often devastating complication for patients with DLBCL. Estimates of CNS relapse incidence vary, occurring overall in approximately 5% of DLBCL patients but with subgroups in which the risk is significantly higher.1,2 Most CNS relapse events occur Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 Matthew R. Wilson,1 Sabela Bobillo,2 and Kate Cwynarski3 Table 1. Summary of consensus guideline recommendations for CNS prophylaxis in DLBCL Patient selection Method for CNS prophylaxis suggested British Society for Haematology (2021)8 Offer to: • High (4-6) CNS-IPI • ≥3 EN sites • High-risk EN site involvement—testicular, renal/adrenal, intravascular Consider in: • Breast involvement • Uterine involvement • HD-MTX (≥3g/m2 for 2-3 cycles) as early as possible as part of first-line therapy without compromising dose and time intensity of R-CHOP-like treatment • IT prophylaxis not recommended if HD-MTX successfully delivered • Consider IT as well as systemic prophylaxis in testicular DLBCL NCCN (2022)48 Consider in: • High (4-6) CNS-IPI • Double/triple-hit HGBL • High-risk EN site involvement—testicular, breast, primary cutaneous, renal/adrenal • HD-MTX (3-3.5g/m2 for 2-4 cycles) during or after the course of treatment and/or • IT methotrexate and/or cytarabine (4-8 doses) during or after the course of treatment ESMO (2018)49 Consider in: • High IPI • High-risk EN site involvement—testicular, renal/adrenal, breast, bone marrow, bone • HD-MTX is “an option . . . even though the level of supporting evidence is low” • “Little or no role” for IT therapy ESMO, European Society for Medical Oncology; HGBL, high-grade B-cell lymphoma; NCCN, National Comprehensive Cancer Network. either during or closely following frontline immunochemotherapy, with a median time in recent prospective clinical trials of 6 to 8 months.1,3 Management of SCNSL is often challenging, with historically poor outcomes. As a result, much attention has focused on both the identification of patients at highest risk for this complication, as well as prophylactic treatments aimed at abrogating risk as much as possible. Although our understand ing of which patients are at highest risk of SCNSL has improved, particularly with the introduction of the CNS-IPI and increased under stand ing of the molec u lar biol ogy of DLBCL,4 decisionmaking around prophylactic interventions continues to be based either on retrospective analyses or data extrapolated from other disease subtypes, with no prospective randomized trials performed aimed at addressing CNS prophylaxis efficacy directly. Clinicians often are faced with the dilemma of trying to prevent such a feared complication whilst ensuring that the patient is not exposed to additional therapy with associated risk of toxicity and a limited evidence-base to demonstrate its efficacy. The limitations of the evidence to inform decision-making are reflected in the variation between national guidelines on the topic (Table 1), as well as the significant disparity in practice between centers within the same health care system. A 2017 American Society of Hematology Educational Program review gave a comprehensive overview of the risk factors for CNS relapse and evidence to guide prophylactic interventions at that time.5 In this article we focus on updates in the field in the last 5 years, with particular attention to the advances in molecular diagnostics and implications for SCNSL, as well as new evidence to question the efficacy of high-dose methotrexate (HD-MTX). How do we identify patients at high risk of CNS relapse? Clinical risk factors Numerous stud ies have inves ti gated poten tial risk fac tors for CNS relapse in DLBCL.5 In 2016 the German High-Grade NonHodgkin Lymphoma Study Group (DSHNHL) developed a prog nostic model (CNS-IPI) incorporating the 5 standard IPI factors as well as involvement of the kidneys or adrenal glands, stratifying patients into 3 risk categories (Table 2).4 Notably, patients with 5 or 6 risk factors had a much higher risk of CNS relapse of 15% and 32.5%, respectively. Although the CNS-IPI is a robust model and has been validated in subsequent studies, it lacks specificity, and half of events occur among patients with low to intermedi ate scores. It should also be noted that although a small number of patients in the DSHNHL trials used to formulate the CNS-IPI had Burkitt lymphoma, the final model is validated for patients with DLBCL only, and CNS prophylaxis strategies for Burkitt lym phoma should be considered separately. Certain EN sites have been associated with a higher risk of CNS recurrence, with kidney/adrenal involvement included in the CNS-IPI model and intravascular lymphoma a distinct entity with a well-established risk of CNS involvement at baseline or at relapse. Testicular involvement has long been recognized as a risk factor, in the context of both limited and advanced stage, with a 10-year CNS relapse risk of 10% to 25% (see section Testicular DLBCL).6 Breast involvement has been associated with a higher risk of CNS relapse (~15%) in retrospective series,7 whereas other EN sites such as the uterus, blood, bone marrow, or epi dural area showed more inconsistent results and are unlikely to be independently predictive of CNS relapse.8 Finally, a large ret rospective study reported that the involvement of 3 or more EN sites as determined by PET-computed tomography conferred a 3-year cumulative risk of CNS relapse of 15%.9 Biological risk factors The dual overexpression of MYC and BCL2, deter mined by immunohistochemistry (double-expressor DLBCL), has not been consistently associated with a high risk of CNS relapse.3,10 However, most double-expressor cases are classified as the activated B-cell (ABC) subtype, which, when determined by gene expres sion profiling, has been associated with a CNS relapse risk of 7% to 9% and 15% when combined with a high CNS-IPI.3,10 Recently, multiplatform analysis defined new molecular sub groups, or clusters.11,12 The MCD and C5 clusters, characterized by a high frequency of MYD88L265P and CD79 mutations, occur almost exclusively in the ABC subtype. Genetic alterations defin ing these sub types are also recur rently mutated in pri mary Dr Prakash Singh Shekhawat CNS prophylaxis in aggressive B-cell lymphoma | 139 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 Guideline Table 2. CNS-IPI risk categories with corresponding 2 year rates of CNS relapse and proportion of patients in each category from training (DSHNHL) and validation (BCCA) cohorts DSHNHL cohort Risk group Risk factors Low Intermediate BCCA cohort N (%) 2-year risk of CNS relapse N (%) 2-year risk of CNS relapse 0-1 1002 (46) 0.6% 463 (31) 0.8% 2-3 896 (41) 3.4% 694 (46) 3.9% High* 4 188 (9) 7.4% 344 (23) 12% High 5 62 (3) 15% High 6 13 (1) 32.5% EN lym pho mas orig i nat ing in the CNS, tes tes, breasts, skin, and intravascular spaces. Interestingly, a recent series of SCNSL (n=13) confirmed a higher prevalence of the MCD subtype than a reference cohort of relapsed DLBCL with no CNS involvement (38% vs 8%; P=.003).13 Furthermore, the hcMCD subtype defined by MYD88L265P mutation or more than 3 mutations in CD79, PIM1, ETV6, BTG1, PRDM1, or PBL1XR1 constituted almost half of the patients with CNS recurrence (46%). The remaining cases were either dou ble-hit lym phoma (DHL) or asso ci ated with TP53 mutations. Although these data need to be validated, there is clear potential for next generation sequencing analysis to help identify patients at risk of CNS relapse. High-grade B-cell lymphomas harboring MYC translocation along with BCL2 and/or BCL6 translocation (DHL or triple-hit lymphomas] have historically been associated with a high risk of CNS involvement). However, there is accumulating evidence to suggest that early data overestimated this risk, as FISH was not performed consistently,10 and such patients often meet other clinical criteria. Baseline screening Baseline screening with brain imaging and lumbar puncture/ cerebrospinal fluid (CSF) analysis is increasingly used to iden tify high-risk patients with CNS involvement who may benefit from CNS-directed therapies. Several studies have shown that CSF analysis with flow cytometry is more sensitive than cytol ogy for the detection of occult CNS involvement.14 However, a proportion of patients with a negative flow cytometry result relapse in the CNS shortly after treatment, suggesting the need for more sensitive techniques. Cell-free circulating tumor DNA (ctDNA) has recently appeared as a prog nos tic bio marker in patients with CNS lymphoma, with good correlation between ctDNA levels (MYD88L265P mutation) and treatment response and outcomes.15-17 Two studies have assessed the role of CSF ctDNA analysis in patients with high-risk B-cell lymphoma.17,18 The first ana lyzed spe cific tumor-derived muta tions in sequen tial CSF sam ples from 12 patients receiv ing front line treat ment, and CSF ctDNA was detected 3 months before CNS relapse in 1 of 2 patients in whom this occurred.17 More recently, Olszewski et al analyzed CSF from 22 patients with aggressive B-cell lymphoma 140 | Hematology 2022 | ASH Education Program using a next generation sequencing-minimal residual assay.18 At diagnosis, CSF ctDNA was identified in 8 patients, 2 of whom relapsed in the CNS, with a 12-month cumulative risk of CNS recurrence of 29% in patients with a positive analysis vs a 0% risk for patients with negative CSF.18 Taken together, acknowledging the limitation of the small number of patients, these results sug gest the potential utility of CSF ctDNA to identify patients with a higher risk of CNS relapse. Further studies are ongoing to vali date these findings before the technology can be incorporated into routine clinical practice. How do we manage patients with CNS relapse/SCNSL? Historically, SCNSL has been associated with a dismal progno sis and median overall survival (OS) of approximately 6 months.19 Identifying effective therapeutic approaches remains challeng ing, and the majority of patients still progress or relapse shortly after treatment. In recent years, combinations of intensive chemotherapies including HD-MTX followed by autologous stem cell transplan tation (ASCT) have been adopted, with 2-year OS of 25% to 68% reported.20,21 Recently, the MARIETTA phase 2 study examined the efficacy of 3 courses of MATRix (rituximab, MTX, cytarabine, thio tepa) plus 3 courses of RICE (rituximab, ifosfamide, etoposide, carboplatin) followed by carmustine and thiotepa-conditioned ASCT in 75 patients with CNS involve ment at diag no sis or relapse. Two-year OS for the intention-to-treat population was 46%,22 while those undergoing ASCT (37/75 patients) had 2-year OS of 83%. Two-year progression-free survival (PFS) of 71% was reported in patients with SCNSL at initial diagnosis, but PFS was only 28% in patients previously treated with R-CHOP. Although this study was restricted to patients under the age of 70, an ECOG PS of 3 or lower, and adequate organ function, thiotepabased ASCT is increasingly utilized in older patients.23 However, induction chemotherapy regimens are intense and less well tol erated in older patients in the real-world setting.24 Chimeric antigen receptor (CAR) T-cell therapy has shown prom is ing results in relapsed/refrac tory DLBCL, includ ing in older and unfit patients. There is accumulating data demonstrat ing the efficacy and safety of CAR T cells in CNS lymphoma.25 In SCNSL, the TRANSCEND study dem on strated com plete Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 One point is scored for any of the following: age >60 years, LDH > normal, ECOG performances status >1, stage III/IV disease, extranodal involvement ≥2 sites, kidney and/or adrenal involvement. BCCA, British Colombia Cancer Agency. *High risk group (4-6 factors) overall 2-year risk of CNS relapse of 10.2% in DSHNHL cohort remission (CR) in 3 of 6 patients, with grade 3 neurological events in 2 cases (27).26 Similarly, small (≤8 patients) series of patients with highly refractory SCNSL treated with commercial agents have been reported as having CR rates of approximately 50% with no significant toxicity.27,28 These findings suggest that CAR T cells may be a viable salvage treatment for this challenging pop ulation, especially for patients not fit enough to receive inten sive immunochemotherapy. Furthermore, a number of phase 1/2 studies evaluating CAR T cells in CNS lymphoma are currently ongoing (NCT04608487, NCT04464200, NCT03484702). delivery of IT therapy can be challenging and uncomfortable for the patient, with some evidence to suggest an association with infection-related hospitalization in older patients.30 With an increasing recognition that the majority of CNS relapses in DLBCL involve the brain parenchyma, an area not penetrated by IT therapy alone, IT use has diminished in this setting, with a move toward systemic antimetabolite therapy instead. An exception to this is in testicular DLBCL, where IT therapy may continue to have a role based on data from prospective IELSG trials (see below). Methods for delivery of CNS prophylaxis Intrathecal chemotherapy HD-MTX Approximately 70% to 80% of CNS relapses in DLBCL involve the brain parenchyma,31 and therefore there is a rationale for prophy lactic therapies that cross the blood-brain barrier and penetrate allCNS com part ments. Intravenous HD-MTX has increas ingly been used over the last 10 years as CNS prophylaxis in DLBCL, with initial supporting evidence mainly derived from its efficacy in primary CNS lymphoma. Over the years several studies that have the common theme of being nonrandomized, retrospec tive analyses have addressed this area, but they have been of variable size and have produced discrepant results (Table 3).32-40 While there has been widespread incorporation of HD-MTX as Table 3. Summary of recent studies evaluating use of HD-MTX in DLBCL Study (year) n Design Risk factors Lewis et al32 (2022) 2300 Multicenter, retrospective CNS-IPI ≥4 Testicular, breast involvement DHL Wilson et al33 (2022) 1384 Multicenter, retrospective Orellana-Noia et al34 (2022) 1030 Puckrin et al35 (2021) Systemic treatment CNS Prophylaxis CNS relapse Comments R-CHOP (94%) R-EPOCH (6%) 1. HD-MTX (18%) 2. No HD-MTX (82%) 1. 9.2% (5y) 2. 8.1% (5y) No benefit HD-MTX High-risk EN sites CNS-IPI ≥4 ≥2 EN and LDH ↑ R-CHOP 1. HD-MTX (all, intercalated, or EOT) 1. 5.7% (3y) 2. 5.8% (3y) No difference between EOT and intercalated HD-MTX Multicenter, retrospective Not described R-CHOP (48%) R-EPOCH (45%) Other (7%) 1. HD-MTX (20%) 2. IT (77%) 1. 6.8% 2. 5.4% No benefit HD-MTX vs IT 326 Multicenter, retrospective CNS-IPI ≥4 Testicular DHL LDH ↑ + ECOG >1 + >1 EN R-CHOP (85%) Intensive chemo therapy (15%) 1. HD-MTX (35%) 2. No HD-MTX (65%) 1. 12.2% 2. 11.2% No benefit HD-MTX Bobillo et al36 (2021) 585 Single-center, retrospective CNS-IPI ≥4 High-risk EN sites DHL R-CHOP (68%) R-EPOCH (15%) Other (17%) 1. HD-MTX (7%) 2. IT MTX (43%) 3. None (50%) 1. 7.5% (5y) 2. 5.5% (3y) 3. 5% No benefit (IT or HD-MTX) Ong et al37 (2021) 226 Multicenter, retrospective High-risk EN sites CNS-IPI ≥4 R-CHOP 1. HD-MTX (29%) 2. No HD-MTX (71%) 1. 3.1% (3y, isolated) 2. 14.6% (3y, isolated) HD-MTX signifi cantly reduced risk of isolated CNS relapse Wilson et al38 (2020) 334 Multicenter, retrospective CNS-IPI ≥4 High-risk EN sites ≥2 EN sites and LDH ↑ R-CHOP 1. HD-MTX (all, intercalated, or EOT) 1. 6.8% (3y) 2. 4.7% (3y) No difference between EOT and intercalated HD-MTX Lee et al39 (2019) 130 Single-center, retrospective CNS-IPI ≥4 High-risk EN sites ≥2 EN and LDH ↑ R-CHOP 1. HD-MTX (49%) 2. None (51%) 1. 6.9% (2y) 2. 8.1% (2y) No benefit HD-MTX Goldschmidt et al40 (2019) 480 Multicenter, retrospective High-risk EN sites Stage IV, LDH ↑, ≥1 EN CHOP +/−R (80%) 1. HD-MTX (27%) 2. None (73%) 1. 6.9% 2. 6.3% No benefit HD-MTX Dr Prakash Singh Shekhawat CNS prophylaxis in aggressive B-cell lymphoma | 141 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 For many years, intrathecal (IT) cytotoxic chemotherapy was used as CNS prophylaxis in DLBCL, with supporting evidence derived mainly from nonrandomized, retrospective analyses as well as data extrapolated from other B-cell malignancies. More recently, a systematic review of stand-alone IT prophylaxis ana lyzed a total of 7357 patients treated with anti-CD20 monoclo nal antibody–based immunochemotherapy, incorporating 3 post hoc trial analyses and 10 retrospective studies.29 Overall, IT pro phylaxis was not found to be associated with a reduction in CNS relapse rate on univariable or multivariable analyses. The 142 | Hematology 2022 | ASH Education Program gether or, at the very least, delivering at end of treatment (EOT). An alternative approach for some very high-risk patients may be to use intensified systemic regimens that incorporate HD-MTX— for example, R-CODOX-M/IVAC, which has promising data in a phase 2 trial but has not been demonstrated to be superior to R-CHOP in a randomized trial and is associated with significant toxicity.42 Testicular DLBCL Testicular lymphoma has been associated with a high risk of longterm CNS relapse, with a 5-year risk of 10% and 25% for limited (primary testicular lymphoma [PTL]) and advanced disease in the rituximab era, respectively.6 Biologically, over 75% of testicular lymphomas resemble the ABC subtype and are enriched for the somatic mutations commonly seen in CNS lymphoma, such as MYD88L265P and CD79, which are present in up to 70% of cases. Two prospective studies explored the role of CNS prophylaxis in the rituximab era. The IELSG-10 phase 2 study demonstrated that patients with PTL treated with R-CHOP and contralateral radiation therapy plus 4 doses of IT MTX had a lower risk of CNS relapse compared to historical series (5-year cumulative risk of 6% vs 20%).43 More recently, the IELSG-30 trial included a total of 54 patients with PTL receiving R-CHOP, contralateral radiation therapy, and 2 courses of HD-MTX (dose, 1.5g/m2), along with 4 doses of IT liposomal cytarabine. Preliminary results showed no CNS relapses after a median follow-up of 5 years.44 According to these studies, patients with testicular lymphoma may benefit from CNS prophylaxis incorporating HD-MTX and/or IT chemo therapy. CLINICAL CASE (Continued) Due to the presence of high-risk features for SCNSL, the patient had a baseline MRI head/spine and lumbar puncture with CSF anal y sis (flow cytom e try) with no CNS disease evident. He went on to receive 6 cycles of R-CHOP-21, with intercalated HD-MTX (3 g/m2) planned on day 8 of R-CHOP cycle 2 and cycle 4. No IT prophylaxis was administered. Following the first HDMTX treatment after R-CHOP cycle 2, the patient experienced a 7-day hospital admission with grade 2 renal toxicity and line infection. As a result, cycle 3 R-CHOP was delayed by 10 days. No further HD-MTX was given, and he received the remaining cycles of R-CHOP on sched ule, with end-of-treat ment PETcomputed tomography demonstrating a complete metabolic response. At 12 months’ follow-up, he remains well with no evi dence of systemic or CNS disease relapse. How do we approach CNS prophylaxis in 2022? The above case, treated prior to the publication of the most recent large HD-MTX analyses,32,33 demonstrates the difficul ties in decision-making in this area. The patient had a CNS-IPI score of 5, corresponding to a 2-year risk of CNS relapse of 15% according to the trial data sets used in the formulation of the score. Had his ECOG performance status been 2 instead of 1, the CNS-IPI score would have been 6, conferring an esti mated risk of 33%, although it should be emphasized that only 13 patients were in this category in the CNS-IPI trial data set. However, the patient had an inherently greater risk of systemic Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 prophylaxis for high-risk patients, disagreement has arisen about the safest and most effective way to incorporate it into front line ther apy. The prac tice of “inter ca lated” HD-MTX was first described in a single-center retrospective study of 65 patients in which HD-MTX was delivered at days 10 to 15 in between cycles of R-CHOP, resulting in a CNS relapse rate of 3%.41 While this approach delivers early CNS-directed therapy, potentially advan ta geous given the often early onset of CNS relapse, it intro duces poten tial tox ic ity and delays to sys temic R-CHOP therapy, with many choosing to wait and deliver HD-MTX after R-CHOP completion instead. In the last year, a number of studies in this area have been published to date, arguably providing the most robust data to inform our practice in the absence of prospective clinical trials. These studies have addressed 2 separate questions: (1) Is HDMTX effective?, and (2) How should it be incorporated into front line (R-CHOP/R-CHOP-like) therapy? Lewis et al carried out an international multicenter analysis of 2300 patients deemed at high risk for SCNSL on the basis of high CNS-IPI, the presence of double-hit FISH abnormalities, or the involvement of high risk sites (breast or testicular).32 Patients received either HD-MTX (n=410) with or without concurrent IT therapy, IT therapy alone, or no CNS prophylaxis. There was no significant difference in the 5-year cumulative incidence of CNS relapse between the HD-MTX and no–HD-MTX arms (9.1% vs 8.4%, respectively), with results unchanged when analyses were restricted to patients achieving CR at the end of systemic treatment (5.0% vs 6.0%) and in subanalyses of patients with “ultra”–high-risk character istics. These findings are consistent with those of another large retrospective study by Orellana-Noia et al, in which no reduction in CNS relapse was seen in patients receiving HD-MTX (n = 236) compared to those receiving IT prophylaxis alone (n = 894). Wilson et al reported an international multicenter analysis of 1384 patients, allof whom received HD-MTX CNS prophylaxis delivered either intercalated (n = 749) or at the end of R-CHOP therapy (n = 635).33 There was no dif fer ence in CNS relapse between the 2 delivery approaches (3-year rate of 5.7% vs 5.8%, respectively), with intercalated delivery causing significantly higher rates of R-CHOP delay. Notably, in ana lyses restricted to patients with high CNS-IPI (n = 600), the 3-year rate of CNS relapse was 9.1%, very similar to the rates reported in the original CNS-IPI study in which minimal CNS prophylaxis was used.4 Both these studies carry inherent caveats associated with ret rospective data collection. Notably, the Lewis et al study had a relatively low number of patients in the HD-MTX arm, with poten tial for a signal toward benefit in very high-risk patients being missed as a result. The Wilson et al study had wide variation in the criteria used for selection for CNS prophylaxis, and both data sets contained significant numbers of patients receiving concur rent IT prophylaxis. However, both studies add compelling data to the argument that HD-MTX may not significantly reduce rates of CNS relapse for the majority of patients deemed to be “high risk” by traditional criteria. If the absolute risk reduction of 1% with HD-MTX from the Lewis et al study is accurate, 100 high-risk patients would need to be treated to avoid 1 CNS relapse. HDMTX carries a significant risk of toxicity, including acute kidney injury, mucositis, and hepatotoxicity.38 Considering that systemic treatment failure is a greater risk than CNS relapse, it appears likely that the balance of risks for the vast majority of patients favors prioritizing systemic therapy and forgoing HD-MTX alto treatment failure. He experienced significant toxicity follow ing the first intercalated HD-MTX, resulting in delayed R-CHOP therapy that could potentially have had detrimental effect on systemic disease control. If this patient were to present now for treatment, suggested approaches based on recent data are outlined in Figure 1. The deci sion-mak ing involved essen tially places greater empha sis on baseline screening for occult CNS involvement in high-risk patients, as well as more judicious use of HD-MTX. Future directions and conclusions Although we have seen advances in this extremely contentious area of DLBCL management in the last 5 years, it is clear that we need to continue to develop more specific methods of identi fying patients at highest risk of CNS relapse and to investigate more effective prophylactic interventions for those at highest risk. As outlined above, the use of ctDNA as a baseline screening tool carries much potential for improving patient selection for prophylaxis. The incorporation of novel agents a ble to cross the blood-brain barrier is likely to be an area of ongoing research. Although trials thus far have not demonstrated an overall bene fit with the addition of ibrutinib or lenalidomide to R-CHOP,45,46 stud ies such as REMoDL-A (NCT04546620) inves ti gat ing the addition of acalabrutinib are ongoing, and results with regard to CNS relapse rates will be of interest. Improving systemic dis ease control may be an effective way to reduce CNS relapses, particularly those that occur concurrent with systemic relapse. The recent POLARIX trial dem on strated an addi tional agent (polatuzumab vedotin) that can improve PFS over R-CHOP alone for the first time, raising the question of whether broad adoption of such a frontline regimen could have an impact on CNS events over time.47 Until then, we must use currently available risk-strat ification models to carefully select patients for more stringent baseline screening for CNS disease and exercise greater caution in the use of prophylactic HD-MTX in light of recently published data. Conflict-of-interest disclosure Mat thew R. Wilson: speakers’ bureau: Kite/Gilead, Janssen; consultancy/advisor: Veriton; conference/travel support: Takeda, Janssen, Kite/Gilead; research funding: Abbvie. Sabela Bobillo: speakers’ bureau: Janssen, Roche, Gilead; travel support: Gilead. Kate Cwynarski: consultancy/advisor: Roche, Takeda, Celgene, Atara, Gilead, KITE, Janssen, Incyte; speakers’ bureau: Roche, Takeda, KITE, Gilead, Incyte; research funding: Roche, Takeda, KITE, Janssen, Bristol Myers Squibb. Off-label drug use Matthew R. Wilson: nothing to disclose. Sabela Bobillo: nothing to disclose. Kate Cwynarski: nothing to disclose. Dr Prakash Singh Shekhawat CNS prophylaxis in aggressive B-cell lymphoma | 143 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 Figure 1. Proposed algorithm for CNS prophylaxis in DLBCL in 2022. CMR, complete metabolic response; CT, computed tomography; PD, progressive disease; PR, partial response; SD, stable disease. Correspondence Kate Cwynarski, University College London Hospital, 3rd Fl, West, 250 Euston Rd, London NW1 2PG, United Kingdom; e-mail: kate.cwynarski@nhs.net. References 1. 144 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 Gleeson M, Counsell N, Cunningham D, et al. Central ner vous sys tem relapse of diffuse large B-cell lymphoma in the rituximab era: results of the UK NCRI R-CHOP-14 versus 21 trial. Ann Oncol. 2017;28(10):2511-2516. 2. Ghose A, Elias HK, Guha G, Yellu M, Kundu R, Latif T. Influence of rituximab on central nervous system relapse in diffuse large B-cell lymphoma and role of prophylaxis—a systematic review of prospective studies. Clin Lymphoma Myeloma Leuk. 2015;15(8):451-457. 3. Klanova M, Sehn LH, Bence-Bruckler I, et al. Integration of cell of origin into the clinical CNS International Prognostic Index improves CNS relapse pre diction in DLBCL. Blood. 2019;133(9):919-926. 4. Schmitz N, Zeynalova S, Nickelsen M, et al. CNS International Prognostic Index: a risk model for CNS relapse in patients with diffuse large B-cell lym phoma treated with R-CHOP. J Clin Oncol. 2016;34(26):3150-3156. 5. Savage KJ. Secondary CNS relapse in diffuse large B-cell lymphoma: defin ing high-risk patients and optimization of prophylaxis strategies. Hematology Am Soc Hematol Educ Program. 2017;2017(1):578-586. 6. Kridel R, Telio D, Villa D, et al. Diffuse large B-cell lymphoma with testicular involvement: outcome and risk of CNS relapse in the rituximab era. Br J Haematol. 2017;176(2):210-221. 7. Hu S, Song Y, Li Y, et al. Primary breast diffuse large B cell lymphoma in the rituximab era: outcomes of a multicenter retrospective study by the Lymphoma and Leukemia Committee of Chinese Geriatric Oncology Society (LLC-CGOS). Paper presented at: 58th Annual Meeting of the American Society of Hematology; 3-6 December 2016; San Diego, CA. 8. McKay P, Wilson MR, Chaganti S, et al; British Society of Haematology. The prevention of central nervous system relapse in diffuse large B-cell lym phoma: a British Society for Haematology Good Practice Paper. Br J Haematol. 2020;190(5):708-714. 9. El-Galaly TC, Villa D, Michaelsen TY, et al. The number of extranodal sites assessed by PET/CT scan is a pow er ful pre dic tor of CNS relapse for patients with diffuse large B-cell lymphoma: an international multicenter study of 1532 patients treated with chemoimmunotherapy. Eur J Cancer. 2017;75(April):195-203. 10. Savage KJ, Slack GW, Mottok A, et al. Impact of dual expression of MYC and BCL2 by immunohistochemistry on the risk of CNS relapse in DLBCL. Blood. 2016;127(18):2182-2188. 11. Schmitz R, Wright GW, Huang DW, et al. Genetics and pathogenesis of dif fuse large B-cell lymphoma. N Engl J Med. 2018;378(15):1396-1407. 12. Chapuy B, Stewart C, Dunford AJ, et al. Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat Med. 2018;24(5):679-690. 13. Ollila TA, Kurt H, Waroich J, et al. Genomic subtypes may predict the risk of central nervous system recurrence in diffuse large B-cell lymphoma. Blood. 2021;137(8):1120-1124. 14. Wilson WH, Bromberg JE, Stetler-Stevenson M, et al. Detection and out come of occult leptomeningeal disease in diffuse large B-cell lymphoma and Burkitt lymphoma. Haematologica. 2014;99(7):1228-1235. 15. Hiemcke-Jiwa LS, Leguit RJ, Snijders TJ, et al. MYD88 p.(L265P) detection on cell-free DNA in liquid biopsies of patients with primary central nervous system lymphoma. Br J Haematol. 2019;185(5):974-977. 16. Mutter JA, Alig S, Lauer EM, et al. Profiling of circulating tumor DNA for noninvasive disease detection, risk stratification, and MRD monitoring in patients with CNS lymphoma. Blood. 2021;138(suppl 1):6. 17. Bobillo S, Crespo M, Escudero L, et al. Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involve ment of B-cell lymphomas. Haematologica. 2021;106(2):513-521. 18. Olszewski AJ, Chorzalska AD, Petersen M, et al. Detection of clonotypic DNA in the cerebrospinal fluid as a marker of central nervous system inva sion in lymphoma. Blood Adv. 2021;5(24):5525-5535. 19. El-Galaly TC, Cheah CY, Bendtsen MD, et al. Treatment strategies, out comes and prognostic factors in 291 patients with secondary CNS involve ment by diffuse large B-cell lymphoma. Eur J Cancer. 2018;93(April):57-68. 20. Ferreri AJ, Donadoni G, Cabras MG, et al. High doses of antimetabolites followed by high-dose sequential chemoimmunotherapy and autologous stem-cell transplantation in patients with systemic B-cell lymphoma and secondary CNS involvement: final results of a multicenter phase II trial. J Clin Oncol. 2015;33(33):3903-3910. 21. Doorduijn JK, van Imhoff GW, van der Holt B, et al. Treatment of sec ondary central nervous system lymphoma with intrathecal rituximab, high-dose methotrexate, and R-DHAP followed by autologous stem cell transplantation: results of the HOVON 80 phase 2 study. Hematol Oncol. 2017;35(4):497-503. 22. Ferreri AJM, Doorduijn JK, Re A, et al; International Extranodal Lymphoma Study Group. MATRix-RICE therapy and autologous haematopoietic stemcell transplantation in diffuse large B-cell lymphoma with secondary CNS involvement (MARIETTA): an international, single-arm, phase 2 trial. Lancet Haematol. 2021;8(2):e110-e121. 23. Khwaja J, Schorb E, Goradia H, et al. International multi-cen tre ret ro spec tive anal y sis of out comes of thio tepa-based autol o gous stem cell transplantation for secondary CNS lymphoma. European Hematology Association Open Access Library [poster abstract]. 12 June 2020. Abstract EP1376. 24. Schorb E, Fox CP, Kasenda B, et al. Induction therapy with the MATRix reg imen in patients with newly diagnosed primary diffuse large B-cell lym phoma of the central nervous system—an international study of feasibility and efficacy in routine clinical practice. Br J Haematol. 2020;189(5):879887. 25. Frigault MJ, Dietrich J, Gallagher K, et al. Safety and efficacy of tisagenlecleucel in pri mary CNS lym phoma: a phase 1/2 clin i cal trial. Blood. 2022;139(15):2306-2315. 26. Abramson JS, Palomba ML, Gordon LI, et al. Lisocabtagene maraleucel for patients with relapsed or refrac tory large B-cell lym pho mas (TRANSCEND NHL 001): a multicentre seam less design study. Lancet. 2020;396(10254):839-852. 27. Frigault MJ, Dietrich J, Martinez-Lage M, et al. Tisagenlecleucel CAR T-cell therapy in secondary CNS lymphoma. Blood. 2019;134(11):860-866. 28. Ahmed G, Hamadani M, Shah NN. CAR T-cell therapy for secondary CNS DLBCL. Blood Adv. 2021;5(24):5626-5630. 29. Eyre TA, Djebbari F, Kirkwood AA, Collins GP. Efficacy of central nervous system prophylaxis with stand-alone intrathecal chemotherapy in diffuse large B-cell lym phoma patients treated with anthracycline-based che mo ther apy in the rituximab era: a sys tem atic review. Haematologica. 2020;105(7):1914-1924. 30. Eyre TA, Kirkwood AA, Wolf J, et al. Stand-alone intrathecal central ner vous system (CNS) prophylaxis provide unclear benefit in reducing CNS relapse risk in elderly DLBCL patients treated with R-CHOP and is associ ated increased infection-related toxicity. Br J Haematol. 2019;187(2):185194. 31. Kansara R, Villa D, Gerrie AS, et al. Site of central nervous system (CNS) relapse in patients with diffuse large B-cell lymphoma (DLBCL) by the CNSIPI risk model. Br J Haematol. 2017;179(3):508-510. 32. Lewis KL, Jakobsen LH, Villa D, et al. High-dose methotrexate is not asso ciated with reduction in CNS relapse in patients with aggressive B-cell lymphoma: an international retrospective study of 2300 high-risk patients. Blood. 2021;138(suppl 1):181. 33. Wilson MR, Eyre TA, Kirkwood AA, et al. Timing of high-dose methotrex ate CNS prophylaxis in DLBCL: a multicenter international analysis of 1384 patients. Blood. 2022;139(16):2499-2511. 34. Orellana-Noia VM, Reed DR, McCook AA, et al. Single-route CNS prophy laxis for aggressive non-Hodgkin lymphomas: real-world outcomes from 21 US academic institutions. Blood. 2022;139(3):413-423. 35. Puckrin R, El Darsa H, Ghosh S, Peters A, Owen C, Stewart D. Ineffectiveness of high-dose methotrexate for prevention of CNS relapse in diffuse large B-cell lymphoma. Am J Hematol. 2021;96(7):764-771. 36. Bobillo S, Joffe E, Sermer D, et al. Prophylaxis with intrathecal or high-dose methotrexate in diffuse large B-cell lymphoma and high risk of CNS relapse. Blood Cancer J. 2021;11(6):113. 37. Ong SY, de Mel S, Grigoropoulos NF, et al. High-dose methotrexate is effec tive for prevention of isolated CNS relapse in diffuse large B cell lymphoma. Blood Cancer J. 2021;11(8):143. 38. Wilson MR, Eyre TA, Martinez-Calle N, et al. Timing of high-dose metho trexate CNS prophylaxis in DLBCL: an analysis of toxicity and impact on R-CHOP delivery. Blood Adv. 2020;4(15):3586-3593. 39. Lee K, Yoon DH, Hong JY, et al. Systemic HD-MTX for CNS prophylaxis in high-risk DLBCL patients: a prospectively collected, single-center cohort analysis. Int J Hematol. 2019;110(1):86-94. 40. Goldschmidt N, Horowitz NA, Heffes V, et al. Addition of high-dose metho trexate to standard treatment for patients with high-risk diffuse large B-cell lymphoma contributes to improved freedom from progression and survival but does not prevent central nervous system relapse. Leuk Lymphoma. 2019;60(8):1890-1898. cin, vincristine, and prednisone in non-germinal center B-cell diffuse large B-cell lymphoma. J Clin Oncol. 2019;37(15):1285-1295. 46. Nowakowski GS, Chiappella A, Gascoyne RD, et al. ROBUST: a phase III study of lenalidomide plus R-CHOP ver sus pla cebo plus R-CHOP in previously untreated patients with ABC-type diffuse large B-cell lymphoma. J Clin Oncol. 2021;39(12):1317-1328. 47. Tilly H, Morschhauser F, Sehn LH, et al. Polatuzumab vedotin in previously untreated diffuse large B-cell lymphoma. N Engl J Med. 2022;386(4):351-363. 48. National Comprehensive Cancer Network. Clinical prac tice guide lines in oncology: B-cell lymphomas. Version 3.2022. https://www.nccn.org /professionals/physician_gls/pdf/b-cell.pdf. Accessed 25 April 2022. 49. Tilly H, Gomes da Silva M, Vitolo U, et al; ESMO Guidelines Committee. Diffuse large B-cell lym phoma (DLBCL): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015;26 (suppl 5):v116-v125. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000331 Dr Prakash Singh Shekhawat CNS prophylaxis in aggressive B-cell lymphoma | 145 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/138/2021790/138wilson.pdf by guest on 09 December 2022 41. Abramson JS, Hellmann M, Barnes JA, et al. Intravenous methotrexate as central nervous system (CNS) prophylaxis is associated with a low risk of CNS recurrence in high-risk patients with diffuse large B-cell lymphoma. Cancer. 2010;116(18):4283-4290. 42. McMillan AK, Phillips EH, Kirkwood AA, et al. Favourable outcomes for highrisk diffuse large B-cell lymphoma (IPI 3-5) treated with front-line R-CODOXM/R-IVAC chemotherapy: results of a phase 2 UK NCRI trial. Ann Oncol. 2020;31(9):1251-1259. 43. Vitolo U, Chiappella A, Ferreri AJ, et al. First-line treatment for primary testicular diffuse large B-cell lymphoma with rituximab-CHOP, CNS pro phylaxis, and contralateral testis irradiation: final results of an international phase II trial. J Clin Oncol. 2011;29(20):2766-2772. 44. Conconi A, Chiappella A, Orsucci L, et al. Intensified (intravenous and intrathecal) CNS prophylaxis in primary testicular diffuse large B-cell lym phoma: 5-year results of the IELSG30 trial. Hematol Oncol. 2021; 39(suppl S2). 45. Younes A, Sehn LH, Johnson P, et al; PHOENIX Investigators. Randomized phase III trial of ibrutinib and rituximab plus cyclophosphamide, doxorubi CONTROVERSIES IN AGGRESSIVE NHL Sequencing therapy in relapsed DLBCL Department of Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX; and 2Lymphoma and Myeloma, Dana-Farber Cancer Institute, Boston, MA 1 Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy worldwide, comprising approximately 30% of all lymphomas. Currently, 50% to 60% of patients diagnosed with DLBCL are alive at 5 years and cured with modern therapy, but about 10% to 15% of patients are refractory to first-line therapy, and an additional 20% to 30% relapse following a complete response. Patients who have relapses beyond 2 years may experience more favorable outcomes and have forms of DLBCL that can be distinguished biologically. Patients who experience early relapse or who have primary refractory disease (less than a complete response or relapse within 3 to 6 months of initial therapy) have worse outcomes. For decades, the standard of care treatment strategy for fit patients with relapsed DLBCL has been salvage therapy with non–cross-resistant combination chemoimmunotherapy regimens followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT) as stem cell rescue for patients with chemosensitive disease. Recent data suggest that certain patients may benefit from chimeric antigen receptor T-cell therapy (CAR T) in the second-line setting. Additional novel therapies exist for patients who are ineligible, who are unable to access these therapies, or who fail ASCT and/or CAR T. Despite the advent of new therapies for DLBCL and improved outcomes, DLBCL remains a lifethreatening illness. Thus, it is essential for clinicians to engage in serious illness conversations with their patients. Goalsof-care communication can be improved through skills-based training and has been demonstrated to have an impact on patient experiences. LEARNING OBJECTIVES • Recognize the therapeutic options available for patients with relapsed/refractory DLBCL • Understand the triggers and process for goals-of-care discussions for patients with relapsed/refractory DLBCL Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy worldwide, comprising approximately 30% of all lymphomas. Currently, 55% to 60% of patients diagnosed with DLBCL are alive at 5 years and cured with modern therapy,1 but approximately 10% to 15% of patients are refractory to first-line therapy, and an additional 20% to 30% relapse following a complete response (CR).2,3 This high-risk DLBCL population constitutes approximately 11 000 individuals annually in the United States.3,4 Evidence suggests that molecular subgroups of DLBCL exhibit significantly different biology, response to standard therapies, and overall survival (OS) based on the cell of origin.5,6 Standard of care first-line therapy for DLBCL has been based on French and US randomized controlled trials (RCTs) completed in 2001 demonstrating that the addition of rituximab (R) to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) combination chemotherapy improved outcomes over CHOP.7,8 Since then, numerous RCTs have failed to improve upon R-CHOP, including trials targeting 146 | Hematology 2022 | ASH Education Program patients with poor-risk clinical features by cell-of-origin subtype.9-16 Recently, a double-blind, placebo-controlled, international RCT compared a modified regimen of R-CHOP (pola-R-CHP), in which vincristine was replaced with polatuzumab vedotin as a first-line therapy for patients with intermediate-risk or high-risk DLBCL according to the International Prognostic Index (IPI).17 Progression-free survival (PFS) was significantly higher in the pola-R-CHP group than in the R-CHOP group (stratified hazard ratio [HR], 0.73; 95% CI, 0.57-0.95) with 2-year PFS of 76.7% (95% CI, 72.7-80.8) vs 70.2% (95% CI, 65.8-74.6) for pola-R-CHP and R-CHOP, respectively. The safety profile was similar in the 2 regimens. Changes in the frontline management of DLBCL based upon these results would likely have an impact on therapeutic decisions in the relapse setting (Table 1). DLBCL prognosis and social determinants of health The hypothetical stories of Alvin and Wilma are based on real patient scenarios and illustrate some of the key challenges faced by patients with newly diagnosed and Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 Christopher R. Flowers1 and Oreofe O. Odejide2 Table 1. A tale of 2 patients Alvin was born in 1955 in a large city that borders the ocean. In 2017 he was diagnosed with DLBCL, not otherwise specified. His diagnostic tumor block underwent IHC analysis, which revealed this to be a non-GCB subtype without evidence of MYC or BCL2 translocation. He had elevated LDH greater than 2 times the upper limit of normal and kidney and bone marrow involvement. Wilma was born in a rural town in the southern United States in the same year as Alvin. She remembers watching how her small town changed over time after the Fair Housing Act. She was diagnosed with non-GCB DLBCL in the same year as Alvin. She also had elevated LDH and bone marrow but no kidney involvement. First-line therapy He had excellent performance status (ECOG PS 0), was offered a randomized controlled trial as initial therapy, was randomized to an arm where he received R-CHOP, and started treatment 18 days after diagnosis. She was self-employed and did not have insurance at the time of diagnosis. She was unable to conduct any work activities (ECOG PS 2) and was admitted to the hospital to start R-CHOP at 13 days after diagnosis. Treatment at relapse In early 2019 (11 months following R-CHOP), Alvin noticed a lymph node in his left axilla. A biopsy showed relapse of non-GCB DLBCL. He was able to purchase genomic testing using an available solid-tumor panel, but it did not provide information that his treating physician thought relevant to guide therapy. His physician performed HLA testing for Alvin and his siblings, and Alvin underwent salvage therapy with RICE (achieving a partial response) followed by ASCT based on the best available evidence. Last year follow-up imaging revealed evidence of relapsed disease with renal involvement (serum creatinine 2.1), and chemoimmunotherapy was restarted. Alvin was referred for CAR T therapy. He is now in your office awaiting scan results 6 months following completion of treatment. Wilma had difficulty with fatigue, alopecia, and peripheral neuropathy during her treatment but recovered to feeling as good as she had several years prior to her diagnosis. She returned to work for 34 months and then began to feel progressively more tired and unable to perform her usual activities. She went back to her treating physician and had repeat scans that showed evidence of DLBCL relapse. Her physician referred her to a center more than 250 miles away to consider ASCT. She connected Wilma with a social worker who aided in garnering state-based insurance for cancer patients. A patient navigator at the center coordinated visits and recommended places for Wilma and her caregiver to stay around the center for the period immediately following ASCT. She now returns to see you 2.5 years after ASCT feeling well. HLA, human leukocyte antigen; RICE, rituximab, ifosfamide, carboplatin, etoposide. relapsed DLBCL. Both patients were diagnosed with stage IV non–germinal center B-cell (GCB) DLBCL with poor prognostic features and an IPI score of 4, placing both in the high-risk cate gory based on similar features. A recent analysis comparing the IPI, revised IPI, and National Comprehensive Cancer Network IPI involved 2124 DLBCL patients treated with R-CHOP or a variant in 1 of 7 multicenter RCTs. These data demonstrated that DLBCL patients in the high-IPI category had 5-year PFS of 45.8% (95% CI, 41.1%-51.0%) and 5-year OS of 53.9% (95% CI, 49.3%-59.1%).18 Although immunohistochemistry (IHC) algorithms have been available for more than a decade and gene expression–based approaches utilizing formalin-fixed paraffin-embedded tissue have been demonstrated to reliably classify DLBCL into biologically and clinically distinct subgroups,19-23 these tests have had little impact on clin ical management for patients with newly diag nosed or relapsed DLBCL. Based on gene expression, patients with acti vated B-cell (ABC) DLBCL (the majority of non-GCB DLBCL) had 5-year PFS of 48% and 5-year OS of 56%, and the cell of origin was identified in this study to have prognostic significance inde pendent of IPI.23 Additional studies found that significant genomic diversity exists within the ABC and GCB subgroups and defined genomic subtypes with prognostic value and potential therapeu tic implications.24-26 Currently, there are no clinical assays that can comprehensively and accurately evaluate the genomic profile of DLBCLs with a short turnaround so that this molecular information can be incorporated into clinical workflow and practice. Additionally, stud ies indi cate that patients diag nosed with DLBCL from rural areas of the United States and that uninsured patients (HR, 1.39; 95% CI, 1.14-1.70) and Medicaid-insured patients (HR, 1.48; 95% CI, 1.23-1.78) with DLBCL had lower survival com pared with patients who had private insurance.27,28 Moreover, rural patients, less commonly, had private insurance and high socio economic status and were more likely to receive treatment within 14 days of diagnosis.27 Accounting for these social determinants of health is critical in understanding the prognosis for individuals with DLBCL and developing suitable management strategies for first-line therapy and relapse disease. As demonstrated by these patient situations, these sociodemographic factors influence the means to access therapies that can be curative. It is also important to note that even when social factors make therapies more diffi cult to access, patients can be supported using evidence-based approaches like patient nav i ga tion that have a dem on strated impact on outcomes in the first-line and relapse setting.29 Other important studies showed that a shorter diag nosisto-treatment interval (DTI) was associated with poor prognos tic factors. DLBCL patients initiating treatment within 14 days had higher lactate dehydrogenase (LDH) levels, worse Eastern Cooperative Oncology Group Performance Status (ECOG PS), more frequent B symptoms, more bulky disease, and a worse age-adjusted IPI.30 A shorter DTI was associated with worse out comes in both ABC and GCB subgroups, was prognostic even after adjusting for the IPI, and was a pre dic tor of sur vival in cohorts across the United States, Europe, Canada, and Japan.30-33 Interestingly, a recent study showed that higher levels of circu lating tumor DNA (ctDNA) were associated with shorter DTI and similarly predicted such outcomes, providing a biological path way for understanding these worse outcomes and a potential biomarker for intervention.31 However, there is limited applicabil ity of ctDNA in routine clinical practice currently. Conventional management strategies for patients with relapsed DLBCL For the majority of patients who relapse, this occurs within the first 2 years of ini tial ther apy. Patients who expe ri ence early Dr Prakash Singh Shekhawat Sequencing therapy in relapsed DLBCL | 147 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 Initial presentation 148 | Hematology 2022 | ASH Education Program Chimeric antigen receptor T-cell therapy Current approved chimeric antigen receptor T-cell (CAR T) thera pies are genetically engineered cell products targeting the B-cell marker CD19 that are available for patients with R/R DLBCL after at least 2 lines of therapy. These include axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), and lisocabtagene maraleucel (liso-cel). These cellular therapies may differ in their mech anisms of viral transfection, costimulatory domains, and T-cell selection, but each has demonstrated the ability to produce durable remissions in about 30% of patients, including patients who have failed prior ASCT.45-47 Axi-cel uses the CD28 costimulatory domain that interacts with the CD3-zeta activation domain to enhance activation and proliferation of CAR T cells. Tisa-cel and liso-cel have a similar extracellular CD19 single-chain variable fragment attached to a 4-1BB intracellular costimulatory domain connected to a CD3ζ signaling domain. Liso-cel has a distinct manufacturing process involving the separation of CD4 and CD8 T cells after leukapheresis-independent transduction, expansion, and administration to patients at equal target concentrations. JULIET was an international phase 2 study of tisa-cel in 93 patients with R/R DLBCL who were ineligible for or had disease progression after ASCT.47 The best overall response rate (ORR) was 52% (95% CI, 41%-62%). Grade 3 or 4 adverse events of interest included cytokine release syndrome (CRS) in 22%, neu rological events in 12%, and cytopenias lasting more than 28 days in 32%. In a subsequent study of this patient group at a median follow-up of 40.3 months, the ORR was 53% and the PFS at 3 years was 77.8% for patients in CR at 6 months.48 Among 111 patients enrolled in ZUMA-1, axi-cel was manufactured for 110 patients and administered to 101 patients.46 The objective response rate was 82%, and the CR rate was 54%. Grade 3 or higher CRS and neurological events occurred in 13% and 28% of patients, respectively. In a subsequent study of this patient group at a median follow-up of 27 months, the median duration of response for all101 patients was 11 months and the median dura tion of response for par tic i pants achiev ing CR was not reached.49 In the TRANSCEND study, 344 patients underwent leukapheresis for the manufacture of CAR T cells, and 269 patients received at least 1 dose of liso-cel.45 Of 256 patients evaluable for response, an objective response was observed in 73% (95% CI, 66.8%–78.0%) and a CR in 53% (95% CI, 46.8%–59.4%). Grade 3 or greater CRS and neurological events occurred in 2% and 10% of patients, respec tively. Together these results dem on strate meaningful and durable responses for patients with R/R DLBCL, including those who have failed prior ASCT. This remains a via ble alternative for patients who can access these therapies. As described in the patient cases, there may be barriers for certain patient groups, such as those who live in rural areas or who lack insurance coverage for CAR T-cell therapy. Additional measures are necessary to ensure equitable access to potentially curative therapy for allpatients. Common lymphoma-specific bridging therapy administered between leukapheresis and CAR T-cell therapy includes steroids, chemotherapy, targeted therapy, or radiation therapy. The opti mal approach for bridging therapy prior to CAR T-cell therapy remains uncertain.3,50-53 Data from 17 academic institutions involved in the US Lymphoma CAR T Consortium showed that bridging therapy was administered to 53% of 298 patients who underwent leukapheresis prior to planned axi-cel. Among these patients Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 relapse or who have primary refractory disease (less than a CR or a relapse within 3 to 6 months to initial therapy) have worse outcomes.1,34,35 Although patients can be cured with treatments fol low ing these early events, most of these patients die from DLBCL. An IPI for relapsed/refractory DLBCL (R/R IPI) was devel oped using 1234 of 5112 patients treated with front line immunochemotherapy in the SEAL con sor tium who expe ri enced progressive disease.36 This study found that time to progression on immunochemotherapy and age at progression were strongly associated with postprogression OS. A prognostic model involv ing both variables had good concordance in the discovery (0.67) and validation sets (cohort 1, c = 0.64; cohort 2, c = 0.68). A cor re spond ing nomo gram is avail able to aid in esti mat ing prog nosis for R/R DLBCL.36 An electronic calculator of the model is also available at https://qxmd.com/calculate/calculator_682 /prognosis-calculator-for-relapsed-refractory-dlbcl and as a smartphone app. Patients who have relapses beyond 2 years may experience more favorable outcomes and have forms of DLBCL that can be distinguished biologically.37 However, despite providing valuable prognostic and predictive information, molecular classification assays including IHC, gene expression profiling, and sequenc ing panels have faced significant challenges, precluding their transition into routine clinical workflow and practice.21-23,38,39 Even when such biomarkers are available, they have had limited impact on therapeutic decision-making in the relapsed setting. Validated, predictive biomarkers will be an integral component of future personalized medicine strategies for DLBCL.39-41 For decades the standard of care treatment strategy for fit patients with relapsed DLBCL has been salvage therapy with non– cross-resistant combination chemoimmunotherapy regimens followed by high-dose chemotherapy and ASCT as stem cell res cue for patients with chemosensitive disease.42 The pivotal RCTs supporting the modern practice of stem cell transplantation for patients with large B-cell lymphoma include the Parma trial that was conducted in the era before the availability of rituximab and the current World Health Organization lymphoma classifi cation. This study randomized patients with R/R intermediateto high-grade non-Hodgkin lymphoma with chemosensitivity to proceed with either autologous stem cell transplantation (ASCT) or additional chemotherapy with dexamethasone, high-dose cytarabine, and cisplatin (DHAP). It demonstrated that high-dose chemotherapy followed by ASCT was associated with improved event-free survival (EFS) at 5 years of 46%, vs 12% with DHAP.42 Importantly, the CORAL trial and the LY.12 trial designed to com pare DHAP to alternative platinum-containing salvage regimens identified that patients with R/R disease within 12 months of diagnosis and prior exposure to rituximab had poor PFS follow ing salvage therapy.43,44 These findings indicate that the prior demonstrated benefits of ASCT were reduced in the R-CHOP era due to resistance to chemoimmunotherapy, identified by early first-line treatment failure. These poor outcomes associated with early relapse of DLBCL are evident in Alvin’s situation. However, ASCT can provide benefits, particularly for patients like Wilma who experience late relapse and have chemosensitive disease. Recent evidence-based guides suggest that ASCT remains a pre ferred option for patients who experience late relapse.3 Based on the R/R IPI calculator, the predicted 2-year survival from pro gression for Alvin and Wilma are 34% and 60%, respectively.36 bridging regimen as an event if it occurred within the first 12 weeks. Notably, all 3 trials demonstrated very poor out comes for patients with early R/R LBCL who underwent SOC salvage therapy and ASCT. A vibrant, detailed, and timely dis cussion of these trials and their implications for management of patients with early relapsed LBCL was recently presented in Blood by Drs Sehn and Westin.3 They also provide a useful algorithm for second-line therapy decision-making for DLBCL. Patients ineligible for ASCT or CAR T therapy or who relapse after ASCT and CAR T therapy While ASCT and CAR T have demonstrated durable remissions for patients who respond to treatment, patients who are ineligible, unable to access these therapies, or who fail treatment often expe ri ence poor out comes. DLBCL patients in these cir cum stances have multiple options from recently approved thera pies, includ ing loncastuximab tesirine, polatuzumab vedotin, selinexor, and tafasitamab. However, limited data exist regard ing outcomes following CAR T therapy. Loncastuximab tesirine is a CD19-directed antibody-drug conjugate that was evaluated in an international, multicenter phase 2 trial (LOTIS-2) in patients with R/R DLBCL following 2 or more lines of therapy. Among 145 patients who received at least 1 dose of loncastuximab, the ORR was 48.3% (95% CI, 39.9-56.7), including 35 patients (24%) who experienced a CR.60 The most common grade 3 or higher adverse events were neutropenia in 26%, thrombocytopenia in 18%, and increased γ-glutamyltransferase in 17%. A matchingadjusted indirect comparison was performed to evaluate the efficacy of loncastuximab compared with chemoimmunotherapy in R/R DLBCL.61 In this analysis, 80 patients from LOTIS-2 were matched by characteristics to 278 patients from pooled extension studies from CORAL, demonstrating an ORR of 53.4% in the loncastuximab cohort compared with 40.3% for chemoimmunotherapy. As exemplified below, real-world evidence com parisons are becoming more common in settings like R/R DLBCL where randomized studies are uncommon. A detailed discussion of these analyses is beyond the scope of this text, but they merit additional consideration. The antibody-drug conjugate polatuzumab vedotin targets CD79b, a component of the B-cell receptor that is expressed on 95% of DLBCLs. Patients with R/R DLBCL ineligible for ASCT (n = 80) were randomized to receive polatuzumab vedotin com bined with bendamustine and rituximab (pola-BR) or bendamustine and rituximab (BR).62 Patients who received pola-BR had a significantly higher independent review committee–assessed CR rate (40.0% vs 17.5%; P = .026), PFS (median, 9.5 vs 3.7 months; P<.001), and OS (median, 12.4 vs 4.7 months), with a median followup of 22.3 months. Pola-BR was associated with higher rates of grade 3 to 4 neutropenia (46.2% vs 33.3%), anemia (28.2% vs 17.9%), and thrombocytopenia (41% vs 23.1%). In a follow-up single-arm extension cohort, 106 addi tional patients received pola-BR, demonstrating an objective response rate of 41.5% and a CR rate of 38.7%.63 Median PFS and OS were 6.6 months and 12.5 months, respectively. No new safety signals with pola-BR were identified, and differences between the randomized arms persisted with additional follow-up. A biomarker analysis led by Dr Herrera ran a ctDNA assay based on a customized panel of recurrently mutated genes in DLBCL at baseline and end of treatment.64 Higher baseline ctDNA levels were associated with poor prognostic factors and were an independent predictor of Dr Prakash Singh Shekhawat Sequencing therapy in relapsed DLBCL | 149 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 23% received ste roids alone, 54% received che mo ther apy, 12% received radi a tion ther apy, and 10% received targeted therapy.54 A multivariable analysis in this data set revealed that bridging was associated with inferior OS. However, the need for bridging may be a surrogate for greater disease burden or more aggressive lymphoma. A study by Pinnix and colleagues sug gests that radiation may be an effective approach when bridg ing is required,50 but additional studies are needed in this area. Recent data suggest that patients such as Alvin may bene fit from CAR T-cell therapy in the second-line setting. Given the results discussed above and real-world comparisons to cohorts of patients with R/R DLBCL,55,56 3 RCTs compared CAR T therapy in the second-line setting to the standard of care (SOC) strategy of salvage chemoimmunotherapy and ASCT for patients with chemosensitive disease: ZUMA-7 (axi-cel), Belinda (tisa-cel), and TRANSFORM (liso-cel).57-59 All trials enrolled patients with large B-cell lymphoma who relapsed within 12 months from comple tion of or refractory to first-line therapy and were considered candidates for ASCT by the treating physician. In ZUMA-7, 180 patients were randomized to receive axi-cel and 179 to receive SOC.58 The ORR and CR rate were signif icantly greater in the axi-cel group (83% and 65%) compared with the SOC group (50% and 32%). At a median follow-up of 24.9 months, the median EFS was 8.3 months for patients who received axi-cel and 2.0 months for patients who received SOC, and the 24-month EFS was 41% and 16%, respectively. Estimated OS at 2 years was 61% in the axi-cel group and 52% for SOC. Grade 3 or higher adverse events occurred in 91% of the patients who received axi-cel and in 83% of those who received SOC. In the TRANSFORM trial, 92 patients were randomized to receive liso-cel and 92 patients to receive SOC.59 The median EFS was 10.1 months for liso-cel vs 2.3 for SOC (HR, 0.349; P<.0001), the median PFS was 14.8 months and 5.7 months, and the CR rate was 66% and 39%, respec tively. In the group receiv ing lisocel, CRS grade 1 occurred in 37%, grade 2 in 11%, and grade 3 in 1 patient. Unlike the other 2 trials, the Belinda study found that CAR T-cell therapy was not superior to SOC salvage ther apy.57 Patients were randomly assigned to receive tisa-cel with optional bridging therapy or salvage chemotherapy and ASCT as SOC. Approximately 26% of the patients in the tisa-cel group experienced progression at week 6, compared with approxima tely 14% of the patients who received SOC. Nearly 96% of the patients in the tisa-cel group received tisa-cel, and 32.5% of the patients in the SOC group received ASCT. Response occurred for 46.3% of patients receiving tisa-cel vs 42.5% of patients receiv ing SOC. The median EFS was 3.0 months in both groups. Some differences in trial design that may explain these results include that Belinda allowed for mul ti ple cycles of bridging therapy, including switching bridging therapy, whereas TRANSFORM allowed for only 1 cycle of bridging, and ZUMA-7 allowed for stabilization with steroids but no bridging therapy. In Belinda bridging chemotherapy was administered to 83% of the patients receiving tisa-cel; 48% received more than 1 cycle, and 12% received more than 1 regimen. Patients enrolled in TRANSFORM and Belinda underwent leukapheresis before randomization and were allowed to cross over from SOC. No crossover was planned in ZUMA-7, but patients were permitted to access approved CAR T-cell therapies outside the protocol. As a statistical nuance, in the assessment of EFS Belinda did not count the initiation of a second salvage or Clinical trials involving bispecific antibodies Bispecific antibodies binding with 2 different cell surface anti gens can direct cytotoxic T cells and other immune effector cells to areas in proximity of lymphoma cells. Several bispecific antibodies binding CD3 and CD20 are in development for DLBCL, including mosunetuzumab, glofitamab, e pcoritamab, plamotamab, and odronextamab, but have some d ifferences in structure, route of administration, and treatment schedule.71-75 Across agents, common adverse events include neutropenia, 150 | Hematology 2022 | ASH Education Program thrombocytopenia, CRS, hypophosphatemia, fatigue, and diarrhea. CRS neurological adverse events were mostly low grade and con fined to cycle 1. Among 129 patients with R/R aggressive B-cell lymphoma (including 82 patients with DLBCL) treated with mosunetuzumab, the ORR was 34.9%, and the CR rate was 19.4%, with a median duration of response for allresponders and complete responders of 7.6 months and 22.8 months, respectively.71 Among 19 patients who had previously undergone CAR T-cell therapy, the ORR and CR rate were 36.8% and 26.3%, respectively, but the duration of response could not be estimated due to the small sample size. In a study of 107 patients who received glofitamab, most patients were refractory to a prior CD20 antibody–containing regimen (85%), many were refractory to their initial therapy (59%), and 32% had prior CAR T. The ORR was 50%, and CR rates were 32% and 37%, respectively, among patients with and with out prior CAR T.76 Epcoritamab, a subcutaneously administered CD3xCD20 bispecific antibody,73 odronextamab (REGN1979), a fully humanized bispecific immunoglob u lin G4 anti body with a 14-day half-life,74 and plamotamab (XmAb13676) also have been examined in early-phase clinical trials, demonstrating meaningful ORRs and relatively similar adverse event profiles.75 For instance, a study of 157 patients who received epcoritamab demonstrated ORRs of 69% for CAR T–naive patients and 54% for patients who received prior CAR T.77 Future studies are needed to clarify the role and sequenc ing of bispecific antibodies rel a tive to the cur rent therapies available for R/R DLBCL. Goals-of-care discussions for patients with DLBCL Although the advent of new therapies for DLBCL has improved survival outcomes, up to 40% of patients develop R/R disease.2,3 This emphasizes that DLBCL is still a life-threatening illness, and it is thus essential for clinicians to engage in serious illness con versations with their patients. Goals-of-care discussions entail eliciting patients’ goals, values, and preferences regarding their overall care as well as end-of-life (EOL) options. Ideally, these discussions should occur early and often in the DLBCL disease trajectory and should not be merely limited to EOL time points.78 Patients who have the opportunity to engage in goals-of-care discussions with their physicians are significantly more likely to receive care that is aligned with their preferences and experi ence higher-quality EOL care.79,80 Hematologic oncologists have an important role in initiating these conversations with patients given the trust built through longitudinal patient-physician rela tionships. In a retrospective study of 383 patients with hemato logic malignancies, of which 37% had lymphoma, patients who had their first goals-of-care discussion with a hematologic oncol ogist (vs other clinicians) were significantly more likely to enroll in hospice more than 3 days before death and had lower odds of admission to the intensive care unit in the last month of life or death in the hospital.81 Such EOL outcomes are associated with a better quality of life for patients and their families, as well as a lower risk of complicated grief for bereaved caregivers.82 Goals-of-care discussions are critical and beneficial for patients with DLBCL, yet these discussions often occur too late or not at all. In a study of patients with aggressive lymphoma relapsed after first- or second-line treatment, while 44.4% had thought of their care preferences in the event of becoming crit ically ill, only a quarter of these patients had discussed these Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 shorter PFS, suggesting that ctDNA may be a tool for identifying patients at higher risk of progression. Selinexor is an oral selective inhibitor of nuclear export that works by binding to exportin 1. An international multicenter phase 2 trial (SADAL) evaluated selinexor given on days 1 and 3 weekly until disease progression or unacceptable toxicity. Among 127 patients who received selinexor at 60 mg and were included in analyses of primary outcome and safety, the ORR was 28%, and the CR rate was 12%.65 The most common grade 3 to 4 adverse events were thrombocytopenia (46%), neutropenia (24%), ane mia (n = 22%), and fatigue (n = 11%). Additional analyses have been performed for biological and prior-treatment subgroups.52,66 Tafasitamab is a humanized, anti-CD19 monoclonal antibody. In the international multicenter phase 2 trial L-MIND, patients with R/R DLBCL ineligible for ASCT received tafasitamab and lenalidomide for up to 12 cycles followed by tafasitamab monotherapy (in patients with stable disease or better) until disease progres sion.67,68 Among 80 patients who received tafasitamab plus lenalidomide, 60% (95% CI, 48%-71%) had an objective response and 43% (95% CI, 32%-54%) experienced CR. Therapy was generally well tolerated, and the most common grade 3 or higher adverse events were neutropenia (48%), thrombocytopenia (17%), and febrile neutropenia (12%). The most common nonhematological adverse effects were rash, diarrhea, asthenia, cough, peripheral edema, and fever. Tafasitamab and lenalidomide was more effec tive as a second-line treatment than as treatment at third line or beyond (median PFS, 23.5 months vs 7.6 months).68 In addition, it is important to note that patients with double- or triple-hit lym phoma (MYC and BCL2 and/or BCL6 rearrangement) or primary refractory disease were excluded from this study. In a follow-up study, the median PFS was 11.6 months, and the median OS was 33.5 months.68 A retrospective observa tional study (RE-MIND) was performed aligning key eligibility criteria with L-MIND using data from 490 patients with R/R DLBCL who received lenalidomide monotherapy; 140 qualified for matching with the L-MIND cohort, and the primary analysis included 76 patients from each cohort.69 The analysis demon strated significantly higher overall response from the matched group from L-MIND. A sec ond study performed matchingadjusted indi rect com par i sons using data from L-MIND to match with described clinical trial cohorts such as pola-BR and gemcitabine-oxaliplatin-rituximab.70 Such approaches may increasingly be used to inform therapeutic decisions as more robust data on treatments and outcomes become avail able for patients with R/R DLBCL. For patients undergoing CAR T-cell therapy prior to this regimen or considering future CAR T-cell therapy following tafasitamab, it remains unclear to what extent anti-CD19 therapy or other treatments might impair the efficacy of future CD19-targeting therapies. Table 2. Triggers for goals-of-care discussions for patients with DLBCL Refractory disease Relapsed disease Declining performance status Organ insufficiency Consideration of ASCT Consideration of CAR T therapy If you “would not be surprised if the patient died within the next year.” Table 3. REMAP framework for patients with DLBCL REMAP Description Reframe Assess the patient’s understanding of his or her disease status; place details of the illness in a larger context, tailoring and titrating information according to patient preference. Expect emotion Acknowledge the patient’s emotions. Map outpatient values Explore what matters most to the patient in the context of DLBCL and the patient’s concerns about the future and goals and the trade-offs they are willing to make. Align with values Verbally reflect what is heard from the patient to ensure clear understanding of the patient’s values. This step offers the opportunity for additional clarification as needed. Propose a plan With permission from the patient, recommend a plan of care that has the best chance of maximizing the patient’s goals using a combination of the patient’s values and your knowledge of feasible medical treatments to help achieve the patient’s goals. Adapted from Childers et al90 with permission. Dr Prakash Singh Shekhawat Sequencing therapy in relapsed DLBCL | 151 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 preferences with their clinician.83 Most hematologic oncologists (56%) in a national survey reported that goals-of-care discus sions typically occur “too late,”84 and the median time between the first documented goals-of-care discussion and death was only 15 days in a study of patients who died of DLBCL or other blood cancers.81 Several barriers contribute to the current state of goals-of-care discussions. Given recent treatment advances and the potential for cure in R/R settings, the high prognostic uncertainty in DLBCL may lead some clinicians to delay discus sions until death is very clearly imminent. Concerns about taking away patients’ hope also contribute to hesitancy in conducting these discussions despite data showing that these conversa tions do not preclude hope.85 Even when clinicians grasp the importance of engaging in goals-of-care discussions, about 40% report that they do not know the right thing to say, which may result in low-quality discussions.86 Taken together, these data suggest an urgent need for strategies to improve goals-of-care discussions for DLBCL patients. To improve time li ness of goals-of-care dis cus sions for patients with DLBCL, it is important to start discussions before decisions are needed to avoid “crisis” communication when patients are very close to death. Having broad conversations to understand patients’ values and goals for their overall care and quality of life during first-line therapy helps to set the foundation for future discussions. Practical triggers to initiate or revisit more specific goals-of-care discussions include the presence of refrac tory or relapsed disease (Table 2). These disease points were identified in a focus group of lymphoma clinicians as critical sign posts for goals-of-care discussions.87 Other triggers identified by lymphoma clinicians include organ insufficiency or declining performance status, even in the absence of lymphoma relapse. Consideration of intensive therapies such as ASCT or CAR T-cell therapy should also prompt goals-of-care discussions. Answering no to the question “Would you be surprised if this patient died in the next year?” is also an effective trigger for engaging in these discussions.88 This question was demonstrated to cor rectly estimate death within 12 months in 68.3% of patients with hematologic malignancies.89 Goals-of-care dis cus sions should not be con sid ered one-time con ver sa tions because patients’ preferences may change as their disease evolves. Communication skills train ing to con duct nuanced dis cus sions that effec tively bal ance the poten tial prom ise of disease-directed treatments with their risks and limitations are necessary to optimize goals-of-care discussions. Tools such as the serious illness conversation guide and REMAP (reframe, expect emotion, map out patient values, align with values, and propose a plan) framework (Table 3) can help clinicians incorpo rate key elements of goals-of-care discussions when engaging in these conversations with DLBCL patients.77,88,90 In using these tools, clinicians should assess how much the patient under stands and desires to know about the disease trajectory and prognosis and then compassionately share information tailored to the patient’s preferences. It is also essential to engage the patient’s loved ones in these conversations to promote align ment of goals across the family unit. These discussions should be documented in the medical record to ensure that patients’ preferences are honored during care transitions. Goals-of-care skills–based training, such as VitalTalk and the Serious Illness Care Program, have effectively incorporated these communica tion tools.91,92 In a cluster randomized trial of the Serious Illness Care Program (a serious illness conversation guide combined with 2.5 hours of skills-based training) among 278 patients with advanced cancer, the program resulted in more frequent, ear lier, and higher-quality goals-of-care discussions.93 Moreover, this intervention was associated with significant reductions in depression and anxiety symptoms.92 These findings illustrate the potential impact of optimizing goals-of-care discussions through out the DLBCL dis ease course. Clinicians can apply this skill set to the discussions and care plans for each of our example patients. Conflict-of-interest disclosure Off-label drug use The bispecific antibodies mentioned in this article, mosunetuzumab, glofitamab, epcoritamab, plamotamab, and odronextamab, are not approved agents and their use was discussed within the context of completed and ongoing clinical trials. Correspondence Christopher R. Flowers, Department of Lymphoma/Myeloma, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: crflowers@mdanderson.org. References 1. Sehn LH, Salles G. Diffuse large B-cell lym phoma. N Engl J Med. 2021;384(9):842-858. 2. Sarkozy C, Sehn LH. Management of relapsed/refrac tory DLBCL. Best Pract Res Clin Haematol. 2018;31(3):209-216. 3. Westin J, Sehn LH. CAR T cells as a second-line therapy for large B-cell lym phoma: a paradigm shift? Blood. 2022;139(18):2737-2746. 4. Teras LR, DeSantis CE, Cerhan JR, Morton LM, Jemal A, Flowers CR. 2016 US lymphoid malignancy statistics by World Health Organization subtypes. 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Flowers: consultancy: AstraZeneca, Bayer, BeiGene, BioAscend, Bristol Myers Squibb, Celgene, Curio Sciences, Denovo Biopharma, Epizyme/Incyte, Foresight Diagnostics, Genentech/Roche, Genmab, MEI Pharmaceuticals, MorphoSys AG, Pharmacyclics/Janssen, SeaGen; stock options: Foresight Diagnostics, N Power; research funding: 4D, Abbvie, Acerta, Adaptimmune, Allogene, Amgen, Bayer, Celgene, Cellectis, EMD, Gilead, Genentech/Roche, Guardant, Iovance, Janssen Pharmaceutical, Kite, Morphosys, Nektar, Novartis, Pfizer, Pharmacyclics, Sanofi, Takeda, TGTherapeutics, Xencor, Ziopharm, Burroughs Wellcome Fund, Eastern Cooperative Oncology Group, National Cancer Institute, V Foundation, Cancer Prevention and Research Institute of Texas CPRIT Scholar in Cancer Research. Oreofe O. Odejide: no competing financial interests to declare. 55. Crump M, Neelapu SS, Farooq U, et al. Outcomes in refractory diffuse large B-cell lymphoma: results from the international SCHOLAR-1 study. 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Lisocabtagene maraleucel (lisocel), a CD19-directed chimeric antigen receptor (CAR) T cell therapy, ver sus standard of care (SOC) with salvage chemotherapy (CT) followed by autologous stem cell transplantation (ASCT) as second-line (2L) treatment in patients (Pts) with relapsed or refractory (R/R) large B-cell lymphoma (LBCL): results from the ran dom ized phase 3 trans form study. Blood. 2021;138(suppl 1):91. 60. Caimi PF, Ai W, Alderuccio JP, et al. Loncastuximab tesirine in relapsed or refractory diffuse large B-cell lymphoma (LOTIS-2): a multicentre, openlabel, single-arm, phase 2 trial. Lancet Oncol. 2021;22(6):790-800. 61. Hamadani M, Chen L, Song Y, et al. Matching-adjusted indirect comparison of the efficacy of loncastuximab tesirine versus treatment in the chemoimmunotherapy era for relapsed/refractory diffuse large B-cell lymphoma. Clin Lymphoma Myeloma Leuk. 2022;22(8):e738-e744. 62. Sehn LH, Herrera AF, Flowers CR, et al. Polatuzumab vedotin in relapsed or refractory diffuse large B-cell lymphoma. J Clin Oncol. 2020;38(2):155165. 63. Sehn LH, Hertzberg M, Opat S, et al. Polatuzumab vedotin plus bendamustine and rituximab in relapsed/refractory DLBCL: survival update and new extension cohort data. Blood Adv. 2022;6(2):533-543. 64. Herrera AF, Tracy S, Croft B, et al. Risk profiling of patients with relapsed/ refractory diffuse large B-cell lymphoma by measuring circulating tumor DNA. Blood Adv. 2022;6(6):1651-1660. 65. Kalakonda N, Maerevoet M, Cavallo F, et al. Selinexor in patients with relapsed or refractory diffuse large B-cell lymphoma (SADAL): a single-arm, mul ti na tional, multicentre, open-label, phase 2 trial. Lancet Haematol. 2020;7(7):e511-e522. 66. Casasnovas RO, Follows G, Zijlstra JM, et al. Comparison of the effective ness and safety of the oral selective inhibitor of nuclear export, selinexor, in diffuse large B cell lymphoma subtypes. Clin Lymphoma Myeloma Leuk. 2022;22(1):24-33. 67. Salles G, Duell J, González Barca E, et al. Tafasitamab plus lenalidomide in relapsed or refractory diffuse large B-cell lymphoma (L-MIND): a multicentre, prospective, single-arm, phase 2 study. Lancet Oncol. 2020;21(7):978-988. 68. Duell J, Maddocks KJ, González-Barca E, et al. Long-term outcomes from the phase II L-MIND study of tafasitamab (MOR208) plus lenalidomide in patients with relapsed or refractory diffuse large B-cell lymphoma. Haematologica. 2021;106(9):2417-2426. 69. Zinzani PL, Rodgers T, Marino D, et al. RE-MIND: comparing tafasitamab + lenalidomide (L-MIND) with a real-world lenalidomide monotherapy cohort in relapsed or refractory diffuse large B-cell lymphoma. Clin Cancer Res. 2021;27(22):6124-6134. 70. Cordoba R, Prawitz T, Westley T, et al. Tafasitamab plus lenalidomide ver sus 3 rituximab-based treat ments for non-trans plant eli gi ble relapsed/ refrac tory dif fuse large B-cell lym phoma: a matching-adjusted indi rect comparison. Adv Ther. 2022;39(6):2668-2687. 71. Budde LE, Assouline S, Sehn LH, et al. Single-agent mosunetuzumab shows durable complete responses in patients with relapsed or refractory B-cell lymphomas: phase I dose-escalation study. J Clin Oncol. 2022;40(5):481-491. 72. Hutchings M, Morschhauser F, Iacoboni G, et al. Glofitamab, a novel, biva lent CD20-targeting T-cell-engaging bispecific antibody, induces durable complete remissions in relapsed or refractory B-cell lymphoma: a phase I trial. J Clin Oncol. 2021;39(18):1959-1970. 73. Hutchings M, Mous R, Clausen MR, et al. Dose escalation of subcutaneous epcoritamab in patients with relapsed or refractory B-cell non-Hodgkin lym phoma: an open-label, phase 1/2 study. Lancet. 2021;398(10306):1157-1169. 74. Bannerji R, Arnason JE, Advani RH, et al. Odronextamab, a human CD20xCD3 bispecific anti body in patients with CD20-pos i tive B-cell malignancies (ELM-1): results from the relapsed or refractory non-Hodgkin lymphoma cohort in a single-arm, multicentre, phase 1 trial. Lancet Haematol. 2022;9(5):e327-e339. Dr Prakash Singh Shekhawat Sequencing therapy in relapsed DLBCL | 153 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 35. Maurer MJ, Habermann TM, Shi Q , et al. Progression-free survival at 24 months (PFS24) and subsequent outcome for patients with diffuse large B-cell lymphoma (DLBCL) enrolled on randomized clinical trials. Ann Oncol. 2018;29(8):1822-1827. 36. Maurer MJ, Jakobsen LH, Mwangi R, et al. Relapsed/Refractory International Prognostic Index (R/R-IPI): an international prognostic calculator for relapsed/refrac tory dif fuse large B-cell lym phoma. Am J Hematol. 2021;96(5):599-605. 37. Wang Y, Farooq U, Link BK, et al. Late relapses in patients with diffuse large B-cell lymphoma treated with immunochemotherapy. J Clin Oncol. 2019;37(21):1819-1827. 38. Robetorye RS, Ramsower CA, Rosenthal AC, et al. Incorporation of digital gene expression profiling for cell-of-origin determination (Lymph2Cx test ing) into the routine work-up of diffuse large B cell lymphoma. J Hematop. 2019;12(1):3-10. 39. Scott DW. Cell-of-ori gin in dif fuse large B-cell lym phoma: are the assays ready for the clinic? Am Soc Clin Oncol Educ Book. 2015;36(14 May):e458-466. 40. Harkins RA, Chang A, Patel SP, et al. Remaining challenges in predicting patient outcomes for diffuse large B-cell lymphoma. Expert Rev Hematol. 2019;12(11):959-973. 41. Kurtz DM, Esfahani MS, Scherer F, et al. Dynamic risk pro fil ing using serial tumor biomarkers for personalized outcome prediction. Cell. 2019;178(3):699-713.e19713e19. 42. Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone mar row transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med. 1995;333(23):1540-1545. 43. Gisselbrecht C, Glass B, Mounier N, et al. Salvage regimens with autolo gous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol. 2010;28(27):4184-4190. 44. Crump M, Kuruvilla J, Couban S, et al. Randomized comparison of gemcitabine, dexamethasone, and cisplatin versus dexamethasone, cytarabine, and cisplatin chemotherapy before autologous stem-cell transplantation for relapsed and refractory aggressive lymphomas: NCIC-CTG LY.12. J Clin Oncol. 2014;32(31):3490-3496. 45. Abramson JS, Palomba ML, Gordon LI, et al. Lisocabtagene maraleucel for patients with relapsed or refrac tory large B-cell lym pho mas (TRANSCEND NHL 001): a multicentre seam less design study. Lancet. 2020;396(10254):839-852. 46. Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell ther apy in refrac tory large B-cell lym phoma. N Engl J Med. 2017;377(26):2531-2544. 47. Schuster SJ, Bishop MR, Tam CS, et al; JULIET Investigators. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med. 2019;380(1):45-56. 48. Schuster SJ, Tam CS, Borchmann P, et al. Long-term clinical outcomes of tisagenlecleucel in patients with relapsed or refractory aggressive B-cell lymphomas (JULIET): a multicentre, open-label, single-arm, phase 2 study. Lancet Oncol. 2021;22(10):1403-1415. 49. Locke FL, Ghobadi A, Jacobson CA, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1-2 trial. Lancet Oncol. 2019;20(1): 31-42. 50. Pinnix CC, Gunther JR, Dabaja BS, et al. Bridging therapy prior to axicabtagene ciloleucel for relapsed/refractory large B-cell lymphoma. Blood Adv. 2020;4(13):2871-2883. 51. Jain MD, Jacobs MT, Nastoupil LJ, et al. Characteristics and out comes of patients receiv ing bridg ing ther apy while awaiting man u fac ture of standard of care axicabtagene ciloleucel CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory large B-cell lymphoma: results from the US Lymphoma CAR-T Consortium. Blood. 2019;134(suppl 1): 245. 52. Schuster M, Zijlstra J, Casasnovas RO, et al. Effect of prior therapy and dis ease refractoriness on the efficacy and safety of oral selinexor in patients with diffuse large B-cell lymphoma (DLBCL): a post-hoc analysis of the SADAL study. Clin Lymphoma Myeloma Leuk. 2022;22(7):483-494. 53. Sim AJ, Jain MD, Figura NB, et al. Radiation therapy as a bridging strategy for CAR T cell therapy with axicabtagene ciloleucel in diffuse large B-cell lymphoma. Int J Radiat Oncol Biol Phys. 2019;105(5):1012-1021. 54. Nastoupil LJ, Jain MD, Feng L, et al. Standard-of-care axicabtagene ciloleucel for relapsed or refractory large B-cell lymphoma: results from the US Lymphoma CAR T Consortium. J Clin Oncol. 2020;38(27):3119-3128. 154 | Hematology 2022 | ASH Education Program 85. Loggers ET, Lee S, Chilson K, Back AL, Block S, Loberiza FR. Advance care plan ning among hema to poi etic cell trans plant patients and bereaved caregivers. Bone Marrow Transplant. 2014;49(10):1317-1322. 86. Odejide OO, Cronin AM, Condron NB, et al. Barriers to quality end-of-life care for patients with blood cancers. J Clin Oncol. 2016;34(26):31263132. 87. Odejide OO, Salas Coronado DY, Watts CD, Wright AA, Abel GA. End-of-life care for blood cancers: a series of focus groups with hematologic oncolo gists. J Oncol Pract. 2014;10(6):e396-e403. 88. Bernacki RE, Block SD; American College of Physicians High Value Care Task Force. Communication about serious illness care goals: a review and synthesis of best prac tices. JAMA Intern Med. 2014;174(12):19942003. 89. Hudson KE, Wolf SP, Samsa GP, Kamal AH, Abernethy AP, LeBlanc TW. The surprise question and identification of palliative care needs among hospi talized patients with advanced hematologic or solid malignancies. J Palliat Med. 2018;21(6):789-795. 90. Childers JW, Back AL, Tulsky JA, Arnold RM. REMAP: a framework for goals of care conversations. J Oncol Pract. 2017;13(10):e844-e850. 91. VITALtalk. Address goals of care: smoothing discussions about prognosis and treatment. Vol. 2020. https://www.vitaltalk.org/topics/reset-goals-of -care/. Accessed 3 October 2022. 92. Bernacki R, Paladino J, Neville BA, et al. Effect of the Serious Illness Care Program in outpatient oncology: a cluster randomized clinical trial. JAMA Intern Med. 2019;179(6):751-759. 93. Paladino J, Bernacki R, Neville BA, et al. Evaluating an inter ven tion to improve communication between oncology clinicians and patients with life-limiting cancer: a cluster randomized clinical trial of the Serious Illness Care Program. JAMA Oncol. 2019;5(6):801-809. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000332 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/146/2021680/146flowers.pdf by guest on 09 December 2022 75. Patel K, Michot JM, Chanan-Khan A, et al. Safety and anti-tumor activity of plamotamab (XmAb13676), an anti-CD20 x anti-CD3 bispecific antibody, in sub jects with relapsed/refrac tory non-Hodgkin’s lym phoma. Blood. 2021;138(suppl 1):2494. 76. Dickinson M, Carlo-Stella C, Morschhauser F, et al. S220: glofitamab induces durable complete remissions and has favorable safety in patients with relapsed/refractory diffuse large B-cell lymphoma and ≥2 prior ther a pies: piv otal phase II expan sion results. HemaSphere. 2022;6(June): 121-122. 77. Thieblemont C, Phillips T, Ghesquieres H, et al. Primary results of subcuta neous epcoritamab dose expansion in patients with relapsed or refractory large B-cell lymphoma: a phase 2 study. Paper presented at: 2022 Euro pean Hematology Association Congress; 2022; Vienna, Austria. Abstract LB2364. 78. Dans M, Smith T, Back A, et al. NCCN guidelines insights: palliative care, Version 2.2017. J Natl Compr Canc Netw. 2017;15(8):989-997. 79. Mack JW, Weeks JC, Wright AA, Block SD, Prigerson HG. End-of-life dis cussions, goal attainment, and distress at the end of life: predictors and outcomes of receipt of care consistent with preferences. J Clin Oncol. 2010;28(7):1203-1208. 80. Sheridan R, Roman E, Smith AG, et al. Preferred and actual place of death in haematological malignancies: a report from the UK Haematological Malignancy Research Network. BMJ Support Palliat Care. 2021;11(1):7-16. 81. Odejide OO, Uno H, Murillo A, Tulsky JA, Abel GA. Goals of care discussions for patients with blood cancers: association of person, place, and time with end-of-life care utilization. Cancer. 2020;126(3):515-522. 82. Wright AA, Zhang B, Ray A, et al. Associations between end-of-life dis cussions, patient mental health, medical care near death, and caregiver bereavement adjustment. JAMA. 2008;300(14):1665-1673. 83. Trevino KM, Rutherford SC, Marte C, et al. Illness under stand ing and advance care planning in patients with advanced lymphoma. J Palliat Med. 2020;23(6):832-837. 84. Odejide OO, Cronin AM, Condron N, Earle CC, Wolfe J, Abel GA. Timeliness of end-of-life discussions for blood cancers: a national survey of hemato logic oncologists. JAMA Intern Med. 2016;176(2):263-265. CONTROVERSIES IN AGGRESSIVE NHL Anita Kumar Department of Medicine, Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, NY Up-front autologous stem cell transplantation (ASCT) is the established standard of care for younger, transplant-eligible MCL patients and is associated with a prolonged progression-free survival (PFS) benefit. However, there is no randomized controlled trial data, with therapy including rituximab and cytarabine, that has established a PFS and overall survival (OS) benefit with ASCT in the modern era. Multiple retrospective studies have failed to identify an OS benefit associated with ASCT in younger MCL patients. The high-risk patient subgroup with evidence of baseline TP53 mutation has a dismal outcome with intensive chemoimmunotherapy followed by ASCT, thus up-front ASCT is not optimal for this patient subset. Ongoing randomized clinical trials will help to clarify the role of up-front ASCT in the future. For example, the ongoing European MCL Network Triangle study incorporating ibrutinib into chemoimmunotherapy induction and maintenance with and without ASCT will help define the role of ASCT in the era of novel biologically targeted agents (ClinicalTrials.gov identifier: NCT02858258). Additionally, minimal residual disease (MRD) assessment is a powerful prognostic tool in MCL, and the ongoing Eastern Cooperative Oncology Group-American College of Radiology Imaging Network E4151 study is comparing maintenance rituximab alone vs ASCT consolidation in MCL patients who achieve remission and MRD-undetectable status post induction (ClinicalTrials.gov identifier: NCT03267433). ASCT remains a highly efficacious initial therapy for younger MCL patients; however, ultimately the decision to pursue ASCT requires discussion of risks vs benefits, incorporating patient preferences and values. LEARNING OBJECTIVES • Understand the data that support and challenge the use of ASCT in first remission in younger, fit MCL patients • Understand that the presence of TP53 mutation is associated with a poor response to standard chemoimmunotherapy, including ASCT • Review the use of novel therapies and MRD assessment in clinical trials that will help define the future role of ASCT in the modern era CLINICAL CASE 1 A 55-year-old otherwise healthy woman presented with intermittent left lower quadrant abdominal pain. A computed tomographic scan of the abdomen and pelvis revealed intussusception of the descending colon, and a colonoscopy showed a 5-cm partially obstructing ulcerated mass in the descending colon. Biopsy of the colonic mass was consistent with mantle cell lymphoma (MCL). Neoplastic cells expressed by immunohistochemistry (IHC) included CD20, CD5, BCL2, and cyclin D1 but did not express TP53. Ki-67 was 25% to 40%. Mutational analysis revealed no TP53 mutation. A positron emission tomographic (PET) scan revealed diffuse fluorodeoxyglucose (FDG)-avid lymphadenopathy and FDG avidity throughout the gastrointestinal (GI) tract. The CBC and lactate dehydrogenase were normal, consistent with the low-risk clinical Mantle Cell Lymphoma International Prognostic Index (MIPI) score. The patient underwent 4 cycles of RDHAX chemotherapy (rituximab, dexamethasone, cytarabine, and oxaliplatin) and achieved a complete remission (CR). She then received ASCT consolidation followed by maintenance rituximab. The standard of care frontline therapy for MCL patients is chemoimmunotherapy. For younger MCL patients (≤65 years) who are transplant eligible, a widely accepted Dr Prakash Singh Shekhawat Autotransplant in mantle cell lymphoma | 155 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 What is the role of up-front autologous stem cell transplantation in mantle cell lymphoma? 156 | Hematology 2022 | ASH Education Program 12.7 and 8.5 years, respec tively.10,11 Although there was a con tin u ous pat tern of relapse over time, for low-risk MIPI patients the median PFS was over 12 years with the Nordic MCL2 treatment approach. This prolonged initial duration of response likely has a greater impact on OS in younger vs older MCL patients given the fewer treatment-related toxicities and com peting health risks. Given the successive shortening of remis sion length with subsequent lines of therapy in MCL, optimizing initial treatment is of value.17 Another reason to utilize ASCT in frontline is that MCL can evolve over time, and the acquisition of high-risk mutations, genomic instability, and/or transforma tion to blastoid or pleomorphic disease are increasingly com mon in the relapsed setting and often associated with disease refrac tory to stan dard chemoimmunotherapy.18 An extended “treat ment-free” inter val after front line ther apy also affords time for advances in the field to develop; for example, between 2013 and 2022, 6 novel ther a pies were US Food and Drug Administration or National Comprehensive Cancer Network approved for R/R MCL, including lenalidomide, ibrutinib, acalabrutinib, zanubrutnib, venetoclax, and brexucabtagene autoleucel.19-24 In summary, frontline intensive induction followed by ASCT and rituximab maintenance for younger, fit MCL patients is a highly efficacious treatment approach and associated with a prolonged PFS benefit. CLINICAL CASE 2 A 64-year-old man with a history of well-controlled hyperten sion and hyperlipidemia presented with fatigue and drenching night sweats. A PET scan revealed diffuse lymphadenopathy and splenic involvement. Biopsy of a lymph node revealed MCL. Neoplastic cells expressed CD20, CD5, BCL2, and cyclin D1 and did not express TP53 by IHC. Ki-67 was 20%. Mutational analysis revealed ATM mutation alone. Bone marrow (BM) biopsy and aspiration showed 50% to 70% involvement with MCL. Labs revealed mild anemia and thrombocytopenia. The MIPI score was intermediate risk. The patient expressed a strong desire to receive outpatient chemoimmunotherapy, balked at the idea of temporarily relocating to New York City for an ASCT, and wanted to maintain his quality of life to continue working and caring for his elderly parent. The patient received treatment with bendamustine and rituximab for 6 cycles followed by rituximab maintenance in his local community. Although ASCT in CR1 established the standard of care for youn ger MCL patients, it is not the only acceptable treatment. It is becoming increasingly clear based on retrospective and realworld data that only a fraction of younger patients with MCL receive up-front ASCT despite national and international guide lines supporting its use. Recently published data from the Flatiron Health data base describ ing treat ment pat terns for MCL patients treated in predominantly community-based practices from 2011 to 2021 showed that only approximately 1 in 4 patients under 65 years of age received an up-front ASCT.25 Data from the Center for International Blood and Marrow Transplant Research database and estimated MCL incidence rates from the National Cancer Institute’s Surveillance, Epidemiology, and End Results database between 2000 and 2017 showed that in patients aged Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 treatment approach consists of 3 phases: (1) cytarabine-containing induc tion chemoimmunotherapy, (2) ASCT, and (3) main te nance rituximab. This treatment paradigm is supported by var i ous national and inter na tional guide lines and expert pan els, including the National Comprehensive Cancer Network, Amer ican Society of Transplantation and Cellular Therapy, Center for International Blood and Marrow Transplant Research, European Society for Blood and Marrow Transplantation, European Society for Medical Oncology, and European Hematology Association.1-4 The pivotal study that established the role of consolidative ASCT in first remission for fit MCL patients was the open-label, multicenter, randomized phase 3 trial performed by the Euro pean Mantle Cell Lymphoma Network enrolling patients between September 1996 and March 2004.5 In this study, patients with advanced-stage MCL aged 65 years or younger were random ized to receive either ASCT or interferon alfa (IFNα) maintenance therapy after cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-like induction therapy with or without rituximab (ie, R-CHOP). In the initial report with a median follow-up of 25 months, patients in the ASCT arm vs the IFNα arm had a significantly longer progression-free survival (PFS; median of 39 vs 17 months; P=.0108) but no significant difference in overall sur vival (OS). Recently published long-term results with a 14-year median follow-up continued to demonstrate a PFS benefit and also showed an OS benefit favoring ASCT consolidation vs IFNα maintenance.6 Building upon this approach, the treatment par adigm for younger, fit MCL patients has been further refined to include rituximab and cytarabine in the initial induction regimen and to include rituximab maintenance post ASCT.7,8 The thera peutic importance of high-dose cytarabine was established by an open-label, phase 3 trial from the European Mantle Cell Lymphoma Network in which MCL patients aged 65 years or younger were randomized to receive R-CHOP vs alternating R-CHOP and R-DHAP (rituximab plus dexamethasone, high-dose cytarabine, and cisplatin) for 6 cycles followed by ASCT.7 After a median follow-up of 6 years, the time to treatment failure (TTF) was sig nificantly longer in the R-CHOP/R-DHAP group vs the R-CHOPalone group (5-year TTF was 65% vs 40%, respectively), thus supporting the inclusion of cytarabine as part of initial induc tion chemoimmunotherapy. In the initial report, there was no OS difference between the R-DHAP-containing vs R-CHOP-alone treatment arms, but with 5 additional years of follow-up, when adjusted for MIPI score without and with Ki-67, OS was signif i cantly supe rior in the R-DHAP arm (haz ard ratio [HR], 0.74; P = .038 and 0.60; P=.0066).9 The LYMA study established rituximab maintenance post ASCT as a standard of care.8 In this study, MCL patients aged 65 years or younger received 4 courses of R-DHAP (any platinum) followed by ASCT consolidation and then were randomized to receive rituximab maintenance vs observa tion post transplant. The PFS and OS at 4 years were superior in the rituximab maintenance vs observation group (PFS, 83% vs 64%; P<.001; OS, 89% vs 80%; P=.04), respectively. Multiple phase 2 studies have demonstrated long-term dura ble remissions with cytarabine-containing induction chemoimmunotherapy followed by ASCT consolidation (Table 1).7,8,10-16 For example, with a median follow-up of 11.4 years, the sec ond Nordic MCL trial (MCL2) with patients under 66 years of age who received alternating courses of R-maxi-CHOP (dose- intensified rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) and high-dose cytarabine (HIDAC) and ASCT consolidation was associated with a median OS and PFS of Table 1. Selected prospective studies including ASCT in younger MCL patients Study/regimen author (ref) Details of regimen Primary end point Patients Outcomes European Mantle Cell Lymphoma Network (MCL Younger): phase 3 study of R-CHOP vs R-CHOP alternating with R-DHAP → ASCT Hermine et al7 (2016) Untreated MCL Age 18-65y, stage II-IV, transplant eligible Arm 1: R-CHOP×6 cycles followed by HDT/ASCT Arm 2: R-CHOP alternating with R-DHAP followed by HDT/ASCT Investigator-assessed TTF n=497 TTF significantly longer in cytarabine group: median 9.1y (95% CI, 6.3-not reached) vs control group: median 3.9y (3.2-4.4); HR, 0.56; P=.038. 5-year TTF 65% vs 40%. No OS difference. LYMA phase 3 study of R-DHAP → ASCT → rituximab maintenance vs other Le Gouill et al8 (2017) Untreated MCL Age >65y, stages II-IV, transplant eligible Arm 1: R-DHAP (platinum derivative)×4 cycles, if CR/PR then HDT/ASCT then rituximab maintenance every 2 mo for 3y *If <CR/PR, then R-CHOP Arm 2: R-DHAP×4 cycles, if CR/PR then HDT/ASCT then observation EFS after ASCT n=257 4-year EFS was 79% (95% CI, 70-86) in the rituximab group vs 61% (95% CI, 51-70) in the observation group (P=.001) 4-year OS was 89% (95% CI, 81-94) in the rituximab group vs 80% (95% CI, 72-88) in the observation group (P=.04). Second Nordic MCL study (MCL2): phase 2 study of R-maxi-CHOP alternating with RHIDAC → ASCT Geisler et al10 (2008) Eskelund et al11 (2016) Untreated MCL Age <66y, stages II-IV, transplant eligible R-maxi-CHOP alternating with R-high-dose cytarabine then HDT/ASCT *Pts in CR who converted from PCR-negative to PCR-positive were offered preemptive rituximab 375mg/m2/wk for 4 weeks to prevent clinical relapse OS, EFS, and PFS n=160 6-year OS, EFS, and PFS were 70%, 56%, and 66%, respectively. GELA phase 2 study: R-CHOP×3 followed by R-DHAP×3 → ASCT Delarue et al12 (2013) Untreated MCL Stage III-IV, transplant eligible Blastoid variants excluded CHOP×2, R-CHOP×1, R-DHAP×3 → ASCT Primary end point EFS n=60 With median follow-up of 67 mo, median EFS 83 mo and median OS not reached. 5-y OS is 75%. Phase 2 study of RB/RC induction followed by ASCT 2 sites: DFCI and WUSTL Merryman et al14 (2020) DFCI: 18-69y, untreated MCL, transplant eligible WUSTL: age 18-65y, untreated MCL, transplant eligible DFCI: BR×3 cycles then R-high-dose cytarabine×3 cycles → ASCT WUSTL: BR alternating with R-HIDAC, total 6 cycles → ASCT Primary end point ORR n=88 End-of-induction ORR 97%. After a median follow-up of 33 mo, 3-y PFS 83% and 3-y OS 92%. Phase 2 study of RHCVAD regimen alternating with high-dose methotrexate and cytarabine Massaro et al16 (2021) Chihara et al15 (2016) Age <70y, untreated MCL, transplant eligible R-HCVAD regimen alternating with highdose methotrexate and cytarabine×4 cycles. If CR → no further tx PR → consolidative ASCT Primary end point complete response rate n=63 With median follow-up of 10.5 years, 10-year PFS and OS of 35% and 61% BR, bendamustine-rituximab; EFS, event-free survival; HCT, high-dose chemotherapy; PR, partial response; Pts, patients; RB/RC, rituximab bendamustine/rituximab cytarabine; tx, treatment. 65 years or younger, ASCT utilization increased from 5.4% in 2001 to a peak of 22.3% in 2013 and plateaued at 18.1% in 2017.26 In addition, an analysis from the National Cancer Database esti mated ASCT utilization to be approximately 30% for patients aged 60 years or younger.27 Rates of ASCT utilization for younger patients appear higher at US academic medical centers (67%) and in certain geographies, like Sweden and Denmark.28-30 A number of factors may contribute to why ASCT utilization rates are lower than anticipated in younger MCL patients. In the Surveillance, Epidemiology, and End Results database study, private health insurance, care at an academic/research medical center, and geographic location were significantly associated with ASCT utilization.27 A Swedish registry study found that MCL patients who had never married, were divorced, or had a lower educational level underwent transplantation less often.29 In the multicenter retrospective study that characterized outcomes and treatment patterns for younger MCL patients treated at US academic medical centers, the reasons for not selecting ASCT consolidation included physician choice, 67%; patient preference, 18%; other reason (eg, mobilization failure), 3%; and unknown, 12%.28 Overall, factors that influence whether up-front ASCT is pursued include patient and/or physician pref erence, academic vs community-based care, and socioeco nomic factors. Dr Prakash Singh Shekhawat Autotransplant in mantle cell lymphoma | 157 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 Key eligibility criteria Table 2. Comparing up-front intensive chemoimmunotherapy with ASCT consolidation vs bendamustine-rituximab Bendamustine-rituximab followed by rituximab maintenance (2y) Median progression-free survival 7-10y *Nordic MCL2 study11 4.4-5.3y *E1411 and SHINE studies49,50 Median overall survival ~10-13y ~10-13y Key toxicities Alopecia, fatigue, severe myelosuppression, infection, graft failure (<1%), mucositis, GI toxicity, B-cell depletion, cardiopulmonary toxicity, liver toxicity, renal insufficiency, cognitive impairment, secondary malignancy Fatigue, myelosuppression, infection, GI toxicity (mild), B-cell depletion, secondary malignancy Quality-of-life considerations *Hospitalization for transplant (or close proximity to hospital) *Seek care at a transplant center *Maintain performance status without requirement for hospitalization *Can receive community-based care One of the reasons why physicians may not recommend upfront ASCT in MCL is because of emerging data that fail to con vincingly demonstrate an associated OS benefit. The survival advan tage pre vi ously described with up-front ASCT may be diminished in the context of improved frontline induction pro grams (including rituximab and cytarabine) with incorporation of maintenance post induction, as well as with improved salvage ther a pies for relapsed MCL, such as Bruton’s tyro sine kinase inhibitors, CD19-directed chimeric-antigen receptor T-cell ther apy, and other novel agents. One critical question that underlies the controversy surrounding ASCT in MCL is: Would the same PFS and OS benefit of ASCT reported in the only randomized pivotal study from the European Mantle Cell Lymphoma Network remain in the current era given access to rituximab, cytarabine, and better salvage therapies? To explore this question, a post hoc subgroup exploratory analysis was performed from the pivotal study, focusing on patients who received rituximab-containing induction regimens. Neither PFS nor OS were significantly different between the ASCT vs IFNα maintenance groups, with a median PFS of 3.4 years vs 1.7 years (P=.087) and a median OS of 9.6 years vs 5.5 years (P=0.68), respectively.6 However, this post hoc subgroup analysis included a small number of patients who received rituximab-containing induction therapy (41 patients who received ASCT consolidation and 27 patients who received IFNα maintenance) and thus was underpowered to detect a significant difference between the 2 approaches.6 Another important retrospective study aiming to assess the benefit of ASCT in the rituximab era was a multicenter analysis from several North American academic medical centers that included 1029 patients aged 65 years or younger.28 In this study, 64% of patients (n=657) underwent ASCT consolidation, and after a median follow-up of 76 months, ASCT was associ ated, using a multivariable regression analysis, with a statistically significant improvement in PFS and a trend toward improved OS. In a propensity score-weighted analysis aimed to address confounders, the median PFS was more prolonged with ASCT (78 vs 48.5 months), but an improvement in OS was not observed. In addition, several real-world analyses also do not show a clear survival benefit to be associated with ASCT. For example, the Flatiron Health database examined ASCT-eligible patients (<65 years who were alive and did not initiate subsequent treat ment within 6 months of first-line treatment) and found no signifi cant improvement in real-world time-to-next-treatment or OS with ASCT (vs without).25 In a real-world study of patients diagnosed 158 | Hematology 2022 | ASH Education Program in Sweden between 2007 and 2017, for patients under 70 years of age there was no significant difference in OS with intensi fied MCL2 pro to col or R-CHOP com pared to bendamustinerituximab.31 Additionally, it may be possible to consider the deferral of ASCT to the relapsed/refractory (R/R) setting. There are data to suggest that ASCT can be effective in CR2, although it is unclear whether outcomes are equivalent to up-front ASCT.32,33 In light of these data, for the patient in Clinical Case 2 I rec om mend discussing the out come data that sup port up-front ASCT, the emerging data that challenge this therapeutic strat egy in the modern era, and the short- and long-term side effects of an intensive approach vs other options. Ultimately, the oncol ogist and the patient can collaborate to devise a care plan based on clinical evidence that balances risks and expected outcomes with patient preferences and values (Table 2). For this patient, the significant time commitment and need for hospitalization, the cost of travel to an urban transplant center, and the potential toxicities associated with ASCT represented an undue burden not offset by the likely prolonged remission duration post trans plant, particularly given the potential lack of OS benefit associ ated with up-front ASCT in the modern era. CLINICAL CASE 3 A 40-year-old otherwise healthy woman presented with wors ening left lower quadrant pain requiring opioid therapy, and a com puted tomo graphic scan of the abdo men and pel vis revealed a bulky retroperitoneal mass measuring 12 cm, caus ing left-sided hydronephrosis. Biopsy of the mass revealed MCL, blastoid variant. Neoplastic cells expressed CD20, CD5, BCL2, and cyclin D1 by IHC. TP53 expression by IHC was not evaluated. Ki-67 was 60% to 70%. Mutational analysis was sent and pending at the time of the initial presentation. A PET scan revealed diffuse FDG-avid lymphadenopathy, including a bulky lymph node con glom er ate in the retroperitoneum, and BM biopsy and aspiration showed 10% to 15% involvement with MCL. The MIPI score was high risk. The patient was admitted for pain control and the initiation of RDHAX with tumor lysis monitoring. She achieved a CR after 4 cycles. During therapy, mutational profiling results returned and revealed the presence of a missense mutation in the TP53 gene (R249T). Although she Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 Cytarabine-containing induction and ASCT consolidation followed by rituximab maintenance (3y) had been scheduled for ASCT consolidation, this was aborted, and the patient proceeded to receive rituximab maintenance. CLINICAL CASE 1 (Cont inu ed) We return again to the 55-year-old woman with MCL, Ki-67 25% to 40%, TP53 wild-type stage IV MIPI low-risk disease involving the GI tract who received 4 cycles of RDHAX and achieved a CR. In this case, the patient enrolled in the ECOG-ACRIN EA4151 trial titled Rituximab With or Without Stem Cell Transplant in Treating Patients With Minimal Residual Disease-Negative Mantle Cell Lymphoma in First Complete Remission (ClinicalTrials The lack of clear OS benefit associated with ASCT in MCL in the modern era has paved the way for the development of clinical trials testing alternate therapeutic strategies omitting ASCT. These clinical trials have tested different approaches including the use of maintenance therapy post induction, the incorpora tion of novel agents, and the use of MRD assessment to develop risk-adapted treatment paradigms. For example, the E1405 study utilized a modified rituximab, hyperfractionated cyclophospha mide, doxorubicin, vincristine, and dexamethasone (R-HCVAD) regimen incorporating bortezomib with maintenance rituximab, and the primary end point of the study was overall response rate (ORR) to this combination.41 The ORR was 95%, and a CR was achieved in 68% of patients. Interestingly, in this study patients could receive either maintenance rituximab or ASCT per physi cian and patient preference, and with a median follow-up of 4.5 years, no substantial difference in PFS or OS was found between patients treated with main te nance rituximab (n=44) vs ASCT (n=22). Although this was only hypothesis generating given the lack of a randomized study design, short follow-up time, and small sample sizes, this study set the stage for future clinical trials to more definitively compare maintenance rituximab and ASCT post induction in a larger randomized context. The prognostic role of MRD assessment, utilizing different assays including flow cytometry, reverse-transcription poly merase chain reaction (PCR)–based techniques, and immunosequencing platforms, has been well established in MCL.42 MRD status post induction chemotherapy and pre-ASCT has been shown to be an important predictor of PFS and a poten tial biomarker for risk-adapted treatments.43,44 Thus, the ongo ing EA4151 study is comparing maintenance rituximab alone vs ASCT consolidation in MCL patients who achieve a remission and an MRD-undetectable status post induction. The primary end point of this study is 6-year OS. The study has the poten tial to estab lish an MRD-guided treat ment strat egy, allowing for therapy de-escalation in selected MRD-undetectable patients. Other stud ies have incor po rated novel agents, such as ibrutinib, into up-front reg i mens. The phase 2 WINDOW-1 study minimized chemotherapy for previously untreated MCL patients 65 years or younger.45 Patients were initially treated with ibrutinib and rituximab, and after achieving CR, patients received R-HCVAD alternating with methotrexate/cytarabine for 4 cycles. The ORR was 98% (n = 131 patients treated), the 3-year estimated PFS was 79%, and the 3-year OS was 95%. Additionally, in the ongoing European MCL Network Triangle study, transplant-eligible patients under 65 were randomized to 1 of 3 arms (rituximab maintenance can be added to all arms): (1) R-CHOP/R-DHAP induction with ASCT, (2) R-CHOP plus ibrutinib/R-DHAP with ASCT and then 2 years of ibrutinib maintenance, and (3) R-CHOP plus ibrutinib/R-DHAP followed by ibrutinib maintenance for 2 years without ASCT (ClinicalTrials.gov identifier: NCT02858258).46 The primary end point is superiority of 1 of the 3 study arms by investigator-assessed fail ure-free sur vival. This study will deter mine the value of Dr Prakash Singh Shekhawat Autotransplant in mantle cell lymphoma | 159 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 The presence of TP53 mutation is associated with a very poor prognosis when treated with conventional chemoimmunotherapy, including ASCT consolidation. In a pooled analysis from the Nordic MCL2 and MCL3 clinical trials, the prognostic value of TP53 mutation at baseline was assessed in 183 patients (of the total 320 enrolled) who had available DNA from diagnostic BM samples. In the MCL2 and MCL3 studies, patients under 66 years of age with stage II to IV disease who were transplant eligible received alternating R-maxi-CHOP and R-high-dose-cytarabine followed by ASCT.34 TP53 mutations were observed in 11%, del17p in 16%, NOTCH1 muta tions in 4%, and CDKN2A muta tions in 20%. All these genetic subgroups, in addition to the MIPI, MIPI-c, blastoid morphology, and Ki-67% over 30% groups, were each associated with inferior survival on univariable analysis. However, on multivariable analysis, only the MIPI-c high-risk group (HR, 2.6; P=.003) and TP53 mutation (HR, 6.2; P<.0001) retained independent prognostic significance for OS. Patients with TP53 mutation had a percentage of Ki-67 greater than 30%, blastoid morphology, and a high-risk MIPI. The outcomes for patients with TP53 mutation were dismal, with a median PFS of 0.9 years and a median OS of 1.8 years. TP53 mutation with or without del17p was associated with inferior outcomes compared to the presence of del17p alone. Increased TP53 expression by IHC also has prognostic value in MCL and is highly correlated with the presence of TP53 mutation.35-37 Given the minimal benefit and potential toxicity of ASCT in this high-risk subset of patients, ASCT consolidation is not recommended in MCL patients with evidence of TP53 mutation. There exists no opti mal front line treat ment approach for this high-risk subset of patients, and clinical trial enrollment is highly recommended. Emerging treatment options for high-risk patients include combinations of biologically targeted ther apies, such as dual inhibition of Bruton’s tyrosine kinase and BCL2 in conjunction with anti-CD20 monoclonal antibody ther apy, which has been, for example, investigated in the OASIS and BOVen studies.38,39 Encouragingly, brexucabtagene autoleucel (KTE-X19), an anti-CD19 autologous CAR-T cell US Food and Drug Administration approved for R/R MCL, has shown prom is ing efficacy even among high-risk MCL patients with elevated pro liferative indices, blastic or pleomorphic morphology, and TP53 mutation.24,40 In the future, novel treatment options, including anti-CD19 chimeric-antigen receptor T-cell therapy or CD20/CD3 bispecific antibody therapy, will likely be incorporated into clini cal trials in earlier lines, particularly for high-risk patients. .gov identifier: NCT03267433). At the end of induction chemo therapy, she was minimal residual disease (MRD) undetectable by an immunosequencing assay (clonoSEQ®) and was random ized to receive rituximab maintenance alone (ASCT omitted). ibrutinib during induction and maintenance and determine if ASCT can be omitted with the addition of ibrutinib. advi sory board: Celgene, Genentech, Kite Pharmaceuticals, Loxo Oncology/Lily, Astra Zeneca. Conclusion Off-label drug use All 4 clinical cases presented highlight the evolving role of upfrontASCTin MCL, and I have summarized my approach in Figure 1. While up-front ASCT remains a well-established and highly effective initial therapeutic strategy for younger MCL patients, there are sub groups of patients in whom this approach is not warranted, such as patients with TP53 mutation. Although ASCT consolidation has been utilized in patients older than 65 years who are fit without signif i cant comorbidity, I do not sup port this approach since the grand major ity of clin i cal trial data supporting upfront ASCT in MCL have focused on younger patients, and the already uncertain OS benefit of ASCT is even less well established in older patients who have an increased risk for transplant-associated toxicities and competing risks for sur vival with advanced age. 47,48 Ultimately, after discussion of benefits vs risks, it is critical to develop individualized treat ment plans that incorporate patient preferences and values. Key clinical trials, including EA4151 and the European Triangle study, will help to further clarify the role of ASCT in the future. Conflict-of-interest disclosure Anita Kumar: research funding: Abbvie Pharmaceuticals, Adaptive Biotechnologies, Celgene, Pharmacyclics, Seattle Genetics, Astra Zeneca, Loxo Oncology/Lily; honoraria: Celgene, Genentech, Kite Pharmaceuticals, Loxo Oncology/Lily, Astra Zeneca; 160 | Hematology 2022 | ASH Education Program Anita Kumar: nothing to disclose. Correspondence Anita Kumar, Department of Medicine, Lymphoma Service, Memorial Sloan Kettering Cancer Center, 1275 York Ave, Box 468, New York, NY 10065; e-mail: kumara2@mskcc.org. References 1. 2. 3. 4. 5. 6. Zelenetz AD, Gordon LI, Chang JE, et al. NCCN Guidelines® insights: B-cell lymphomas, Version 5.2021. J Natl Compr Canc Netw. 2021;19(11):12181230. Munshi PN, Hamadani M, Kumar A, et al. ASTCT, CIBMTR, and EBMT clinical practice recommendations for transplant and cellular therapies in mantle cell lymphoma. Bone Marrow Transplant. 2021;56(12):2911-2921. Dreyling M, Campo E, Hermine O, et al. Newly diagnosed and relapsed mantle cell lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2017;28(suppl 4):iv62-iv71. Linton K, Dreyling M. EHA endorsement of ESMO Clinical Practice Guidelines for Newly Diagnosed and Relapsed Mantle Cell Lymphoma. Hemasphere. 2020;4(5):e464. Dreyling M, Lenz G, Hoster E, et al. Early consolidation by myeloablative radiochemotherapy followed by autologous stem cell transplantation in first remission significantly prolongs progression-free survival in mantlecell lymphoma: results of a prospective randomized trial of the European MCL Network. Blood. 2005;105(7):2677-2684. Zoellner AK, Unterhalt M, Stilgenbauer S, et al; European Mantle Cell Lymphoma Network. Long-term survival of patients with mantle cell lymphoma after autologous haematopoietic stem-cell transplantation in first remis sion: a post-hoc analysis of an open-label, multicentre, randomised, phase 3 trial. Lancet Haematol. 2021;8(9):e648-e657. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 Figure 1. Treatment algorithm for untreated MCL patients. 7. 26. Riedell PA, Hamadani M, Ahn KW, et al. Outcomes and utilization trends of front-line autologous hematopoietic cell transplantation for mantle cell lymphoma. Transplant Cell Ther. 2021;27(11):911.e1-911911.e7. 27. Sawalha Y, Radivoyevitch T, Jia X, et al. The impact of socioeconomic disparities on the use of upfront autologous stem cell transplantation for man tle cell lymphoma. Leuk Lymphoma. 2022;63(2):335-343. 28. Gerson JN, Handorf E, Villa D, et al. Survival outcomes of younger patients with mantle cell lymphoma treated in the rituximab era. J Clin Oncol. 2019;37(6):471-480. 29. Glimelius I, Smedby KE, Albertsson-Lindblad A, et al. Unmarried or lesseducated patients with mantle cell lymphoma are less likely to undergo a transplant, leading to lower survival. Blood Adv. 2021;5(6):1638-1647. 30. Abrahamsson A, Albertsson-Lindblad A, Brown PN, et al. Real world data on pri mary treat ment for man tle cell lym phoma: a Nor dic Lymphoma Group observational study. Blood. 2014;124(8):1288-1295. 31. Albertsson-Lindblad A, Palsdottir T, Smedby KE, Weibull CE, Glimelius I, Jerkeman M. Survival in mantle cell lymphoma after frontline treatment with R-bendamustine, R-CHOP and the Nor dic MCL2 reg i men—a real world study on patients diagnosed in Sweden 2007-2017. Haematologica. 2022;107(3):740-743. 32. Cassaday RD, Guthrie KA, Budde EL, et al. Specific fea tures iden tify patients with relapsed or refrac tory man tle cell lym phoma benefit ting from autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2013;19(9):1403-1406. 33. Fenske TS, Zhang MJ, Carreras J, et al. Autologous or reduced-intensity conditioning allogeneic hematopoietic cell transplantation for chemother apy-sensitive mantle-cell lymphoma: analysis of transplantation timing and modality. J Clin Oncol. 2014;32(4):273-281. 34. Eskelund CW, Dahl C, Hansen JW, et al. TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy. Blood. 2017;130(17):1903-1910. 35. Aukema SM, Hoster E, Rosenwald A, et al. Expression of TP53 is associated with the outcome of MCL independent of MIPI and Ki-67 in trials of the European MCL Network. Blood. 2018;131(4):417-420. 36. Rodrigues JM, Hassan M, Freiburghaus C, et al. p53 is associated with highrisk and pinpoints TP53 missense mutations in mantle cell lymphoma. Br J Haematol. 2020;191(5):796-805. 37. Ferrero S, Rossi D, Rinaldi A, et al. KMT2D mutations and TP53 disruptions are poor prognostic biomarkers in mantle cell lymphoma receiving highdose therapy: a FIL study. Haematologica. 2020;105(6):1604-1612. 38. Le Gouill S, Morschhauser F, Chiron D, et al. Ibrutinib, obinutuzumab, and venetoclax in relapsed and untreated patients with mantle cell lymphoma: a phase 1/2 trial. Blood. 2021;137(7):877-887. 39. Kumar A, Soumerai JD, Abramson JS, et al. Preliminary safety and efficacy from a multicenter, investigator-initiated phase II study in untreated TP53 mutant mantle cell lymphoma with zanubrutinib, obinutuzumab, and venetoclax (BOVen). Blood. 2021;138(suppl 1):3540. 40. Wang M, Munoz J, Goy A, et al. Three-year follow-up of KTE-X19 in patients with relapsed/refractory mantle cell lymphoma, including high-risk sub groups, in the ZUMA-2 study [published online 4 June 2022]. J Clin Oncol. 2022;JCO2102370. 41. Chang JE, Li H, Smith MR, et al. Phase 2 study of VcR-CVAD with mainte nance rituximab for untreated mantle cell lymphoma: an Eastern Cooperative Oncology Group study (E1405). Blood. 2014;123(11):1665-1673. 42. Ladetto M, Tavarozzi R, Pott C. Minimal residual disease in mantle cell lym phoma: methods and clinical significance. Hematol Oncol Clin North Am. 2020;34(5):887-901. 43. Pott C, Hoster E, Delfau-Larue MH, et al. Molecular remission is an indepen dent predictor of clinical outcome in patients with mantle cell lymphoma after combined immunochemotherapy: a European MCL intergroup study. Blood. 2010;115(16):3215-3223. 44. Liu H, Johnson JL, Koval G, et al. Detection of minimal residual disease fol lowing induction immunochemotherapy predicts progression free survival in mantle cell lymphoma: final results of CALGB 59909. Haematologica. 2012;97(4):579-585. 45. Wang ML, Jain P, Zhao S, et al; Mantle Cell Research Group. Ibrutinibrituximab followed by R-HCVAD as frontline treatment for young patients (≤65 years) with mantle cell lymphoma (WINDOW-1): a single-arm, phase 2 trial. Lancet Oncol. 2022;23(3):406-415. 46. Dreyling M, Ladetto M, Doorduijn JK, et al. Triangle: autologous transplan tation after a rituximab/ibrutinib/ara-c containing induction in generalized Dr Prakash Singh Shekhawat Autotransplant in mantle cell lymphoma | 161 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 Hermine O, Hoster E, Walewski J, et al; European Mantle Cell Lymphoma Network. Addition of high-dose cytarabine to immunochemotherapy before autol o gous stem-cell trans plan ta tion in patients aged 65 years or youn ger with man tle cell lym phoma (MCL Younger): a randomised, open-label, phase 3 trial of the European Mantle Cell Lymphoma Network. Lancet. 2016;388(10044):565-575. 8. Le Gouill S, Thieblemont C, Oberic L, et al; LYSA Group. Rituximab after autologous stem-cell transplantation in mantle-cell lymphoma. N Engl J Med. 2017;377(13):1250-1260. 9. Hermine O, Jiang L, Walewski J, et al. Addition of high-dose cytarabine to immunochemotherapy before autologous stem-cell transplantation in patients aged 65 years or younger with mantle cell lymphoma (MCL Younger): a long-term follow-up of the randomized, open-label, phase 3 trial of the European Mantle Cell Lymphoma Network. Blood. 2021; 138(suppl 1):380. 10. Geisler CH, Kolstad A, Laurell A, et al; Nordic Lymphoma Group. Long-term progression-free survival of mantle cell lymphoma after intensive frontline immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma Group. Blood. 2008;112(7):2687-2693. 11. Eskelund CW, Kolstad A, Jerkeman M, et al. 15-year follow-up of the Second Nordic Mantle Cell Lymphoma trial (MCL2): prolonged remissions without survival plateau. Br J Haematol. 2016;175(3):410-418. 12. Delarue R, Haioun C, Ribrag V, et al; Groupe d’Etude des Lymphomes de l’Adulte (GELA). CHOP and DHAP plus rituximab followed by autolo gous stem cell transplantation in mantle cell lymphoma: a phase 2 study from the Groupe d’Etude des Lymphomes de l’Adulte. Blood. 2013;121(1): 48-53. 13. Romaguera JE, Fayad LE, Feng L, et al. Ten-year follow-up after intense chemoimmunotherapy with rituximab-HyperCVAD alter nat ing with rituximabhigh dose methotrexate/cytarabine (R-MA) and without stem cell trans plantation in patients with untreated aggressive mantle cell lymphoma. Br J Haematol. 2010;150(2):200-208. 14. Merryman RW, Edwin N, Redd R, et al. Rituximab/bendamustine and rituximab/cytarabine induction therapy for transplant-eligible mantle cell lym phoma. Blood Adv. 2020;4(5):858-867. 15. Chihara D, Cheah CY, Westin JR, et al. Rituximab plus hyper-CVAD alter nating with MTX/Ara-C in patients with newly diagnosed mantle cell lym phoma: 15-year follow-up of a phase II study from the MD Anderson Cancer Center. Br J Haematol. 2016;172(1):80-88. 16. Massaro F, Stepanishyna Y, Manni M, et al. Long-term results of the MCL01 phase II trial of rituximab plus HyperCVAD alter nat ing with high-dose cytarabine and methotrexate for the initial treatment of patients with man tle cell lymphoma. Br J Haematol. 2021;192(6):1011-1014. 17. Kumar A, Sha F, Toure A, et al. Patterns of survival in patients with recurrent mantle cell lymphoma in the modern era: progressive shortening in response duration and survival after each relapse. Blood Cancer J. 2019;9(6):50. 18. Jain P, Wang M. Blastoid mantle cell lymphoma. Hematol Oncol Clin North Am. 2020;34(5):941-956. 19. Goy A, Sinha R, Williams ME, et al. Single-agent lenalidomide in patients with mantle-cell lymphoma who relapsed or progressed after or were refractory to bortezomib: phase II MCL-001 (EMERGE) study. J Clin Oncol. 2013;31(29):3688-3695. 20. Wang ML, Rule S, Martin P, et al. Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N Engl J Med. 2013;369(6):507-516. 21. Wang M, Rule S, Zinzani PL, et al. Acalabrutinib in relapsed or refractory mantle cell lymphoma (ACE-LY-004): a single-arm, multicentre, phase 2 trial. Lancet. 2018;391(10121):659-667. 22. Tam CS, Opat S, Simpson D, et al. Zanubrutinib for the treat ment of relapsed or refractory mantle cell lymphoma. Blood Adv. 2021;5(12):25772585. 23. Davids MS, Roberts AW, Seymour JF, et al. Phase I first-in-human study of venetoclax in patients with relapsed or refractory non-Hodgkin lymphoma. J Clin Oncol. 2017;35(8):826-833. 24. Wang M, Munoz J, Goy A, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med. 2020;382(14):13311342. 25. Martin P, Cohen JB, Wang M, et al. Treatment out comes and roles of transplantation and maintenance rituximab in patients with previously untreated mantle cell lymphoma: results from large real-world cohorts [published online 28 June 2022]. J Clin Oncol. 2022;JCO2102698. mantle cell lymphoma—a randomized European MCL Network trial. Blood. 2019;134(suppl 1):2816. 47. Dahi PB, Tamari R, Devlin SM, et al. Favorable outcomes in elderly patients undergoing high-dose therapy and autologous stem cell transplantation for non-Hodgkin lymphoma. Biol Blood Marrow Transplant. 2014;20(12):20042009. 48. Dahi PB, Lee J, Devlin SM, et al. Toxicities of high-dose chemotherapy and autologous hematopoietic cell transplantation in older patients with lym phoma. Blood Adv. 2021;5(12):2608-2618. 49. Smith MR, Jegede O, Martin P, et al. ECOG-ACRIN E1411 randomized phase 2 trial of bendamustine-rituximab (BR)-based induction followed by rituximab (R)±lenalidomide (L) consolidation for Mantle cell lymphoma: effect of adding bortezomib to front-line BR induction on PFS. JCO. 2021;39(suppl 15):7503. 50. Wang ML, Jurczak W, Jerkeman M, et al; SHINE Investigators. Ibrutinib plus bendamustine and rituximab in untreated mantle-cell lymphoma. N Engl J Med. 2022;386(26):2482-2494. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000333 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/155/2021743/155kumar.pdf by guest on 09 December 2022 162 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat IMMUNOTHERAPY IN MULTIPLE MYELOMA Antibodies and bispecifics for multiple myeloma: effective effector therapy Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada The therapeutic landscape in multiple myeloma (MM) has changed dramatically over the last 2 decades. With the introduction of novel immunotherapies, patients with MM can expect deeper responses, longer remissions, and improved overall survival. Since its approval by the US Food and Drug Administration in 2015, the monoclonal antibody specific for CD38, daratumumab, has been incorporated into both frontline and relapsed treatment regimens. Its role as a maintenance therapy is currently being explored. Subsequently, a variety of novel antibody therapeutics have evolved from the success of daratumumab, using similar concepts to target the malignant plasma cell clone. Noteworthy naked monoclonal antibodies include isatuximab, another agent directed against CD38, and elotuzumab, an agent directed against SLAM family member 7. Antibody-drug conjugates, complex molecules composed of an antibody tethered to a cytotoxic drug, target malignant cells and deliver a lethal payload. The first to market is belantamab mafodotin, which targets B-cell maturation antigen (BCMA) on malignant plasma cells and delivers a potent microtubule inhibitor, monomethyl auristatin F. Additionally, bispecific T-cell antibodies are in development that engage the immune system directly by simultaneously binding CD3 on T cells and a target epitope—such as BCMA, G-protein coupled receptor family C group 5 member D (GPRC5d), and Fc receptor homologue 5 (FcRH5)—on malignant cells. Currently, teclistamab, an anti-BCMA bispecific, is closest to approval for commercial use. In this review, we explore the evolving landscape of antibodies in the treatment of MM, including their role in frontline and relapse settings. LEARNING OBJECTIVES • Review the use of MoAbs in NDMM (transplant eligible and ineligible) and RRMM • Understand the evolving role of ADCs in RRMM • Explore the current landscape and future directions of BsAbs in RRMM Introduction CLINICAL CASE A 54yearold woman is diagnosed with immunoglobulin G kappa (IgGκ) multiple myeloma (MM). She undergoes induction therapy with triplet combination, followed by autologous stem cell transplant (ASCT) and lenalidomide maintenance. She experiences several relapses and is cycled through various lines of therapy. She has now had 4 lines of therapy and is considered tripleclass refractory (TCR), having become refractory to a proteosome inhibi tor, an immunomodulatory drug, and an antiCD38 mono clonal antibody (MoAb). Her treating physician offers to send her to an academic center for a clinical trial, but she opts to stay close to home and is started on belantamab mafodotin. Despite decades of scientific advancement, MM remains incurable in the vast majority of patients.1 Currently, the pri mary goal of treatment is to increase survival and maintain a reasonable quality of life. This is accomplished through therapeutic suppression of the malignant plasma cell (PC) clone, which subsequently mitigates diseaserelated com plications.1 Since their approval, MoAbs have been widely incor porated into both frontline and relapsed settings. Still, patients who become TCR remain challenging to treat and require therapies with novel mechanisms of action. In the recent LocoMMotion study, TCR MM patients who prospec tively received widely available standard of care (SOC) ther apies demonstrated an overall response rate (ORR) of 25%, a median duration of response (DOR) of 4.5 months, and Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 163 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Christopher Cipkar, Christine Chen, and Suzanne Trudel Monoclonal antibodies In the last decade, three MoAbs were approved for the treat ment of MM. Daratumumab (anti-CD38) and elotuzumab (antiSLAM family member 7 [SLAMF7]) were approved for commercial use in 2015 while isatuximab (anti-CD38) was approved in 2020. Daratumumab is a humanized IgG1κ MoAb that binds to CD38 expressed on malig nant PCs.4 It effectively eliminates CD38- expressing PCs through several mechanisms: antibody-dependent T-cellular cytotoxicity, antibody-dependent T-cellular phago cytosis, complement-dependent cytotoxicity, induction of apop tosis via Fcγ receptor–mediated cross-linking, and by various immunomodulatory effects.4 Several randomized controlled tri als have observed the clinical benefits of adding daratumamab to immunomodulator drugs and proteosome inhibitors in RRMM, establishing its use as an SOC agent in this setting (Table 1).5-8 In transplant-eligible patients, the addition of daratumamab to bortezomib-thalidomide-dexamethasone (phase 3 CASSIOPEIA trial) improved depth of response and led to a significant progres sion-free survival (PFS) benefit.9 Similarly, the addition of daratu mumab to lenalidomide-bortezomib-dexamethasone (RVd; phase 2 GRIFFIN trial) in transplant-eligible patients improved depth of response, including stringent complete remission and minimal residual disease (MRD) (10−5), and showed PFS benefit (HR 0.45; p = 0.324), while median OS was not reached in either group with limited follow-up.10 In both CASSIOPEIA and GRIFFIN, there was a benefit to adding daratumumab across allsubgroups except those with high-risk cytogenetics or International Scoring System stage III disease. Table 1. Pivotal randomized phase 3 trials of monoclonal antibody treatment for MM Arm (n=number of patients) Overall response (%) Median progression-free survival (months) Hazard ratio (P value) Transplant-ineligible NDMM D-VMP (n=350) 91 36.4 0.42 (P<.001) VMP (n=356) 74 19.3 MAIA11 Transplant-ineligible NDMM D-Rd (n=368) 92.9 NR Rd (n=369) 81.6 34.4 ELOQUENT 124 Transplant-ineligible NDMM E-Rd (n=374) 83 31.4 Rd (n=374) 79 29.5 CASSIOPEIA9 Transplant-eligible NDMM D-VTd (n=543) 93 NR VTd (n=542) 90 NR GRIFFIN10,a Transplant-eligible NDMM D-RVd (n=99) 99 NR RVd (n=97) 92 NR GMMG-HD623 Transplant-eligible NDMM E-RVd (n=555) B1 81.5%; B2 80.7% (≥VGPR) B1 66.2%; B2 67.2% (3y PFS) RVd (n=559) A1 78.9%; A2 78.2% (≥VGPR) A1 68.8%; A2 68.5% (3y PFS) GMMG-HD719 Transplant-eligible NDMM I-RVd (n=329) MRD 50.1% NR RVd (n=331) MRD 35.6% NR POLLUX6 RRMM, at least 1 prior line D-Rd (n=286) 93 44.5 Rd (n=283) 76 17.5 ELOQUENT 220 RRMM, 1-3 prior lines E-Rd (n=321) 79 19.4 Rd (n=325) 66 14.9 CANDOR8 RRMM, 1-3 prior lines D-Kd (n=312) 84 NR Kd (n=154) 75 15.8 IKEMA18 RRMM, 1-3 prior lines I-Kd (n=179) 87 NR Kd (n=123) 83 19.2 CASTOR5 RRMM, at least 1 prior line D-Vd (n=251) 83 16.7 Vd (n=247) 63 7.1 Clinical trial Patient population ALCYONE12 164 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat 0.53 (P<.001) 0.93 (P =.44) 0.47 (P<.001) Not available Not available (P =.86; no significant difference) Not available (OR=1.82; P<.001) 0.44 (P<.001) 0.71 (P<.001) 0.62 (P =.003) 0.53 (P<.001) 0.31 (P<.001) Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 overall survival (OS) of 11.1 months.2 To address this area of unmet need, the focus has shifted toward new approaches, such as chi meric antigen receptor (CAR) T-cell therapies, antibody-drug conjugates (ADCs), and bispecific antibodies (BsAbs).3 Although CAR T-cell ther apy has shown remark able over all effi cacy of greater than 70% across various early-phase studies—leading to the approvals of idecabtagene vicleucel and ciltacabtagene autoleucel—delays in drug administration due to manufacturing time and grade 3/4 adverse events (AEs) from cytokine release syn drome (CRS), neu ro tox ic ity, and cytopenias remain major concerns.3 The desire for safe “off-the-shelf” immunotherapies has strengthened interest in ADCs and BsAbs, which themselves have demonstrated promising efficacy in relapsed/refractory MM (RRMM).3 In this review we present the state of development of each class of antibody-directed therapy in MM, with an emphasis on the rapidly advancing ADC and BsAb therapeutic armamentarium. Table 1. Pivotal randomized phase 3 trials of monoclonal antibody treatment for MM (Continued ) Clinical trial Patient population ELOQUENT 321 RRMM, at least 2 prior lines APOLLO7 ICARIA-MM17 RRMM, at least 1 prior line RRMM, at least 2 prior lines Arm (n=number of patients) Overall response (%) Median progression-free survival (months) Hazard ratio (P value) E-Pd (n=60) 53 10.3 0.54 (P=.008) Pd (n=57) 26 4.7 D-Pd (n=151) 69 12.4 Pd (n=153) 46 6.9 I-Pd (n=154) 63 11.5 Pd (n=153) 32 6.5 0.63 (P=.0018) 0.6 (P=.001) Randomized phase 2 study Note: This list is not exhaustive for allrandomized trials including monoclonal antibodies. D, daratumumab; d, dexamethasone; E, elotuzumab; I, isatuximab; K, carfilzomib; M, melphalan; OR, odds ratio; P, pomalidomide; p, prednisone; R, lenalidomide; T, thalidomide; V, bortezomib. a in transplant-eligible NDMM patients (phase 2 SWOG-1211 and phase 3 GMMG-HD6 trials) or to Rd (phase 3 ELOQUENT-1 trial) in transplant-ineligible patients, no improvements in outcomes were demonstrated.22-24 Antibody-drug conjugates Despite the success of naked MoAbs, the vast majority of MM patients ultimately relapse and require novel interventions. Con sequently, researchers have cleverly combined the specificity of MoAbs with a cytotoxic drug, creating a sophisticated delivery system that transports a lethal payload directly to the antigenexpressing cell (Figure 1). This technology has already shown success in other hematologic malignancies, including lymphoma (brentuximab vedotin) and acute mye loid leu ke mia (gemtu zumab ozogamicin).25 Several ADCs have been evaluated in clinical trials in RRMM (Table 2), the most promising of which target B-cell maturation antigen (BCMA). BCMA has emerged as an attractive target, as it is expressed at high levels on PCs and plasmablasts but not on other tissues.26 This selectivity, in addition to the fact that BCMA undergoes internalization, makes it an ideal target for ADCs.26 Belantamab mafodotin (GSK2857916), a first-in-class human ized IgG1, afucosylated ADC conjugated to monomethyl auri statin-F (MMAF), was the first ADC to demonstrate significant therapeutic benefit in MM. Preclinical studies demonstrate that belantamab mafodotin eliminates myeloma cells through sev eral mechanisms of action: direct cell killing via the inhibition of microtubule polymerization, classical IgG effector functions through an enhanced fragment crystallizable region (Fc) domain, and immunogenic cell death, a process in which dying cells elicit an adaptive immune response.27 Belantamab mafodotin was eval u ated in the piv otal DREAMM-2 study.28 This phase 2 study explored doses of 2.5 mg/kg or 3.4 mg/kg admin is tered intra ve nously every 3 weeks until progression. In patients receiving the US Food and Drug Administration–approved 2.5-mg/kg dose (n = 97), all were TCR and had a median of 7 (3-12) prior lines of therapy. The ORR was 31% (97.5% CI, 20.8–42.6), and the median PFS and OS were 2.8 (95% CI, 1.6–3.6) and 13.7 months (95% CI, 9.9-not reached), respec tively. For responding patients, the DOR was 11 months (95% CI, 4.2-not reached). Post hoc ana Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 165 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 A PFS benefit is also seen for frontline daratumamab in com bination with bortezomib-melphalan-prednisone (VMP; phase 3 ALCYONE trial) or lenalidomide-dexa meth a sone (Rd; phase 3 MAIA trial) in transplant-ineligible patients.11,12 Further, daratu mumab has been com bined with carfilzomib/Rd (KRd) as an induction therapy, with or without ASCT, in the phase 2 MASTER and MANHATTAN studies. Early results show impressive MRDnegative (10−5) remissions; 80% in the MASTER trial and 71% in the MANHATTAN trial.13,14 The ques tion of daratumumab main te nance was first addressed in the second randomization of the CASSIOPEIA trial, in which patients were further randomized after ASCT/consoli dation to daratumumab for up to 2 years or active surveillance. The median PFS was not reached (NR) in the daratumumab maintenance arm and was 46.7 months in the observation arm (hazard ratio [HR], 0.53; 95% CI, 0.42–0.68; P < .0001).15 Unex pectedly, those patients who had received daratumumab during induction and consolidation derived no incremental benefit from daratumumab maintenance alone vs observa tion alone (HR, 1.02; 95% CI, 0.71–1.47; P = .91), suggesting that depth of response, rather than continuous therapy, may have been the determining factor.15 Ongoing tri als such as GRIF FIN (NCT02874742), DRAMMATIC (NCT04071457), PERSEUS (NCT03710603), and AURIGA (NCT03901963) will help elucidate the optimal use of daratumumab or daratumamab plus lenalid omide as maintenance therapy. Isatuximab is another IgG1κ MoAb that binds to CD38. One nota ble dif fer ence from daratumumab is that isatux imab can induce direct cyto tox ic ity via caspase-depen dent apoptosis and lysosome-mediated nonapoptotic cell killing.16 Isatuximab has been successfully combined with pomalidomidedexamethasone (Pd; phase 3 ICARIA-MM trial) and Kd (phase 3 IKEMA trial) in RRMM, with superior PFS demonstrated in the isatuximab arms (Table 1).17,18 In the GMMG-HD7 phase 3 study in newly diagnosed multiple myeloma (NDMM), isatuximab added to RVd improved MRD (10−5) negativity rates (50.1% vs 35.6%) prior to ASCT, further supporting the incorporation of an antiCD38 MoAb in frontline treatment.19 Elotuzumab, a human ized IgG1 MoAb directed against SLAMF7, has shown improved PFS and OS in combination with Rd (phase 3 ELOQUENT-2 trial) and Pd (phase 3 ELOQUENT-3 trial) in RRMM.20,21 Disappointingly, when added to frontline RVd lyses demonstrated similar response and OS in patients with high-risk cytogenetics and those with impaired renal function, although patients with extramedullary disease did not appear to derive the same benefit.28 Consistent with MMAF-containing ADCs, the most common AEs (any grade/grade ≥3) were ker atopathy (72%/46%), change in best corrected visual acuity (54%/31%), thrombocytopenia (38%/22%), anemia (27%/21%), and blurred vision (25%/4%). Dose reductions and delays due to toxicity occurred in 54% and 35%, respectively, and were less common in the 2.5-mg/kg arm of the study. Belantamab mafodotin is US Food and Drug Administration approved for use in RRMM patients who have received 4 or more lines of therapy. The approval came with a boxed warning, indi cating that toxicity to the cornea may result in vision loss, corneal ulcers, or dry eyes. Although the mechanism is yet unclear, pre clinical data suggest that ocular toxicity is related to the receptorindependent uptake of the intact ADC into the epithelial limbal stem cells of the cornea.29 In DREAMM-2, 72% of patients in the 2.5-mg/kg cohort dem on strated keratopathy on oph thal mo logic exam; however, only 56% experienced symptoms (most commonly blurred vision and/or dry eyes), and only 3 patients (3%) discontinued therapy for corneal AEs.28 Importantly, expe rience from DREAMM-2 indicates that patients recover from cor neal toxicity with dose holds for grade 2 events or higher, with the majority of patients (88%) maintaining responses despite prolonged dose delays. Based on this experience, the recom mendations for management of corneal events include the fre quent use of pre ser va tive-free lubri cant eye drops, eye care 166 | Hematology 2022 | ASH Education Program pro fes sional assess ments before each dose, and timely dose holds and modifications. Numerous studies are now evaluating belantamab mafodotin in different drug combinations, dosing schedules, and treatment settings (Table 2). This includes studies combining belantamab mafodotin with pembrolizumab (DREAMM-4),30 with novel agents (DREAMM-5),31 with lenalidomide or bortezomib (DREAMM-6),32 and with RVd in NDMM (DREAMM-9).33 Notably, in the Algonquin study, belantamab mafodotin was combined with Pd to identify the optimal dose and schedule in pomalidomide-naive patients.34 Across cohorts of patients receiv ing belantamab mafodotin, 1.92 mg/kg every 4 weeks or 2.5 mg/kg every 4, 8, or 12 weeks (n=56), the ORR was 88.9% (≥ very good partial response [VGPR], 72%) and PFS, 17 months (14.5-not reached); nota bly, 48% of patients were TCR.34 Additionally, studies exploring lower doses and extended schedules of belantamab mafodotin as a strategy to reduce the incidence and severity of corneal toxicity are ongo ing (DREAMM-9, DREAMM-14, and NCT04808037). Emerging data from these studies are encouraging, demonstrating a reduction in the incidence and severity of corneal toxicity with good clinical efficacy.34,35 Several other ADCs targeting BCMA have been devel oped. AMG 224 is a humanized IgG1 anti-BCMA mertansine- conjugated ADC. In a phase 1 study, at the expansion dose of 3 mg/kg (n = 11) the ORR was 27%.36 Mild ocular events were observed in 30% of patients with no reports of keratopathy. MEDI2228 is another humanized pyrrolobenzodiazepine (PBD)conjugated anti-BCMA ADC. In a first-in-human study, an ORR Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Figure 1. Characteristics of ADCs. The components of the ADC and its target antigen influence the efficacy and safety profile. Pref erably, target antigens should only be found on malignant cells, be abundantly expressed, be capable of internalization, and not be shed from the cellular membrane. The cytotoxic drug (payload or warhead) is the ultimate effector component, inducing direct cell killing either by inhibiting microtubule formation or directly damaging cellular DNA. It should be highly potent in the subnanomolar range and preferably nonpermeable to avoid damage to surrounding tissues. The linker connects the warhead to the antibody. These should be stable in circulation and cleavable upon lysosomal degradation. The conjugation chemistry of the linker determines the drug:antibody ratio, which critically influences the ADC potency. ADCC, antibody-dependent T-cellular cytotoxicity, Fab, fragment antigen-binding region. Table 2. Summary of antibody-drug conjugates for MM Target Payload Combination Trial phase: number of patients (n) Response/ activity Current status (ClinicalTrials.gov) Belantamab mafodotin (belamaf) BCMA MMAF Monotherapy28 Phase 1: n=35 ORR 60% DREAMM-1 (completed) NCT02064387 Phase 2: n=196 ORR 31% DREAMM-2 (completed) NCT03525678 B (Q3W) vs Pd Phase 3: n=380 (E) N/A DREAMM-3 (recruiting) NCT04162210 B (Q3W) + Pemb30 Phase 1/2: n=41 ORR 47% DREAMM-4 (active, not recruiting) NCT03848845 B + novel agent31 Phase 1/2: n=464 (E) ORR 53% with feladilimab DREAMM-5 (recruiting) NCT04126200 B-Vd OR Rd32 Phase 1/2: n=152 (E) ORR 78% in BVd arm DREAMM-6 (active, not recruiting) NCT03544281 B-Pd34 Phase 1/2: n=96 (E) ORR 88.9% ALGONQUIN (recruiting) NCT03715478 B-Rd (transplantineligible NDMM) Phase 1/2: n=66 (E) N/A NCT04808037 (recruiting) B-Vd vs D-Vd Phase 3: n=575 (E) N/A DREAMM-7 (active, not recruiting) NCT04246047 B-Pd vs V-Pd Phase 3: n=450 (E) N/A DREAMM-8 (recruiting) NCT04484623 B-VRd (transplantineligible NDMM33 Phase 1: n=144 (E) ORR 100% (n=12) DREAMM-9 (recruiting) NCT04091126 Monotherapy in renal impairment Phase 1: n=36 (E) N/A DREAMM-12 (recruiting) NCT04398745 Monotherapy in liver impairment Phase 1: n=28 (E) N/A DREAMM-13 (recruiting) NCT04398680 Monotherapy (varying doses and schedules) Phase 2: n=180 (E) N/A DREAMM-14 (recruiting) NCT05064358 AMG 224 BCMA Mertansine Monotherapy36 Phase 1: n=42 (E) ORR 27% (3 mg/kg) NCT02561962 (active, not recruiting) CC 99712 BCMA Maytansinoid-like Monotherapy Phase 1: n=160 (E) N/A NCT04036461 (recruiting) MEDI2228 BCMA PBD Monotherapy Phase 1: n=82 ORR 66% at 0.14 mg/kg NCT03489525 (completed) Indatuximab ravtansine CD138 Maytansinoid DM4 In combination with R or P Phase 1/2: n=64 ORR 71.7% with R and 70.6% with P NCT01001442 (completed) Lorvotuzumab mertansine CD56 Mertansine Monotherapy Phase 1: n=37 ORR 5.7% NCT00346255 (completed) Milatuzumab CD74 Doxorubicin Monotherapy Phase 1: n=25 26% SD NCT00421525 (completed) 37 STRO-001 CD74 MMAF Monotherapy Phase 1: n=N/A N/A NCT03424603 (recruiting) DFRF4539A FcRH5 MMAE Monotherapy Phase 1: n=39 ORR 5%; 49% SD NCT01432353 (completed) SGN-CD48A CD48 MMAE Monotherapy Phase 1: n=14 N/A NCT03379584 (terminated) ABBV-838 SLAMF7 MMAE Monotherapy Phase 1/1b: n=75 ORR 10.7% NCT02462525 (terminated) Note: This list is not exhaustive for allADCs developed for MM. B, belantamab; belamaf, belantamab mafodotin; D, daratumumab; d, dexamethasone; E, estimate; MMAE, monomethyl auristatin E; N/A, not avail able; Pemb, pembrolizumab; P, pomalidomide; Q3W, once every 3 weeks; R, lenalidomide; SD, stable disease; V, bortezomib. of 65.9% was reported at the maximum tolerated dose (MTD) of 0.14 mg/kg (n = 41).37 The safety profile was consistent with PBD-containing ADCs and included rash (31.7%), thrombocyto penia (31.7%), pleural effusions (24.4%), and increased gammaglutamyl transferase (24.4%). Unexpectedly, ocular AEs in the form of photophobia were reported in 58.5% of patients. Phase 1 studies of CC-99712, an anti-BCMA ADC conjugated to 4 may tansinoid molecules, and HDP-101, an anti-BCMA ADC conju gated to an amanitin derivative, are recruiting (NCT04036461 and NCT04879043, respectively). Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 167 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Name ADCs targeting antigens other than BCMA have shown less promising efficacy in MM. Table 2 summarizes the current state of development of indatuximab ravtansine (anti-CD138), lorvotu zumab mertansine (anti-CD56), STRO-001 (anti-CD74), and several other ADCs with novel targets, most having had their develop ment halted due to disappointing efficacy. Bispecific antibodies Figure 2. Characteristics of BsAbs. IgG-like BsAbs include an Fc region, while non–IgG-like BsAbs consist of only Fab (fragment antigen-binding) variable regions and linkers. Since the Fc portion provides stability and longevity to the molecule in circulation, most non–IgG-like BsAbs require more frequent dosing to maintain therapeutic plasma levels; they also lack the Fc-mediated effector functions such as antibody-dependent T-cellular cytotoxicity and complement-dependent cytotoxicity. Several BsAbs, both IgG-like and non–IgG-like, are under development for the treatment of MM. BiAb, bispecific antibodies. 168 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 BsAbs are unique antibody constructs that simultaneously bind 2 antigens. In cancer therapeutics, this usually involves target ing an anti gen on the tumor and a mol e cule on an immune cell, resulting in immune effector cell activation and tumor lysis (Figure 2). Similar to the ADCs, BCMA has been the selected anti gen target for the development of BsAbs, although others have emerged (Table 3). AMG 420, which targets BCMA on PCs and CD3 on T cells, was the first BsAb to show efficacy in human trials. Coined a BiTE® (bispecific T-cell engager) because it lacks an Fc region, AMG 420 justified the development of bispecifics in MM, demonstrat ing a 70% ORR at the MTD (400 µg/d; n=10).38 The BiTE® format offers the advantage of better tissue penetrance and access to epitopes but with the caveat of a short half-life, necessitating a continuous intravenous infusion. This has been remedied by the development of an extended half-life version, AMG 701,39 and numerous Fc-containing BsAbs that can be administered every 1 to 3 weeks, with the majority moving to subcutaneous formula tions (Table 3).40-46 Fc-containing bispecifics are larger and there fore more stable in circulation, while the Fc function may or may not be silenced depending on the agent. Thus far, BsAbs have been evaluated in heavily pretreated patients, with the major ity being TCR; a good number have also included older patients (>80 years). All have demonstrated encouraging clinical activ ity (Table 3), although data on durability of responses are still immature. Teclistamab, an anti-BCMA/CD3 BsAb, is furthest along in clin ical development, with an anticipated commercial approval in 2022. In the phase 1/2 MajesTEC-1 study, 165 patients received teclistamab (77.8% TCR) at the recommended tar get dose of 1.5 mg/kg.40 The ORR was 63% (≥complete remission, 39.4%), with 44 patients (26.7%) achieving MRD (10−5). Responses were maintained across dif fer ent sub groups, includ ing poor-risk groups, with the exception of those with extramedullary disease, those with stage III disease, or those with PC marrow involve ment of 60% or greater. The median DOR was 18.4 months (95% CI, 14.9-not estimable), while the PFS was 11.3 months (95% CI, 8.8-17.1). CRS, neutropenia, infection, and neurotoxicity of any grade/grade higher than or equal to 3 occurred in 72.1%/0.6%, 70.9%/64.2%, 76.4%/44.8%, and 14.5%/0%, respec tively; 19 patients died from AEs, including 12 deaths due to COVID-19. In the phase 1 MagnetisMM-1 study, elranatamab (PF06863135), another anti-BCMAxCD3 BsAb, dem on strated an ORR of 64% among 55 patients (91% TCR) receiv ing doses of 215 µg/kg or higher.41 Remarkably, 7 of 10 patients treated with prior BCMA-targeted therapy achieved a partial response or better.41 The incidence of CRS at the recommended dose (1000 µg/kg or 76 mg) was 67% (allgrade 1/2).41 Studies of Table 3. Summary of bispecific T-cell antibodies for MM Triple-class refractory (median LoT) Trial phase Preliminary response/ activity Current status (ClinicalTrials .gov) Target Antibody construct AMG 42038 BCMA-CD3 BiTE® N/A (29% prior antiCD38, median 5 LoT) Phase 1 Continuous infusion for 4 wk (out of 6) ORR=31% ORR MTD=70% 38% CRS (6.25% ≥ gr 3) 5% ≥ gr 3 polyneuropathy 24% ≥ gr 3 infection Active, not recruiting NCT03836053 AMG 70139 BCMA-CD3 HLE-BiTE® 68% (median 6 LoT) Phase 1/2 Weekly IV ORR=36% ORR=83% at 9 mg 75% CRS (10.5% ≥ gr 3) 8% neurotoxicity (gr 1-2) 13% ≥ gr 3 infection Recruiting NCT03287908 Elranatamab41 BCMA-CD3 Humanized IgG2a Fc 91% (median 6 LoT; 22% prior antiBCMA) Phase 1 Weekly or every 2 wk Sc ORR=64% for doses ≥215 µg/kg 67% CRS (gr 1-2) MagnetisMM-1 Recruiting NCT03269136 REGN545842 BCMA-CD3 Fc Fab arms 97.1% (median 5 LoT) Phase 1/2 Weekly IV ORR=73.3% at 96-200-mg doses 38.2% CRS (gr 1-2) 4% neurotoxicity (gr 1-2) 23% pneumonia (11% ≥ gr 3) Recruiting NCT03761108 Teclistamab40 BCMA-CD3 Humanized IgG4 Fc 77.8% (median 5 LoT; prior anti-BCMA not permit ted) Phase 1/2 Weekly Sc ORR=63% 72.1% CRS (gr 3, 0.6%; no gr 4) 14.5% neurotoxicity (1 gr 4 event) 44.8% ≥ gr 3 infection MajestTEC-1 Recruiting NCT03145181 CC-9326944 BCMA-CD3 Asymmetric 2-arm IgG 66.7% (median 6 LoT) Phase 1 Weekly IV ORR=83.3% in 10 pts with doses ≥6 mg 89.5% CRS (1 gr 5 event) 26.3% infection Recruiting NCT03486067 TNB-383B44 BCMA-CD3 IgG4 Fc CD3 activating T effector cells 62% (median 5 LoT) Phase 1 Q21d IV ORR=79% at doses ≥40 mg 52% CRS (3% ≥ gr 3 at RP2D) 28% infection Recruiting NCT03933735 Cevostamab45 FcRH5CD3 Humanized IgG1 Fc 85% (median 6 LoT; 33.5% prior antiBCMA) Phase 1 Q21d IV ORR=54.5% at 160-mgdose level 80.7% CRS (1.3% ≥ gr 3) 18.8% ≥ gr 3 infection 14.3% neurotoxicity (0.3% ≥ gr 3) Recruiting NCT03275103 Talquetamab46 GPRC5DCD3 Humanized IgG4 Fc Weekly: 77% (median 6 LoT; 30% prior antiBCMA) Biweekly: 65% (median 5 LoT; 17% prior antiBCMA) Phase 1/2 Weekly or biweekly Sc Weekly: ORR=70% Biweekly: ORR=71% Weekly: 73% CRS (1 gr 3) Biweekly: 78% CRS (gr 1-2) MonumenTal-1 Recruiting NCT03399799 Schedule Safety Note: This list is not exhaustive for allbispecific T-cell antibodies developed for MM. Fab, fragment antigen-binding; gr, grade; HLE, half-life extended; IV, intravenous; LoT, lines of therapy; pts, patients; Q21d, every 21 days; RP2D, recommended phase 2 dose; Sc, subcutaneous; wk, week. teclistamab and elranatamab in combination with immunomod ulatory drugs and anti-CD38s and in earlier lines of treatment are ongoing (Table 4). Other nota ble anti-BCMA BsAbs include REGN5458, CC93269, and TNB-383B (Table 3). In a phase 1/2 trial of REGN5458, the ORR was 73.3% among patients treated at the 96-mg- and 200-mg-dose lev els; no patients expe ri enced CRS of grade 3 or higher.42 Of the 12 patients treated with 6 mg or more of CC-93269, the ORR was 83.3%.43 CRS was reported in 89.5% of patients, mostly grade 1/2 (57.9%/26.3%); however, one patient died in the setting of CRS, with infection as a potential contrib utor.43 In the dose-escalation cohort of TNB-383B (doses ≥40 mg every 3 weeks), the ORR was 79% (n=19/24) with CRS reported as mainly grade 1/2.44 Fc receptor homolog 5 (FcRH5) is a cell surface antigen of unknown func tion whose expres sion is restricted to B cells, Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 169 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Name Table 4. Summary of bispecific T-cell antibody combinations for MM Name Patient population Trial phase Combination drugs Elranatamab RRMM Phase 1b/2 Arm 1: elranatamab + nirogacestat (GSI) Arm 2: elranatamab + lenalidomide + dexamethasone Phase 3 Arm 1: elranatamab Arm 2: elranatamab + daratumumab Current status (ClinicalTrials.gov) MagnetisMM-4 (recruiting) NCT05090566 MagnetisMM-5 (recruiting) NCT05020236 Arm 3: earatumumab + pomalidomide + dexamethasone Teclistamab + talquetamab RRMM Phase 3 Arm 1: teclistamab + daratumumab Arm 2: daratumumab + pomalidomide + dexamethasone MajestTEC-3 (recruiting) NCT05083169 Phase 1 Arm 1: teclistamab + talquetamab NCT04586426 (recruiting) Arm 2: teclistamab + talquetamab + daratumumab Phase 1b Arm 1: daratumumab + teclistamab Arm 2: daratumumab + talquetamab TRIMM-2 (recruiting) NCT04108195 Arm 3: daratumumab + talquetamab + pomalidomide Arm 4: daratumumab + teclistamab + pomalidomide Cevostamab RRMM Phase 1 Arm 1: cevostamab Recruiting NCT04910568 Arm 2: cevostamab + pomalidomide + dexamethasone Arm 3: cevostamab + daratumumab + dexamethasone Note: This list is not exhaustive for allbispecific T-cell antibody combinations developed for MM. GSI, gamma secretase inhibitor. with the highest expression on PCs. BFCR4350A (cevostamab) is a humanized IgG Fc antibody targeting FcRH5 and CD3. In an ongoing phase 1 study, cevostamab was administered for a fixed duration of 17 cycles, unlike other BsAb trials that treat to progression.45 Among 160 patients (85% TCR), the ORR was 56.7% and 36.1% for those patients receiving doses of 132 to 198 mg (n=60) or 20 to 90 mg (n=83), respectively. The ORR among patients who had prior CAR T-cell or anti-BCMA ther apy was 44.4% and 36.4%, respectively. CRS was observed in 80.7% (128/160) of patients (mainly grade 1/2; 2 cases grade 3). In the single step-up dose cohorts (n=86), the median DOR was 11.5 months (95% CI, 6.0, 18.4) at a follow-up of 14.3 months. Thus, cevostamab appears to be a beneficial treatment option, with a unique therapeutic target, for RRMM patients. Talquetamab is an IgG4 Fc-containing BsAb targeting Gprotein–coupled receptor family C group 5 member D (GPRC5D).46 GPRC5D is highly expressed on PCs but also on keratinized tis sues. In the MonumenTal-1 study, patients received subcutaneous talquetamab at doses of 405µg/kg (n=30) weekly or 800 µg/kg biweekly (n=23).44 Unique AEs, owing to the expres sion of GPRC5D on keratinized tissues, include dysgeusia, palmar/plantar desquamation, nail dystrophy, and systemic rash, which were reported in 75% of patients (mostly grade 1/2; 7.5% grade 3).46 Mitigation strategies for these toxicities included the use of emol lient creams and, for oral AEs, saliva-substitute sprays and rinses at the onset of symptoms. The ORR and CRS in patients receiving the 405-µg/kg or 800-µg/kg dose was 70% and 73% (1 grade 3) and 71% and 78% (allgrade 1/2), respectively.46 Trials explor ing talquetamab in combination with teclistamab, as well as with SOC antimyeloma agents, are actively recruiting (Table 4). 170 | Hematology 2022 | ASH Education Program CLINICAL CASE (Continued) The patient responds to belantamab mafodotin; however, she experiences multiple dose interruptions for corneal AEs and progresses after 9 months. She is then referred to an academic center and is enrolled in a clinical trial evaluating talquetamab (anti-GPRC5D) monotherapy. In cycle 1 she experiences grade 1 CRS but no neurotoxicity. After 6 months of therapy, she con tinues to have an ongoing response with manageable grade 1 dysgeusia and dermatologic symptoms. Conclusion Alongside CAR T-cell therapy, antibody-based immunotherapies have emerged as important off-the-shelf therapeutic options for allpatients with RRMM (Table 5). The sequencing and duration of these therapies remain ongoing clinical questions, although emerging data are encouraging. In a heavily pretreated RRMM population relapsing on a BsAb (n=64), the ORR to subsequent treatment (second BsAb, n=20; CAR T, n=15) was 58%, with an OS of 17.6 months (95% CI, 21.6-not reached).47 Further, in the Mag netisMM-1 study, 7 of 13 patients previously treated with an antiBCMA targeted therapy (5/7 for those treated with anti-BCMA ADCs) responded to the BCMAxCD3 BsAb, elranatamab.41 Mean while in the ongoing phase 1 study of cevostamab (FCRH5xCD3 BsAb) that limited treatment to 17 cycles, 6 patients continued to maintain responses for 6 months or longer after the cessation of treatment.45 While ADCs and BsAbs demonstrate promising efficacy, future efforts need to focus on the optimal timing and duration Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Arm 3: daratumumab + bortezomib + dexamethasone Table 5. Comparison of novel immunotherapy approaches in multiple myeloma Advantages Disadvantages Bispecific T-cell engagers CAR T-cell therapy Off-the-shelf therapy Off-the-shelf therapy - Immune and nonimmune mechanisms of action - - Infrequent dosing (every 3 wk-12 wk) - One-time therapy Encouraging response rates Deep responses Deep responses No CRS/ICANS Mostly grade 1-2 CRS/ICANS - Outpatient administration Only initial dosing as inpatient Vacation from continuous therapy Continuous therapy until progression Continuous therapy until progression - Frequent dose interruptions Weekly or biweekly dosing Administration delays due to manufacturing time Ocular toxicity Significant immunosuppression Potential for severe CRS/ICANS; prolonged cytopenias Ophthalmic exams prior to dosing Specialized centers required Complex infrastructure required Cost ($$) Cost ($$) Cost ($$$) ICANS, immune effector cell-associated neurotoxicity syndrome; wk, week. of therapy, on combinational strategies, on mitigation of the risk of immunosuppression/infection (BsAbs) and corneal tox icity (ADC), on the mechanisms of resistance, and on equita ble access. Although much work is still to be done, these novel approaches offer new hope for a yet incurable disease. Conflict-of-interest disclosure Christopher Cipkar: no competing financial interests to declare. Christine Chen: consultancy: Forus Therapeutics; advisory board: Amgen, Bristol Myers Squibb, Janssen Pharmaceuticals. Suzanne Trudel: research funding: Amgen, Bristol Myers Squibb, Genentech, GlaxoSmithKline, Janssen Pharmaceuticals, Pfizer, Roche; con sul tancy: Bristol Myers Squibb, Forus, GlaxoSmith Kline, K36 Therapeutics, Roche; advisory board: Amgen, Bristol Myers Squibb, GlaxoSmithKline, Janssen Pharmaceuticals, Pfizer, Sanofi. Off-label drug use Christopher Cipkar: nothing to disclose. Christine Chen: nothing to disclose. Suzanne Trudel: nothing to disclose. Correspondence Suzanne Trudel, Princess Margaret Cancer Centre, Department of Medical Oncology and Hematology, 700 University Ave, Ste 6-322, Toronto, ON M5G 1Z5, Canada; e-mail: Suzanne.trudel@ uhn.ca. References 1. 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Daratumumab plus lenalidomide and dexamethasone in relapsed/refractory multiple myeloma: extended follow-up of POLLUX, a randomized, open-label, phase 3 study. Leukemia. 2020;34(7):1875-1884. 7. Dimopoulos MA, Terpos E, Boccadoro M, et al; APOLLO Trial Investigators. Daratumumab plus pomalidomide and dexa meth a sone ver sus poma lidomide and dexamethasone alone in previously treated multiple mye loma (APOLLO): an open-label, randomised, phase 3 trial. Lancet Oncol. 2021;22(6):801-812. 8. Usmani SZ, Quach H, Mateos MV, et al. Carfilzomib, dexamethasone, and daratumumab ver sus carfilzomib and dexa meth a sone for patients with relapsed or refractory multiple myeloma (CANDOR): updated outcomes from a randomised, multicentre, open-label, phase 3 study. Lancet Oncol. 2022;23(1):65-76. 9. Moreau P, Attal M, Hulin C, et al. Bortezomib, thalidomide, and dexametha sone with or without daratumumab before and after autologous stem-cell transplantation for newly diagnosed multiple myeloma (CASSIOPEIA): a randomised, open-label, phase 3 study. Lancet. 2019;394(10192):29-38. 10. Sborov D, et al. Daratumumab (DARA) plus lenalidomide, bortezomib, and dexamethasone (RVd) in patients (Pts) with transplant-eligible newly diag nosed multiple myeloma (NDMM): final analysis of GRIFFIN after ≥ 1y follow up. Presented at: The International Myeloma Society 2022 Annual Meeting; August 27, 2022; Los Angeles, CA. 11. Facon T, Kumar S, Plesner T, et al. Daratumumab, lenalidomide and dexa methasone alone in newly diagnosed multiple myeloma (MAIA): overall survival results from a randomized, open-label, phase 3 trial. Lancet Oncol. 2021;22(11):1582-1596. doi: 10.1016/S1470-2045(21)00466-6. 12. Mateos MV, Cavo M, Blade J, et al. Overall survival with daratumumab, bortezomib, melphalan, and prednisone in newly diagnosed multiple mye loma (ALCYONE): a randomised, open-label, phase 3 trial. Lancet. 2020;395(10218):132-141. 13. Costa LJ, Chhabra S, Medvedova E, et al. Daratumumab, carfilzomib, lena lidomide, and dexamethasone with minimal residual disease responseadapted therapy in newly diagnosed multiple myeloma. J Clin Oncol. 2022;40(25):2901-2912. 14. Landgren O, Hultcrantz M, Diamond B, et al. Safety and effectiveness of weekly carfilzomib, lenalidomide, dexa meth a sone, and daratumumab combination therapy for patients with newly diagnosed multiple myeloma: the MANHATTAN nonrandomized clinical trial. JAMA Oncol. 2021;7(6):862868. 15. Moreau P, Hulin C, Perrot A, et al. Maintenance with daratumumab or observation following treatment with bortezomib, thalidomide, and dexa methasone with or without daratumumab and autologous stem-cell trans Dr Prakash Singh Shekhawat MRD testing in AML 2022 | 171 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 Antibody drug conjugates 172 | Hematology 2022 | ASH Education Program 32. Popat R, Nooka A, Stockerl-Goldstein K, et al. DREAMM-6: safety, tolerabil ity and clinical activity of belantamab mafodotin (belamaf) in combination with bortezomib/dexamethasone (BorDex) in relapsed/refractory multi ple myeloma (RRMM) [abstract]. Blood. 2020;138(suppl 1);1419. Abstract 1419. 33. Usmani S, Alonso A, Quach H, et al. DREAMM-9: phase I study of belantamab mafodotin plus standard of care in patients with transplant-ineligible newly diagnosed multiple myeloma. Blood. 2021;138(suppl 1):2738. 34. Trudel S, McCurdy A, Sutherland H, et al. Part 1 results of a dose-finding study of belantamab mafodotin in combination with pomalidomide and dexamethasone for the treatment of relapsed/refractory multiple mye loma (RRMM) [abstract]. Blood. 2021;138(suppl 1):1653. Abstract 725. 35. Terpos E, Gavriatopoulou M, Ntanasis-Stathopoulos I, et al. Safety and efficacy of belantamab mafodotin in combination with RD in newly diag nosed, transplant ineligible multiple myeloma patients: a phase 1/2 study by the Hel lenic Society of Hematology [ab stract]. HemaSphere. 2022; 6:79-80. Abstract S178. 36. Lee HC, Raje NS, Landgren O, et al. Phase 1 study of the anti-BCMA anti body-drug conjugate AMG 224 in patients with relapsed/refractory multi ple myeloma. Leukemia. 2021;35(1):255-258. 37. Kumar S, Migkou M, Bhutani M, et al. Phase 1, first-in-human study of MEDI2228, a BCMA-targeted ADC in patients with relapsed/refractory mul tiple myeloma [abstract]. Blood. 2020;138(suppl 1);179. Abstract 179. 38. Topp MS, Duell J, Zugmaier G, et al. Anti-B-cell maturation antigen BiTE molecule AMG 420 induces responses in multiple myeloma. J Clin Oncol. 2020;38(8):775-783. 39. Harrison SJ, Minnema MC, Lee HC, et al. A phase 1 first in human (FIH) study of AMG 701, an anti-B-cell maturation antigen (BCMA) half-life extended (HLE) BiTE (bispecific T-cell engager) molecule, in relapsed/refractory (RR) multiple myeloma (MM). Blood. 2020;136(suppl 1):181. 40. Moreau P, Garfall AL, van de Donk NWCJ, et al. Teclistamab in relapsed or refractory multiple myeloma. N Engl J Med. 2022;387(6):495-505. 41. Jakubowiak AJ, Bahlis NJ, Raje NS, et al. Elranatamab, a BCMA-targeted T-cell redirecting immu no ther apy, for patients with relapsed or refrac tory multiple myeloma: updated results from MagnetisMM-1. J Clin Oncol. 2022;40(suppl 16):8014. 42. Zonder JA, Richter J, Bumma N, et al. Early, deep and durable responses and low rates of cytokine release syndrome with REGN5458, a BCMAxCD3 bispecific mono clo nal anti body, in a phase 1/2 first-in-human study in patients with relapsed/refractory multiple myeloma (RRMM). Blood. 2021; 138(suppl 1);160. 43. Costa LJ, Wong SW, Bermúdez A, et al. First clinical study of the B-cell maturation antigen (BCMA) 2+1 T cell engager (TCE) cc-93269 in patients (pts) with relapsed/refractory multiple myeloma (RRMM): interim results of a phase 1 multicenter trial. Blood. 2019;134 (suppl 1):143. 44. Kumar SK, D’Souza A, Shah N, et al. A first-in-human study of TNB383B, a BCMA x CD3 $$. Blood. 2021;138(suppl 1):900. doi:10.1182/bl ood-2021-150757.45. Trudel S, Cohen A, Krishnan A, et al. Cevostamab monotherapy continues to show clinically meaningful activity and man ageable safety in patients with heavily pre-treated relapsed/refractory multiple myeloma (RRMM): updated results from an ongoing phase I study. Blood. 2021;138(suppl 1):157. doi:10.1182/blood-2021-147983.46. Krishnan AY, Minnema MC, Berdeja JG, et al. Updated phase 1, results from monu mental: first-in-human study of talquetamab, a G protein-coupled recep tor family C group 5 member D x CD3 bispecific antibody, in patients with relapsed and/or refractory multiple myeloma (RRMM). Blood. 2021; 138(suppl 1):158. doi:10.1182/blood-2021-146868.47. Mouhieddine TH, Van Oekelen O, Pan D, et al. Clinical outcomes of relapsed/refractory multiple myeloma patients following treatment with bispecific antibodies (BiAbs). Blood. 2021;138(suppl 1):821. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000334 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/163/2021838/163cipkar.pdf by guest on 09 December 2022 plant in patients with newly diagnosed multiple myeloma (CASSIOPEIA): an open-label, randomised, phase 3 trial. Lancet Oncol. 2021;22(10):13781390. 16. Richardson PG, Beksaç M, Špička I, Mikhael J. Isatuximab for the treat ment of relapsed/refractory multiple myeloma. Expert Opin Biol Ther. 2020;20(12):1395-1404. 17. Attal M, Richardson PG, Rajkumar SV, et al; ICARIA-MM Study Group. Isat uximab plus pomalidomide and low-dose dexamethasone versus poma lidomide and low-dose dexa meth a sone in patients with relapsed and refrac tory mul ti ple mye loma (ICARIA-MM): a randomised, multicentre, open-label, phase 3 study. Lancet. 2019;394(10214):2096-2107. 18. Moreau P, Dimopoulos MA, Mikhael J, et al; IKEMA Study Group. Isatux imab, carfilzomib, and dexa meth a sone in relapsed mul ti ple mye loma (IKEMA): a multicentre, open-label, randomised phase 3 trial. Lancet. 2021;397(10292):2361-2371. 19. Goldschmidt H, Mai EK, Nievergali E, et al. Addition of isatuximab to lena lidomide, bortezomib and dexamethasone as induction therapy for newlydiagnosed, transplant-eligible multiple myeloma patients: the phase III GMMG-HD7 trial. Blood. 2021;138(suppl 1):463. 20. Dimopoulos MA, Lonial S, Betts KA, et al. Elotuzumab plus lenalidomide and dexamethasone in relapsed/refractory multiple myeloma: extended 4-year follow-up and analysis of relative progression-free survival from the randomized ELOQUENT-2 trial. Cancer. 2018;124(20):4032-4043. 21. Dimopoulos MA, Dytfeld D, Grosicki S, et al. Elotuzumab plus pomalidomide and dexamethasone for multiple myeloma. N Engl J Med. 2018;379(19):18111822. 22. Usmani SZ, Hoering A, Ailawadhi S, et al; SWOG1211 Trial Investigators. Bor tezomib, lenalidomide, and dexamethasone with or without elotuzumab in patients with untreated, high-risk multiple myeloma (SWOG-1211): primary analysis of a randomised, phase 2 trial. Lancet Haematol. 2021;8(1):e45e54. 23. Goldschmidt H, Mai EK, Bertsch U, et al. Elotuzumab in combination with lenalidomide, bortezomib, dexamethasone and autologous transplanta tion for newly-diagnosed multiple myeloma: results from the randomized phase III GMMG-HD6 trial. Blood. 2021;138(suppl 1):486. 24. Dimopoulos MA, Richardson PG, Bahlis NJ, et al; ELOQUENT-1 Investigators. Addition of elotuzumab to lenalidomide and dexamethasone for patients with newly diagnosed, transplantation ineligible multiple myeloma (ELO QUENT-1): an open-label, multicentre, randomised, phase 3 trial. Lancet Haematol. 2022;9(6):e403-e414. 25. Rossi C, Chrétien ML, Casasnovas RO. Antibody-drug conjugates for the treatment of hematological malignancies: a comprehensive review. Target Oncol. 2018;13(3):287-308. 26. Kleber M, Ntanasis-Stathopoulos I, Terpos E. BCMA in multiple myeloma—a promising key to therapy. J Clin Med. 2021;10(18). 27. Tai YT, Mayes PA, Acharya C, et al. Novel anti-B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multi ple myeloma. Blood. 2014;123(20):3128-3138. 28. Lonial S, Lee HC, Badros A, et al. Longer term outcomes with single-agent belantamab mafodotin in patients with relapsed or refrac tory mul ti ple myeloma: 13-month follow-up from the pivotal DREAMM-2 study. Cancer. 2021;127(22):4198-4212. 29. Zhao H, Atkinson J, Gulesserian S, et al. Modulation of macropinocytosismediated internalization decreases ocular toxicity of antibody-drug conju gates. Cancer Res. 2018;78(8):2115-2126. 30. Suvannasankha A, Bahlis NJ, Trudel S, et al. Safety and clin i cal activ ity of belantamab mafodotin with pembrolizumab in patients with relapsed/refrac tory mul ti ple mye loma (RRMM): DREAMM-4 Study [abstract]. J Clin Oncol. 2022;(suppl 16);8018. Abstract 8018. 31. Callander N, Ribrag V, Richardson P, et al. DREAMM-5 study: investigat ing the synergetic effects of belantamab mafodotin plus inducible T-cell co-stimulator agonist (aICOS) combination therapy in patients with relapsed/refractory multiple myeloma [abstract]. Blood. 2021;138(suppl 1);897. Abstract 897. IMMUNOTHERAPY IN MULTIPLE MYELOMA Sarah A. Holstein Division of Oncology and Hematology, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE The development of novel cellular therapies and bispecific T-cell-engaging antibodies is occurring at breakneck speed in multiple myeloma (MM). While groundbreaking, these agents have their unique logistical and toxicity issues and currently do not represent a curative approach. In this context, there continues to be an urgent need to develop novel, off-the-shelf immunotherapy approaches to add to the armamentarium. This article explores novel agents being investigated in combination with standard immunomodulatory drugs as well as next-generation cereblon E3 ligase modulators. These novel agents include drugs being repurposed from their use in other diseases as well as novel monoclonal antibodies. In addition, agents under development such as immunocytokines, immunotoxins, and natural killer-cell activators/engagers are reviewed. These novel therapeutic strategies hold the promise of countermanding the immunosuppressive tumor microenvironment, leading to enhanced anti-MM activity. LEARNING OBJECTIVES • Learn about novel IMiD-based combinations and CELMoDs being tested in R/R MM • Recognize the developing therapeutic landscape of immunocytokines, immunotoxins, and NK-cell activators/ engagers in MM CLINICAL CASE A 64-year-old woman with immunoglobulin G (IgG) lambda relapsed/refractory multiple myeloma (R/R MM) received commercial idecabtagene vicleucel 6 months ago and is now experiencing biochemical relapse. She had previously received 5 prior lines of therapy, including prior autologous stem cell transplant (ASCT), and her disease is refractory to lenalidomide, pomalidomide, bortezomib, carfilzomib, daratumumab, and belantamab mafodotin. Her chimeric antigen receptor (CAR) T-cell course was complicated by cytokine release syndrome and neurotoxicity, and she wishes to avoid any subsequent therapies that could be associated with those adverse events (AEs). What novel non-CAR T-cell/non-bispecific antibody immunotherapy approaches are currently under investigation? Introduction Novel immunotherapies such as CAR T cells and bispecific T-cell-engaging antibodies show remarkable promise for MM. However, given the current logistical constraints of commercial CAR T-cell products, as well as the potential logistical issues with bispecific antibodies that may limit access to these therapies to certain populations, there continues to be a need for alternative approaches. With the remarkable success of small-molecule agents such as immunomodulatory drugs (IMiDs) and proteasome inhibitors, the ongoing substantial focus on developing novel small-molecule agents for MM is not surprising. In addition, the field is exploring the therapeutic potential of novel antibody-based approaches and natural killer (NK)-cell activators/engagers. In this educational session, we discuss the most recent research investigating these novel non-CAR T-cell/bispecific-based approaches for the treatment of R/R MM. IMiDs plus novel agent combinations The IMiDs lenalidomide and pomalidomide have helped transform the management of MM. Lenalidomide is a current standard of care in the newly diagnosed (ND) and maintenance settings, while pomalidomide is exclusively used in the R/R setting. The pleiotropic effects of these agents, which include both direct cytotoxic effects as well as a myriad of effects on the immune microenvironment, Dr Prakash Singh Shekhawat Next generation of novel therapies for myeloma | 173 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 Beyond the cell: novel noncellular immunotherapy approaches to multiple myeloma Table 1. Summary of ongoing trials involving IMiDs and novel agents Phase Study population Treatment Novel agent mechanism Primary end point(s) NCT03702725 1 R/R Ibrutinib + LenDex BTK inhibitor MTD, ORR NCT03110822 1 R/R with ≥2 prior lines Ruxolitinib + Len + steroids Jak inhibitor MTD, TEAE NCT04508790 2 R/R Leflunomide + PomDex Dihydroorate dehydrogenase inhibitor ORR NCT03143985 1 R/R Vactosertib + Pom TGFB1 receptor antagonist MTD NCT04405167 1 R/R Tasquinimod +/− IRD S100A9 inhibitor MTD NCT04942067 1/2 R/R Liasftoclax + PomDex or DaraLenDex BCL2 inhibitor DLT NCT04674514 1/2 R/R with ≥1 prior line Lisaftoclax + LenDex BCL2 inhibitor DLT NCT04025450 3 ND high risk Chidamide + VRD vs VRD HDAC inhibitor CR rate, AE NCT03605056 2 R/R with ≥1 prior line Chidamide + LenDex HDAC inhibitor ORR NCT02400242 1 R/R ACY-241 + PomDex HDAC inhibitor MTD NCT01997840 1/2 R/R with ≥2 prior lines Ricolinostat + PomDex HDAC inhibitor MTD; ORR NCT03031730 1 R/R with 1-3 prior lines KRT-232 + KRD MDM2 inhibitor Safety, TEAE BCL2, B-cell lymphoma 2; CR, complete response; Dara, daratumumab; Dex, dexamethasone; DLT, dose limiting toxicity; HDAC, histone deacetylase; Jak, Janus kinase; Len, lenalidomide; MDM2, mouse double minute 2; MTD, maximum tolerated dose; Pom, pomalidomide. coupled with generally tolerable side-effect profiles, have led to numerous combination-based studies. Substantial research is focused on deter min ing the mech a nisms of resis tance to these agents and whether these resistance mechanisms can be overcome via novel combinations. In this section an overview of some of the most recent IMiD plus novel agent combination strategies are provided, including those involving small mole cules (Table 1) and antibody-based therapies (Table 2). In several cases, agents being used to treat other malignancies or diseases are being repurposed for the treatment of MM. Other ongoing studies are investigating IMiD-based combinations with agents such as lisaftoclax (BCL2 inhibitor), KRT-232 (MDM2 inhibitor), or novel histone deacetylase inhibitors (eg, ricolinostat, ACY-241, chidamide) (Table 1). Not discussed in this chapter but included in Table 2 are a number of ongoing studies evaluating IMiDs with bispecific antibodies. The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is now widely used in the treatment of several B-cell malignancies. BTK is expressed in MM cells, and it may modulate the bone mar row (BM) microenvironment by decreasing cytokine/chemokine secre tion and suppressing mye loid-derived sup pres sor cell activity.1 While single-agent ibrutinib led to an overall response rate (ORR) of 0% in patients with R/R MM (64% lenalidomide refractory),2 sub se quent efforts focused on com bin ing the agent with IMiDs. A phase 1 study of ibrutinib-lenalidomidedexamethasone reported an ORR of 7% with a clinical benefit rate (≥ minimal response) of 80%. Notably, 54% of the patients were refractory to lenalidomide.3 An ongoing phase 1 study is evaluating the combination in patients with at least 1 prior line of therapy (eligible patients must not have progressed on full-dose lenalidomide but could have experienced disease progression on lenalidomide maintenance) (Table 1). A phase 1 study of ibrutinib in combination with pomalidomide-dexamethasone was completed, and the sponsor chose not to pursue the phase 2 component (NCT02548962). The Janus kinase (Jak) inhibitor ruxolitinib has a number of immunomodulatory properties that appear relevant for MM. 174 | Hematology 2022 | ASH Education Program Recent work has shown that ruxolitinib can decrease PD-L1 expression and decrease expression of a variety of genes impli cated in resistance to lenalidomide.4,5 A phase 1 study of ruxolitinib in combination with lenalidomide and methylprednisolone reported an ORR of 38% in a patient population in which 93% were lenalidomide refractory.6 Leflunomide was approved for the treat ment of rheu ma toid arthritis by the US Food and Drug Administration in 1998. Leflunomide is metab o lized to teriflunomide, which inhib its dihydroorate dehydrogenase, an enzyme involved in pyrimidine biosynthesis. While earlier studies suggested that leflunomide induced cytotoxicity in MM cells in part related to the disrup tion of pyrimidine biosynthesis, more recent work has indicated that this agent downregulates c-Myc expres sion by directly inhibiting the PIM family of serine/threonine kinases.7 Preclinical studies showed the anti-MM activity of leflunomide but only in immune-competent mice.7 Increases in T-cell activation markers (LAMP-1, CD69) with a decrease in the T-cell exhaustion marker CTLA4 were observed in treated animals.7 In addition, the com bination of lenalidomide with leflunomide prolonged survival in a mouse model of MM.7 Subsequently a phase 1 study of singleagent leflunomide was conducted in patients with R/R MM. No objective responses were reported; however, 9 in 11 patients achieved stable disease.8 Currently, this agent is being investi gated in patients with smoldering MM as well as in combination with pomalidomide-dexamethasone in patients with R/R MM. Vactosertib is a transforming growth factor beta 1 (TGFB1) receptor antagonist, and as there are data connecting increased TGFB1 secretion by MM cells with impaired immune surveillance, studies have been conducted evaluating the potential therapeu tic use of vactosertib in MM. Preclinical studies with vactosertib demonstrated single-agent activity in a mouse MM model as well as in primary patient samples.9 A phase 1 study was conducted evaluating the combination of vactosertib with pomalidomide (no steroids) and demonstrated the safety of this combination along with a 6-month progression-free survival (PFS) rate of 80% (n=15).10 Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 NCT number Table 2. Summary of ongoing trials involving IMiDs and novel immune-modulating antibodies Phase Study population Antibody target Treatment Primary end point(s) 1 R/R CD47 Lemzoparlimab +/− PomDex, CarDex, DaraDex DLT NCT04892446 2 R/R CD47 Magrolimab + PomDex, CarDex, BorDex, Dara DLT, AE, ORR NCT04150965 1/2 R/R with ≥3 prior lines LAG-3 TIGIT Relatlimab + PomDex; BMS-986207 + PomDex ORR, AE NCT04722146 1 R/R BCMA/CD3 Tec + DaraPom, DaraLenBor, nirogacestat, Len, DaraLen DLT, AE NCT05090566 2 R/R with ≥3 prior lines BCMA/CD3 Elran + LenDex DLT, TEAE NCT05020236 3 R/R with ≥1 prior line including Len + PI BCMA/CD3 Elran vs Elran+ Dara vs Elran +DaraPomDex PFS NCT05137054 1 R/R with ≥3 prior lines BCMA/CD3 REGN5458 + DaraDex, CarDex, LenDex or BorDex DLT, TEAE NCT05317416 3 MRD-positive post–up-front ASCT BCMA/CD3 Len vs Elran maintenance MRD negativity, PFS NCT05243797 3 Post–up-front ASCT BCMA/CD3 Len vs TecLen maintenance PFS NCT04910568 1 R/R FCRH5/CD3 Cevost +/− PomDex, DaraDex AE NCT05050097 1 R/R GPRC5D/CD3 Talq +/− Car, DaraCar, Len, DaraLen, Pom DLT, AE Bor, bortezomib; Car, carfilzomib; Cevost, cevostamab; Dara, daratumumab; Dex, dexamethasone; DLT, dose-limiting toxicity; Elran, elrantamab; MRD, minimal residual disease; PI, proteasome inhibitor; Pom, pomalidomide; Talq, talquetamab; Tec, teclistamab. Tasquinimod is an agent that targets myeloid-derived sup pressor cells via the inhibition of S100A9. While thus far it has primarily been investigated in the setting of prostate cancer, preclinical studies in MM revealed single-agent activity as well as in combination with lenalidomide.11 No direct cyto toxic effect of this agent was observed against MM cells in vitro, suggesting that the mechanism of action is via modulation of the tumor microenvironment.11 A phase 1 study of single-agent tasquinimod in R/R MM was completed and this agent is currently being evaluated in combination with ixazomib-lenalidomidedexamethasone (NCT04405167). While several phase 3 trials studying IMiDs in combination with anti-programmed cell death 1 protein inhibitors such as pembrolizumab and nivolumab failed,12-14 there continues to be interest in investigating the therapeutic potential of alternative checkpoint inhibitors. Several ongoing clinical trials are evaluating combi nations of IMiDs with novel checkpoint inhibitors such as antiLAG3 and anti-TIGIT monoclonal antibodies (mAbs) (Table 2). There is also inter est in targeting CD47, often referred to as the mac ro phage “don’t eat me” sig nal, and recent/ongo ing stud ies are explor ing magrolimab- or lemzoparlimab-based combinations in R/R MM. Whether the toxicity issues encoun tered with IMiDs and anti-programmed cell death 1 inhibitors will also be observed with any of these novel immune-targeting mAbs remains to be deter mined,15 but it is nota ble that the lemzoparlimab-based study was recently ter mi nated by the sponsor for as yet undisclosed reasons while unexpected seri ous AEs were reported with magrolimab in other malignancies. CELMoDs Iberdomide (CC-220) is an orally available agent that, similar to lenalidomide and pomalidomide, binds to the cereblon E3 ubiquitin ligase complex and thus is considered to be a CELMoD (cereblon E3 ligase modulator). Iberdomide more potently binds cereblon, thus leading to greater degradation of Ikaros and Ailos in cell culture studies compared to lenalidomide or pomalidomide.16 Preclinical studies demonstrated the activity of iberdomide in lenalidomide- or pomalidomide-refractory MM cells.17 A phase 1b/2a dose-esca la tion study of iberdomide in R/R MM was conducted.18 Iberdomide (daily on days 1-21 of a 28-day cycle) was administered in combination with weekly dexamethasone. The median number of prior regimens was 5 (2-12), with 100% receiving lenalidomide and 69% receiving prior pomalidomide. Dosing started at 0.3 mg, and the recommended phase 2 dose (RP2D) was determined to be 1.6 mg. The most common grade 3 to 4 AEs were neutropenia (26%), thrombocytopenia (11%), and neuropathy (2%). The ORR was 31% while 88% achieved stable disease or better (disease control rate).18 The results from the dose-expansion phase of this study showed an ORR of 28% with a clinical benefit rate of 36% and a disease control rate of 79% (n=107). In patients who had previously received anti–B-cell mat uration antigen (BCMA) therapy, the ORR was 25% (n=24).19 Mass cytometry analysis of BM aspirate samples from patients treated in the phase 1b/2a study revealed marked changes in the tumor microenvironment, including a reduction in naive and regula tory B cells, a reduction in CD4+ T cells with an increase in CD8+ T-cells, a shift toward the cytotoxic effector-memory phenotype of T cells, a decrease in CD8+ T cells expressing inhibitory check points (TIGIT, KLRG1), and an increase in NK cells (including acti vated NK cells) with a decrease in TIGIT+ NK cells.20 Clinical trials investigating iberdomide in combination with other standard agents are ongoing (Table 3). Preliminary results from a phase 1/2 study evaluating iberdomide in combination with either daratumumab-dexa meth a sone or bortezomibdexamethasone have been reported.21 The RP2D of iberdomide in both combinations was also determined to be 1.6 mg daily. The rate of grade 3 to 4 neutropenia in the iberdomide-daratumumabdexamethasone cohort was 67% and 26% in the iberdomidebortezomib-dexamethasone cohort. The rates of diarrhea (all grades) were 26% (iberdomide-daratumumab-dexamethasone) Dr Prakash Singh Shekhawat Next generation of novel therapies for myeloma | 175 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 NCT number NCT04895410 Table 3. Summary of ongoing trials involving iberdomide or mezigdomide Phase Study population Treatment Primary end point(s) 2 Intermediate or high-risk smoldering IberDex×4 cycles ⟶ Iber until PD vs Iber until PD ORR NCT05272826 2 ND TI IberBorDex sCR rate after 4 cycles NCT02773030 1/2 ND TI, R/R ND, R/R R/R post-BCMA R/R DaraIberDex IberBorDex IberDex IberCarDex MTD, RP2D, ORR NCT05199311 1/2 ND TE IberCarDex×2-4 cycles ⟶ ASCT Rate of TEAEs; CR/sCR rates NCT04934475 (IFM 2020-02) 3 ND TE Isa-KRD induction. Randomization based on MRD status to additional Isa-KRD vs ASCT + IsaKRD consolidation vs tandem ASCT. Maintenance includes randomiza tion to Len×3y vs IberIsa×3y MRD negativity rates NCT05177536 2 Post–up-front ASCT Iber maintenance until PD 1-y completion rate NCT04564703 2 Post–up-front ASCT Iber maintenance until PD Response improvement rate after 6 mo; rate of dose reductions/discontinuations within 6 mo NCT05354557 2 Post–up-front ASCT with ≤VGPR on Len maintenance or post-salvage ASCT after 2-3 prior therapies Iber maintenance×12 cycles 6-mo CR rate NCT04975997 3 R/R with 1-2 prior lines DaraIberDex vs DaraBorDex PFS NCT05289492 1/2 R/R with ≥3 prior lines (prior BCMA targeted therapy allowed) EOS884448, EOS884448-Iber, EOS884448-IberDex Rate of AEs/SAEs, number of DLTs, RP2D of EOS884448 +/− Iber(Dex), ORR NCT04392037 2 R/R with 2-4 prior lines; Len refractory IberCyDex PFS NCT03374085 1/2 R/R with ≥3 prior lines (prior CAR T therapy allowed) MezigDex AEs, PK parameters, MTD, ORR NCT03989414 1/2 ND or R/R R/R R/R R/R R/R MezigBorDex MezigCarDex MezigEloDex MezigIsaDex MezigDaraDex RP2D, DLTs, AEs, ORR Bor, bortezomib; Car, carfilzomib; CR, complete response; Cy, cyclophosphamide; Dara, daratumumab; Dex, dexamethasone; DLT, dose-limiting toxicity; Elo, elotuzumab; Iber, iberdomide; Isa, isatuximab; KRD, carfilzomib, lenalidomide, dexamethasone; Len, lenalidomide; Mezig, mezigdomide; MRD, minimum residual disease; MTD, maximum tolerated dose; PD, progressive disease; PK, pharmacokinetic; SAE, serious adverse event; sCR, strin gent complete response; TE, transplant eligible; TI, transplant ineligible. and 35% (iberdomide-bortezomib-dexa meth a sone). No pa tients developed thrombotic events. Antithrombotic prophy laxis is man da tory in allongo ing stud ies with iberdomide. Other iberdomide-based combination trials include ixazomibiberdomide-dexamethasone, iberdomide-cyclophosphamidedexamethasone, and iberdomide-carfilzomib-dexamethasone, as well as iberdomide in com bi na tion with EOS884448GSK4428859A, an anti-TIGIT monoclonal antibody. In addition, several studies are evaluating iberdomide as maintenance ther apy post ASCT (Table 3). Mezigdomide (CC-92480) is another potent CELMoD. This agent has a sig nifi cantly higher deg ra da tion effi ciency com pared to either lenalidomide or pomalidomide.22 The phase 1 study of CC-92480 in heavily treated R/R MM patients (median prior lines=6; half had triple-class refractory disease) in combi nation with dexamethasone showed an ORR of 55% at the RP2D 176 | Hematology 2022 | ASH Education Program (1.0 mg/d for 21 to 28 days).23 Grade 3/4 treatment-emergent AEs (TEAEs) were reported in 88% of patients, with the most frequent grade 3 and 4 AEs including neutropenia (53%), infec tions (30%), anemia (29%), and thrombocytopenia (17%). Correlative studies have shown Ikaros/Aiolos degradation in peripheral T cells in a dose-dependent manner at doses greater than or equal to 0.6 mg and showed substrate recovery during drug holidays (full recovery with ≥7-day breaks).24 Preliminary results have dem onstrated the safety and feasibility of combining mezigdomide with bortezomib-dexamethasone,25 and ongoing studies include other mezigdomide-based combinations, including carfilzomibdexamethasone, elotuzumab-dexamethasone, and isatuximab (or daratumumab)-dexamethasone (Table 3). In addition, more novel combinations such as mezigdomide-dexamethasone with tazemetostat (an EZH2 inhibitor), BMS-986158 (a BET inhibitor), or trametinib (a MEK inhibitor) are also planned (NCT05372354). Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 NCT number NCT04776395 Immunocytokines and immunotoxins Figure 1. Examples of immunocytokines and immunotoxins undergoing clinical trial evaluation in MM. Immunocytokines are antibody-cytokine fusion proteins, while immunotoxins are fusion proteins containing antibody fragments and a biologic toxin. SLTA, Shiga-like toxin A subunit; VH, heavy chain variable region; VL, light chain variable region. Created with BioRender .com. 38% in patients with a median of 7 prior lines of therapy, as well as an ORR of 38% in patients refractory to anti-CD38 mAb ther apy.30 The most notable AEs included cytopenias (76% thrombo cytopenia, 69% neutropenia, 66% anemia) and infusion-related reactions (31%).30 Currently, a randomized phase 2 study is eval uating 2 different dose levels (flat dosing of 120 or 240 mg every 4 weeks) in R/R MM. TAK-169 is considered an immunotoxin because it is an engineered pro tein containing a deimmunized form of the ribo some-inactivating Shiga-like toxin A subunit (SLTA) fused to an antibody fragment that specifically targets the CD38 cell sur face receptor.32 This agent is internalized by CD38-expressing cells, leading to cell death via the irreversible inhibition of pro tein synthesis. Ex vivo studies using primary BM samples showed lysis of the MM cells, with the highest amount of lysis in samples from ND or daratumumab-naive R/R patients.33 A phase 1 study in patients with R/R MM or non-Hodgkin lymphoma is ongoing (NCT04017130). An initial report after 4 patients were enrolled at the starting dose noted 1 patient experiencing a TEAE of asymp tomatic grade 2 myocarditis that was reversible.34 A number of other immunotoxins have undergone preclinical evaluation thus far, targeting a vari ety of tumor anti gens (eg, CD38, CD138, BCMA) and involving a number of toxins (eg, Shiga-like toxin A subunit SLTA, pseudomonas exotoxin, saporin, ricin).35 NK-cell activators and engagers Determining how to effectively harness the anti-MM activity of NK cells has been an active area of research for decades. While many ongoing studies involve genetic engineering of NK cells (eg, CAR NK cells), interest in alternative strategies by which to enhance the activity of endogenous NK cells continues. Earlier efforts included mAbs targeting killer immunoglobulin-like receptors (KIRs; eg, IPH2101, 1-7F9, lirilumab) and NK group 2 member A ( monalizumab). Currently, no ongoing studies are investigating these agents in MM. An alter na tive strat egy involves agents that enhance IL-15 activity, as IL-15 plays a key role in NK development and survival. ALT-803 is an IL-15 superagonist fusion protein that was evalu ated in a phase 1 study in R/R MM; however, the results have not been disclosed (NCT02099539). A study using ALT-803 in patients relapsing after allogeneic stem cell transplant did dem onstrate that this agent increased NK and CD8+ T-cell numbers and function.36 NKTR-255 is a polymer-conjugated IL-15 receptor agonist reported to have synergistic activity with daratumumab in preclinical models.37 Currently, this agent is being examined in a phase 1 study either as a single agent or in combination with daratumumab in R/R MM (NCT04136756). Finally, an exciting emerging group of therapeutic agents are the bispecific and trispecific killer engagers (BiKEs and TriKEs), as well as the antibody-recruiting molecules (ARMs) (Figure 2). BiKEs include 2 linked single-chain variable frag ments that recognize the tumor antigen (eg, BCMA) and an NK cell activation receptor (eg, NKp30, NK group 2 member A, CD16). TriKEs also incorporate another component, such as IL-15, to further enhance NK cell activation. Half-life extended nanobody-based BiKEs are under development.38 ARMs con tain 2 linked termini; 1 terminus binds to a tumor antigen, while the other binds endogenous IgG antibodies regardless of the antigen-binding specificity.39 This leads to the opsonization of tumor cells with endogenous IgG, resulting in tumor Dr Prakash Singh Shekhawat Next generation of novel therapies for myeloma | 177 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 The US Food and Drug Administration–approved anti-CD38 mAbs daratumumab and isatuximab have found widespread use in the R/R setting as well as increasing use in the ND setting. The pre dominant mechanisms of action for these agents include direct induction of apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity, anti body-dependent cellular phagocytosis, and inhibition of CD38 ectoenzyme activity, although a variety of immunomodula tory effects have been described as well.26,27 As alternatives to “naked” mAbs, newer drug development efforts have focused on the modification of anti-CD38 mAbs, thus leading to the gen eration of immunocytokines and immunotoxins (Figure 1). Modakafusp alfa (formerly known as TAK-573) is an example of an immunocytokine. This agent was designed as a delivery system of interferon alpha (IFNA) 2b to CD38+ cells. Modakafusp is a recombinant humanized IgG4 anti-CD38 mAb that is fused to an attenuated IFNA protein. While IFNA was investigated as an anti-MM agent decades ago due to its cytotoxic and immu nomodulatory properties, the systemic toxicity of the agent limited its use.28 The hypothesis underlying the novel fusion pro tein is that the systemic toxicity is limited as a consequence of both reduced binding affinity to the IFNA receptor as well as an increased localized concentration at CD38+ target cells. In vitro studies demonstrated that modakafusp displayed 10 000fold increased specificity for CD38+ cells while being approx imately 6000-fold less toxic to normal BM cells compared to native IFNA.29 Preclinical studies showed synergy with standard agents such as lenalidomide and bortezomib.29 Due to the lim ited Fc functionality of this fused protein, it is thought to be unlikely to induce ADCC. This agent activates innate and adap tive immune cells, as well as induces apoptotic signals to the MM cells via signaling through the IFNA receptor.30 Interestingly, modakafusp binds to an epitope of CD38 that is unique relative to the commercially available anti-CD38 mAbs.31 A first-in-human phase 1 study in patients with R/R MM has shown that a dose of 1.5 mg/kg (intravenously) every 4 weeks resulted in an ORR of lysis via NK cell–mediated ADCC.39 Preclinical studies have demonstrated the potential of BiKEs, TriKEs, and ARMs,39-41 and a clinical trial evaluation is the next step. Correspondence Sarah A. Hol stein, University of Nebraska Medical Center, 987680 Nebraska Medical Ctr, Omaha, NE 68198; e-mail: sarah .holstein@unmc.edu. References 1. CLINICAL CASE (Continued) A discussion is held with the patient about the many promis ing approaches currently under investigation, including the use of IMiDs with novel agents, CELMoDs, immunocytokines, and NK-cell activators/engagers. As allof these approaches involve modulation of the immune system in some manner, off-target effects are possible. She expresses interest in learning more about the clinical trial options available at your institution. 2. 3. 4. 5. Conflict-of-interest disclosure Sarah A. Holstein: consultancy: Bristol Myers Squibb, Celgene, Janssen, Genentech, Oncopeptides, Sanofi, Secura Bio, Takeda; research funding: Oncopeptides. 7. Off-label drug use Sarah A. Holstein: by definition, everything that is discussed in this chapter is currently experimental and undergoing clinical trial and is therefore off-label. 178 | Hematology 2022 | ASH Education Program 6. 8. Tai YT, Anderson KC. Bruton’s tyrosine kinase: oncotarget in myeloma. Oncotarget. 2012;3(9):913-914. Richardson PG, Bensinger WI, Huff CA, et al. Ibrutinib alone or with dexa meth a sone for relapsed or relapsed and refrac tory mul ti ple mye loma: phase 2 trial results. Br J Haematol. 2018;180(6):821-830. Ailawadhi S, Parrondo RD, Moustafa MA, et al. Ibrutinib, lenalidomide and dexamethasone in patients with relapsed and/or refractory multiple mye loma: phase I trial results [published online ahead of print 30 April 2022]. Hematol Oncol. Chen H, Li M, Ng N, et al. Ruxolitinib reverses checkpoint inhibition by reducing programmed cell death ligand-1 (PD-L1) expression and increases anti-tumour effects of T cells in multiple myeloma. Br J Haematol. 2021;192(3):568-576. Chen H, Li M, Sanchez E, et al. JAK1/2 pathway inhibition suppresses M2 polarization and overcomes resistance of myeloma to lenalidomide by reducing TRIB1, MUC1, CD44, CXCL12, and CXCR4 expression. Br J Haematol. 2020;188(2):283-294. Berenson JR, To J, Spektor TM, et al. A phase I study of ruxolitinib, lenalidomide, and steroids for patients with relapsed/refractory multiple mye loma. Clin Cancer Res. 2020;26(10):2346-2353. Buettner R, Morales C, Caserta E, et al. Leflunomide regulates c-Myc expres sion in myeloma cells through PIM targeting. Blood Adv. 2019;3(7):1027-1032. Rosenzweig M, Palmer J, Tsai NC, et al. Repurposing leflunomide for relapsed/refractory multiple myeloma: a phase 1 study. Leuk Lymphoma. 2020;61(7):1669-1677. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 Figure 2. Novel approaches for improving NK-cell–mediated targeting of myeloma cells. Bispecific killer cell engagers (BiKEs) are composed of 2 antibody fragments that recognize tumor antigens and NK-cell activation receptors, while trispecific killer cell engagers (TriKEs) also include a component such as IL-15 to further enhance NK-cell activation. Antibody-recruiting molecules (ARMs) contain a terminus that binds endogenous IgG antibodies and is linked to a second terminus that binds tumor antigen, thus leading to opsonization of tumor cells with endogenous IgG and resulting in NK-cell–mediated ADCC. Created with BioRender.com multiple myeloma (RRMM): preliminary results from the phase 1/2 study CC-92480-MM-002. Blood. 2021;138(suppl 1):2731. 26. Krejcik J, Casneuf T, Nijhof IS, et al. Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma. Blood. 2016;128(3):384-394. 27. Deckert J, Wetzel MC, Bartle LM, et al. SAR650984, a novel humanized CD38-targeting antibody, demonstrates potent antitumor activity in mod els of multiple myeloma and other CD38+ hematologic malignancies. Clin Cancer Res. 2014;20(17):4574-4583. 28. Fritz E, Ludwig H. Interferon-alpha treat ment in mul ti ple mye loma: meta-analysis of 30 randomised trials among 3948 patients. Ann Oncol. 2000;11(11):1427-1436. 29. Pogue SL, Taura T, Bi M, et al. Targeting attenuated interferon-α to mye loma cells with a CD38 antibody induces potent tumor regression with reduced off-target activity. PLoS One. 2016;11(9):e0162472. 30. Vogl DT, Kaufman JL, Holstein SA, et al. Modakafusp alfa (TAK-573), an immunocytokine, shows clinical activity in patients with relapsed/refrac tory mul ti ple mye loma: updated results from a first-in-human phase 1 study. Blood. 2021;138(suppl 1):898. 31. Vogl DT, Kaufman JL, Holstein SA, et al. TAK-573, an anti-CD38/attenuated IFNα fusion protein, has clinical activity and modulates the IFNα receptor (IFNAR) pathway in patients with relapsed/refractory multiple myeloma. Blood. 2020;136(suppl 1):37-38. 32. Willert EK, Robinson GL, Higgins JP, et al. Abstract 2384: TAK-169, an excep tionally potent CD38 targeted engineered toxin body, as a novel direct cell kill approach for the treatment of multiple myeloma [abstract]. Cancer Res. 2019;79(suppl 13). Abstract 2384. 33. Bruins WSC, Zheng W, Higgins JP, et al. TAK-169, a novel recombinant immunotoxin specific for CD38, induces powerful preclinical activity against patient-derived mul ti ple mye loma cells. Blood. 2020;136(suppl 1):11-12. 34. Kamur S, Mamuye A, Dabovic K, et al. Interim results of a phase 1 study of the novel engineered toxin body TAK-169 in patients with relapsed or refractory multiple myeloma [abstract]. J Immunother Cancer. 2021;9. Abstract 447. 35. Bruins WSC, Zweegman S, Mutis T, van de Donk NWCJ. Targeted ther apy with immunoconjugates for multiple myeloma. Front Immunol. 2020; (20 June)11:1155. 36. Romee R, Cooley S, Berrien-Elliott MM, et al. First-in-human phase 1 clini cal study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation. Blood. 2018;131(23):2515-2527. 37. Fernandez AF, Pierre-Louis L, Xu Y, et al. Restoring NK cell activities in mul tiple myeloma with IL-15 receptor agonist NKTR-255. Abstract presented at: 62nd ASH Annual Meeting and Exposition; 20 December 2020; virtual. Abstract 667. 38. Hambach J, Fumey W, Stähler T, et al. Half-life extended nanobody-based CD38-specific bispecific killercell engagers induce killing of multiple mye loma cells. Front Immunol. 2022;13(16 May):838406. 39. Bunin A, McGrath K, Rossi AM, et al. A novel class of bifunctional immu notherapeutic that exploits a universal antibody binding terminus (uABT) to recruit endogenous antibodies to cell expressing CD38 demonstrate in vivo efficacy in three distinct animal models. Blood. 2019;134(suppl 1): 1820. 40. Wang Y, Li H, Xu W, et al. BCMA-targeting bispecific antibody that simulta neously stimulates NKG2D-enhanced efficacy against multiple myeloma. J Immunother. 2020;43(6):175-188. 41. Watkins-Yoon J, Guzman W, Oliphant A, et al. CTX-8573, an innate-cell engager targeting BCMA, is a highly potent multispecific antibody for the treatment of multiple myeloma. Blood. 2019;134(suppl 1):3182. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000335 Dr Prakash Singh Shekhawat Next generation of novel therapies for myeloma | 179 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/173/2021746/173holstein.pdf by guest on 09 December 2022 9. Malek E, Hwang S, de Lima M, et al. Preclinical studies and phase I trial of vactosertib in combination with pomalidomide in relapsed multiple mye loma: a corticosteroid-free approach by targeting TGF-β signaling path way. Blood. 2019;134(suppl 1):3232. 10. Malek E, Hwang S, Caimi PF, et al. Phase Ib trial of vactosertib in combina tion with pomalidomide in relapsed multiple myeloma: a corticosteroidfree approach by targeting TGF-β signaling pathway. J Clin Oncol. 2021; 39(suppl 15):8039. 11. Lin C, Leso A, Rosenwasser M, Torngren M, Eriksson H, Nefedova Y. Inhibition of S100A9 with tasquinimod demonstrates potent anti-tumor activity in pre-clinical models of multiple myeloma. Paper presented at: European Hematology Association; 19-20 November 2020; virtual meeting. 12. Usmani SZ, Schjesvold F, Oriol A, et al; KEYNOTE-185 Investigators. Pembrolizumab plus lenalidomide and dexa meth a sone for patients with treatment-naive multiple myeloma (KEYNOTE-185): a randomised, openlabel, phase 3 trial. Lancet Haematol. 2019;6(9):e448-e458. 13. Mateos MV, Blacklock H, Schjesvold F, et al; KEYNOTE-183 Investigators. Pembrolizumab plus pomalidomide and dexamethasone for patients with relapsed or refractory multiple myeloma (KEYNOTE-183): a randomised, open-label, phase 3 trial. Lancet Haematol. 2019;6(9):e459-e469. 14. Gormley NJ, Pazdur R. Immunotherapy combinations in multiple myeloma— known unknowns. N Engl J Med. 2018;379(19):1791-1795. 15. Oriol A. A critical evaluation of pembrolizumab in addition to lenalidomide and dexamethasone for the treatment of multiple myeloma. Expert Rev Hematol. 2020;13(5):435-445. 16. Matyskiela ME, Zhang W, Man HW, et al. A cereblon mod u la tor (CC220) with improved deg ra da tion of Ikaros and Aiolos. J Med Chem. 2018;61(2):535-542. 17. Bjorklund CC, Kang J, Lu L, et al. CC-220 is a potent cereblon modulating agent that displays anti-proliferative, pro-apoptotic and immunomodula tory activity on sensitive and resistant multiple myeloma cell lines. Blood. 2016;128(22):1591. 18. Lonial S, van de Donk NWCJ, Popat R, et al. First clinical (phase 1b/2a) study of iberdomide (CC-220; IBER), a CELMoD, in combination with dexa methasone (DEX) in patients (pts) with relapsed/refractory multiple mye loma (RRMM). J Clin Oncol. 2019;37(suppl 15):8006. 19. Lonial S, Popat R, Hulin C, et al. Iberdomide (IBER) in combination with dexamethasone (DEX) in patients (pts) with relapsed/refractory multiple myeloma (RRMM): results from the dose-expansion phase of the CC-220MM-001 trial. Blood. 2021;138(suppl 1):162. 20. Van Oekelen O, Amatangelo M, Guo M, et al. Large-scale mass cytometry reveals significant activation of innate and adaptive immunity in bone mar row tumor microenvironment of iberdomide-treated myeloma patients. Blood. 2021;138(suppl 1):730. 21. Van de Donk N, Popat R, Larsen J, et al. First results of iberdomide (IBER; CC-220) in combination with dexamethasone (DEX) and daratumumab (DARA) or bortezomib (BORT) in patients with relapsed/refractory multi ple myeloma (RRMM). Blood. 2020;136(suppl 1):16-17. 22. Hansen JD, Correa M, Nagy MA, et al. Discovery of CRBN E3 ligase modu lator CC-92480 for the treatment of relapsed and refractory multiple mye loma. J Med Chem. 2020;63(13):6648-6676. 23. Richardson PG, Vangsted AJ, Ramasamy K, et al. First-in-human phase I study of the novel CELMoD agent CC-92480 combined with dexameth asone (DEX) in patients (pts) with relapsed/refractory multiple myeloma (RRMM). J Clin Oncol. 2020;38(suppl 15):8500. 24. Wong L, Lamba M, Jiménez Nuñez MD, et al. Dose- and schedule-dependent immunomodulatory effects of the novel CELMoD agent CC-92480 in patients with relapsed/refractory multiple myeloma. Blood. 2020;136(suppl 1):47-48. 25. Richardson PG, Ocio E, Raje NS, et al. CC-92480, a potent, novel cereblon E3 ligase modulator (CELMoD) agent, in combination with dexamethasone (DEX) and bortezomib (BORT) in patients (pts) with relapsed/refractory Paula Rodriguez-Otero and Jesús F. San-Miguel Clínica Universidad de Navarra, Centro de Investigación Médica Aplicada, Instituto de Investigación Sanitaria de Navarra, Centro de Investigación Biomédica en Red Cáncer, Pamplona, Spain Despite significant improvement in the treatment of multiple myeloma (MM), a cure remains elusive, and patients failing proteasome inhibitors, immunomodulatory drugs, and anti-CD38 monoclonal antibodies remain a challenge due to a lack of standard of care treatment and a dismal survival rate. The development of T-cell redirecting therapies, including bispecific T-cell engagers and chimeric antigen receptor (CAR) T cells, have transformed the outcome of triple-class exposed relapsed and refractory MM (RRMM). B-cell maturation antigen (BCMA) has proven to be an important target in MM, and BCMA-directed CAR T cells have shown unprecedented efficacy with a prolonged duration of response in a population with advanced RRMM, leading to the approval of 2 different BCMA CAR T-cell products. Still, and in contrast to prior experience in the field of CD19-directed CARs, no plateau has been seen in the survival curves, and relapses continue to occur. Therefore, further improvement is needed. Early use in the course of the disease as well as of nextgeneration CARs may further augment the efficacy of these therapies. In this review we address current state-of-the-art approved BCMA-directed CAR T-cell therapy in RRMM, as well as potential future developments focused on optimizing patient care and novel CAR designs. LEARNING OBJECTIVES • Summarize the current evidence regarding BCMA-directed CAR T cells for RRMM, focusing on approved constructs • Discuss potential limitations of current CAR T-cell therapy in myeloma • Describe potential strategies for improving current results, with a focus on the clinical perspective CLINICAL CASE A 56-year-old male was diagnosed in September 2019 with immunoglobulin A lambda multiple myeloma (MM). At diagnosis he presented with diffuse osteolytic lesions and a revised International Staging System (R-ISS) 3 status (high lactose dehydrogenase + t(4;14)). He received frontline treatment with daratumumab in combination with bortezomib, lenalidomide, and dexamethasone (D-VRD) in the context of a randomized clinical trial. He completed 4 planned induction cycles followed by autologous stem cell transplant and 2 additional consolidation cycles. He achieved a very good partial response after consolidation and started maintenance treatment with daratumumab plus lenalidomide in June 2020. Unfortunately, 1 month later he progressed with the presence of a lumbar plasmacytoma. As of this moment, he has been transferred to our department to be considered for a B-cell maturation antigen (BCMA) 180 | Hematology 2022 | ASH Education Program chimeric antigen receptor (CAR) T-cell clinical trial enrolling patients with early relapse after frontline treatment. Introduction The treatment landscape of MM has significantly evolved in recent years, leading to unprecedented survival rates.1 Nevertheless, this improvement has not been uniform, and there are still patient populations with high-risk features who have not benefited enough from recently approved combinations and require innovative strategies to overcome their dismal prognosis. These populations are, among others, patients with extramedullary disease, a high number of circulating plasma cells, high-risk cytogenetic abnormalities (Cas), particularly double-hit myeloma, and patients who relapse early after optimized frontline therapy.2-4 Regarding the latter, patients with R-ISS 3 status at diagnosis who fail to achieve undetectable minimal residual Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 Cellular therapy for multiple myeloma: what’s now and what’s next disease (MRD) after frontline treatment are at a high risk of an early relapse with a median progression-free survival (PFS) of only 14 months and a median overall survival (OS) of fewer than 2 years.5 In addition, patients who have failed proteasome inhib itors, immunomodulatory drugs, and anti-CD38 monoclonal antibodies have very poor outcomes and limited treatment options.6 Therefore, we need new treatments with novel mechanisms of action able to rescue difficult-to-treat populations and improve patient survival. BCMA CAR T cells approved: what is now Figure 1. Several strategies are being evaluated to improve the current clinical efficacy of CAR T-cell therapy in the context of RRMM. These strategies can be divided into patient-related factors, aspects related to the manufacturing process and CAR design, patient management during bridging time and infusion, and finally, understanding the mechanisms of resistance and CAR failure. CCR, chimeric costimulatory receptor; EMD, extramedullary disease; HR-CA, high-risk cytogenetic abnormalities. Dr Prakash Singh Shekhawat CAR T-cell therapy in multiple myeloma | 181 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 In this scenario CAR T-cell therapy has shown unprecedented response rates in heavily pretreated patients, leading to the approval of 2 BCMA-directed CAR T-cell products (ABECMA™ and CARVIKTY™) for the treat ment of tri ple-class exposed relapsed/refrac tory (RR) MM who have received at least 4 prior lines of treatment. In this review we discuss the current state-of-the-art of approved CAR T-cell therapy in RRMM and future developments, with a focus on the clinical perspective (Figure 1). Idecabtagene vicleucel (ide-cel, bb2121, ABECMA™) was the first cellular therapy approved for the treatment of RRMM. Ide-cel is a second-generation CAR with a 4-1BB costimulatory domain approved for the treatment of RRMM based on the data of the phase 2 single-arm pivotal KarMMa trial.7 Overall, 128 patients were infused with 2 different doses of ide-cel 150, 300, and 450×106 CAR T+ cells. The median number of prior lines was 6 (3-18), and 84% of patients were triple-class refractory. All patients received standard lymphodepleting chemotherapy with fludarabine and cyclo phos pha mide. The over all response rate (ORR) was 73% among alltreated patients and 81% in those receiving the higher-dose level (450×106), with 33% and 39% of complete responses (CR), respectively. Responses were rapid, with a median time to first response of 1.0 month (range, 0.5-8.8 months). Median PFS was 8.8 months in the overall population and was longer in patients receiving the target dose (median PFS, 12.1 months) and in those achieving CR or better (median PFS, 20.2 months; 95% CI, 12.3-NE [not evaluable]). Median OS was 24.8 months (95% CI, 19.9-31.2). Safety overall was manageable, with cytokine release syndrome (CRS) reported in 84% of patients across the 3 doses and 96% at the target dose. The majority of events were grade 1 or 2 with a CRS grade higher than or equal to 3 in fewer than 6% of patients, with a median time to onset of 1 day (range, 1-12). Neurological complications were less common, were reported in (95% CI, 57.6-80.9) in sCR patients. Two-year OS was 71.0% (95% CI, 57.6-80.9). Regarding safety, cytopenias and CRS were the most frequent treatment-related adverse events reported. Any grade of neutropenia was present in 96% of patients, with 95% having grade 3 or higher. The incidence of CRS was 95%, mostly grade 1 to 2, with a median time to onset of 7 days (range, 1-12). Neurological events were reported in 20 patients. Among these, 16 presented immune-effector cell–associated neurotoxicity syndrome (ICANS), and 12 patients presented with other neurotoxicities. Of these, 5 experienced a movement and neurocognitive disorder with a median time to onset of 27.0 days (IQR 16.0-73.0) (Table 1).8 Other BCMA-directed CARs are under development, and the status of those studies are summarized in Table 2. Table 1. Safety and efficacy data of the 2 FDA- and EMA-approved CARs Idecabtagene vicleucel KarMMa trial1-3 Ciltacabtagene autoleucel CARTITUDE 1 trial Number of patients infused, n 128 (140 apheresed) 97 (120 apheresed) Phase 2 1b/2 Target/costimulation BCMA/4-1BB BCMA/4-1BB *2 BCMA-targeting heavy-chain antibody scFv Chimeric mouse Chimeric llama Specificity Autologous Autologous Follow-up, median (range) 13.3 mo (0.2-21.2) 21.7 mo (not reported) Prior lines, median (range) 6 (3 to 16) 6 (3-18) Triple-class refractory, n (%) 108 (84) 85 (87.6) Penta-exposed, n (%) 77 (60) 81 (83.5) Bridging therapy, n (%) 112 (88) 73 (75) Response to bridging therapy, n (%) 5/112 (4) 33/73 (45) EMD, n (%) 50 (39) CAR T-cell dose 150, 300, 450×10 CAR+T 0.75×106 CAR T+/kg LD chemotherapy Fludarabine 30mg/m2×3 d Cyclo 300mg/m2×3 d Fludarabine 30mg/m2×3 d Cyclo 300mg/m2×3 d ORR, n (%) 94 (73) At 450×106 (n=54): 44 (81%) 95 (97.9) CR or sCR, n (%) 42 (33) At 450×106 (n=54): 21 (39) 82.5% (sCR) Time to first response, median (range) 1.0 mo (0.5-8.8) 1.0 mo (IQR 0.9-1.0) MRD in CR, n (%) 33/42 MRD neg (10 ) DOR, median (95% CI) 10.7 mo (9.0-11.3) Not reported PFS, median (95% CI) 8.8 mo (5.6-11.6) At 450×106: 12.1 mo (8.8-12.3) NR (16.8-NE) 2-y PFS: 60.5% (48.5-70.4) 13 (13) 6 −5 PFS in high-risk, median (95% CI) 61 evaluable 92% MRD negative (10−5) PFS at 2y, % (95% CI) ISS 3/R-ISS 3 4.9 mo (1.8-8.2) NE (NE-NE) High-risk CA 8.2 mo (4.8-11.9) 48.4% (25.1-68.4) Plasmacytomas 7.9 mo (5.1-10.9) 47.4% (24.4-67.3) OS, median (95% CI) 24.8 mo (19.9-31.2) NR (27.2-NE) 107 (84) 92 (95) a CRS, n (%) Overall 7 (5) 4 (4) Time to CRS onset, median (range) Grade 3-4 1 d (1-12) 7 d (5-8) Duration of CRS, median (range) 5 d (1-63) 4 d (3-6) 182 | Hematology 2022 | ASH Education Program Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 18% of patients, and were mostly grade 1 to 2. On the other hand, cytopenias were frequent and not dose related, with a median time to recovery from grade 3 or higher neutropenia and throm bocytopenia of 2 and 3 months, respectively (Table 1).7 Ciltacabtagene autoleucel (cilta-cel; for mer LCAR-B38M, CARVIKTY™), another second-generation CAR T cell containing 2 BCMA-binding domains, was investigated in the CARTITUDE-1 study (and previously evaluated in the LEGEND-2 phase 1 study). Overall, 97 patients were infused with a median number of 6 prior lines and 87.6% were triple class refractory. The ORR was 97.9% with 82.5% of patients achieving a stringent CR (sCR). With a median follow-up of 2 years, outstanding survival rates have been reported, with a 2-year PFS of 60.5% (95% CI, 48.5-70.4) and 71.0% Table 1. Safety and efficacy data of the 2 FDA- and EMA-approved CARs (Continued) Idecabtagene vicleucel KarMMa trial1-3 Ciltacabtagene autoleucel CARTITUDE 1 trial Overall 23 (18) 20 (21)b Grade 3-4 4 (3) 9 (9) Time to neurotoxicity onset, median (range) 2 d (1-10) 8 d (6-8) for ICANs Other: CAR-T cell neurotoxicites: 26.5 d (11-108)b Grade 3-4 neutropenia, n (%) 114 (89) 92 (95) 1.9 mo (1.2-5.6) Not reported 67 (52) 58 (60) 2.1 mo (1.2-13.8) Not reported Infections, n (%) 88 (69) 56 (58) Grade 3-4 infections, n (%) 28 (22) 19 (20) Death, n (%) 44 (34) 14 Reference (1) Munshi et al9 (2) Anderson et al.34 (3) Oriol et al.35 (4) Berdeja et al8 (5) Martin et al.36 (6) Jakubowiak et al11 Neurotoxicity, n (%) Grade 3-4 thrombocytopenia, n (%) Time to recovery, median (range) Data from the KarMMa and CARTITUDE-1 pivotal trials with ide-cel and cilta-cel, respectively, are summarized. a PFS in R-ISS 3. b ICANS occurred in 16 (17%) patients. Other neurotoxicities occurred in 12 patients, with a median time to onset of 27 days. Five patients had a cluster of movement and neurocognitive treatment–emergent adverse events. EMA: European Medicines Agency; EMD, extramedullary disease; FDA: US Food and Drug Administration. What is next and how to improve current results Despite the impres sive clin i cal results obtained thus far and given the short follow-up in some studies, no plateau has yet been seen in the survival curves, and relapses continue to occur. Therefore, further improvement is needed, and several points remain to be discussed. Improving patient selection Patient selec tion is a crit i cal aspect of CAR T-cell ther apy. Indeed, it is of particular relevance if we consider the present and future availability of other off-the-shelf BCMA-directed ther apies. Although high and deep responses are achieved with both approved therapies, outcomes in selected high-risk pop ulations remain inferior compared to standard-risk patients. In the KarMMa trial, patients with extramedullary disease, high-risk CAs, and R-ISS 3 showed lower CR rates and shorter PFS com pared to patients without high-risk disease (Table 1).9,10 Likewise, in the CARTITUDE-1 trial, patients with high-risk CAs, ISS 3, and the presence of plasmacytomas showed a lower PFS at 2 years compared to the intention-to-treat population, suggesting that further improvement is needed to abrogate the adverse out come of patients with high-risk features even in the context of potent therapies such as CAR T cell (Table 1).11 On top of this, data from the different studies suggest that the achievement of deep responses (CR or MRD negativity) is of utmost importance in the context of “one-shot” CAR T-cell therapy and is associated with prolonged duration of response (DoR) and PFS.8,9 Therefore, understanding the patient charac teristics associated with a higher likelihood of achieving CR is of great importance. A recent analysis of the KarMMa trial showed that the presence of high tumor burden (soluble BCMA) and high inflammation (D-dimer, ferritin) along with the myeloma subtype (immunoglobulin G heavy chain) had a negative asso ciation with CR/sCR achievement, whereas a high vector copy number in the drug product showed a positive correlation with the CR/sCR rate.12 These factors suggest that optimal and timely patient selection may be relevant for CAR T-cell outcomes since it is not an off-the-shelf therapy, and rapidly progressive patients with high-risk features or limited bridging options may be diffi cult to manage given the manufacturing time. In fact, between 10% and 20% of the patients who underwent apheresis in the different trials did not reach infusion due to complications dur ing the manufacturing time or disease progression. In this regard, adequate selection of bridging therapy is of utmost importance to maintain the patient’s condition dur ing manufacturing. Whenever possible, agents to which the patient has not been previously exposed should be prioritized. In this sense, and although the data are limited, BCMA-targeted agents are probably not the best option during the bridging period. Indeed, patients with prior BCMA treatment were excluded from the pivotal CAR T studies, and limited data are therefore available. Recent evidence from the cohort C of the CARTITUDE-2 trial that enrolled patients previously treated with BCMA-antibody drug conjugates (BCMA-ADC) and BCMA biespecific antibodies (BCMA-BsAb) showed that responses to cilta-cel were inferior to that of the CARTITUDE-1 trial (ORR: 62% in the BCMA-ADC and 57% in the BCMA-BsAb groups, respectively). Factors related to response were a shorter duration of prior BCMA treatment and a longer median time between last BCMA treatment and apheresis, suggesting a potential negative impact of prior BCMA treatment in outcome with CAR T-cell therapies.13 On the other hand, in the recently reported real-world experience, 22% of patients had prior anti-BCMA therapy, and the ORR was consistent with that of the KarMMa trial.14 Even so, both the downregulation of BCMA expression and the biallelic loss of the BCMA locus have Dr Prakash Singh Shekhawat CAR T-cell therapy in multiple myeloma | 183 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 Time to recovery, median (range) Dr Prakash Singh Shekhawat Autologous 150-450M Specificity CAR T-cell dose 50 (69) NR Penta-refractory, n (%) 36% 4 (1-10) NCT03915184 NCT03430011 NCT03274219 Identification Data of safety and efficacy are shown. NR, not reached. Chen et al.38 Mailankody et al.37 NR NR 1 patient 2 (DL0) & 1 (DL1) ≈30/20 4 (DL0) & 3 (DL1) 2 (DL0) & 1 (DL1) 0 (0) 15/18 (83.3) Raje et al27 Reference 27.8% NR Ongoing 4 (1-10) NR 50% NCT05066646 Chunrui et al.39 Ongoing 1 10 0 1 (1.3) 20/34.7 5 (1-30) 6 (1-12) 2 (2.5) 72 (92.4) 25.3 mo (3.0-NE) 58.2% 75 (94.9) 13 (16.5) 85% 94% 5 (3-13) 5 (3-11) Not further developed 2d Not further developed Status Duration (d), median (range) 4 (1-6) 2 (3) 3 (4) 7 d (2-24) Grade 3-4, n (%) 8 (13) 76/52 11 (15) Onset (d), median (range) All grade, n (%) ICANS 4d 53/17 Duration (d), median (range) Tocilizumab/steroids %/% 2 (1-4) 2 (3) 3 (2 grade 5) 2 d (1-20) Grade 3-4, n (%) 55 (89) 54 (75) 9.3 mo (300M) 36% 12.8 mo (7.3-18.6) 92% 30 (48) 69% Onset (d), median (range) All grade, n (%) CRS PFS (mo), median (95% CI) CR, n (%) ORR, n (%) Efficacy 6 (3-18) 6 (3-17) Prior lines, median (range) Triple-class refractory, n (%) 58 (94) 61 (33-77) 56 (39-70) Autologous 1.0×106/kg Autologous 1.5-3.0×108 cells Autologous 300-600M 62 (36-78) BCMA/4-1BB Human BCMA/4-1BB 1/2 25.3 mo (4.1-36.7) 1b/2 CT103A FUMANBA-1 (n=79) 6 mo (2-11) CT053 LUMMICAR (n=18) Human BCMA/4-1BB 62 (33-76) Age, median (range) Population Mouse BCMA/4-1BB Mouse Target/costimulation scFv 1 9.5 mo 1 23 (9-46) Phase Follow-up, median (range) Orva-cel EVOLVE (n=62) BB21217 CRB-402 (n=72) Table 2. Main reported clinical trials of BCMA-directed CAR T cells in RRMM NCT05066646 Gan An, et al.40 Ongoing 1 8 0 (0) 1 (4.3) 26/9 5 (2-9) 6 (1-11) 1 (4.3) 21 (91.3) 6 mo PFS: 65.1% 43.5% 22 (95.7) NR NR 4 (2-12) 60 (45-74) 1.0-6.0×106/kg Autologous Human BCMA/4-1BB 6.2 mo (0.7-16.1) 1 C-CAR088 (n=23) 1 BCMA/4-1BB Autologous — de Larrea et al24 NCT04309981 Mailankody et al.42 NCT04093596 Costello et al.41 NCT03288493 0 — 0 (0) NR Ongoing 0 6 (14) NR 4 (1-12) 76/12 0 (0) NR 1 (2) NR NR 87% 24 (56) 23/14 60% 15.8 mo (12.9-NE) 25% NR 100% NR 61% 4 (2-10) 61 (36-74) 3×106/kg 71% At 320×106 42% NR 5 (3-11) 64 (46-77) 40-480×106 Allogeneic BCMA/4-1BB Human Ongoing NR BCMA/4-1BB Human 13 mo (7-13) 1 ARI-002h (n=30) Not further developed NR 4% 4% 7/6 NR NR 0% 17% NR NR 50%-75% NR 60% 8 (2-18) 60 (42-74) 51-1178×106 Autologous Mouse 4 mo 1/2 NA ALLO-715 UNIVERSAL (n=43) P-BCMA-101 PRIME (n=53) Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 184 | Hematology 2022 | ASH Education Program CLINICAL CASE (Cont inu ed) Our patient under went apher e sis after the com ple tion of screen ing tests and received bridg ing ther apy, with pro gressive disease as the best response. The bridging period was complicated with acute kidney failure and several infec tious complications. After recovery, he received standard fludarabine-cyclophosphamide conditioning before CAR T-cell infusion, which was performed in October 2020. He did not develop CRS or ICANs but did have grade 4 cytopenia requiring transfusion support, granulocyte colony-stimulating factor, erythropoietin-stimulating agents, and thrombopoietin analogues. After CAR T-cell treatment, the patient achieved a stringent CR with MRD negativity (10−6) as well as a complete metabolic response (Deauville 3), using an F-fluorodeoxyglucose emission tomography/computed tomography scan, that continues today. His cytopenia improved by month 12, and the patient no longer requires transfusions or medication. Improving the T-cell manufacturing process Several studies across different CAR T-cell products have shown a positive correlation between the depth and DoR and CAR T-cell expansion or persistence.9,17 Therefore, significant effort has been fostered to optimize the final CAR T-cell product. One relevant aspect to consider is the immunogenicity of approved animal-derived constructs. Indeed, in the KarMMa trial, patients with antidrug antibodies (ADAs) increased over time, from 21% (21 of 102) at month 3 to 65% (34 of 52) at month 12.9 Although these antibodies did not seem to influence CAR concentration or response, only patients without negative ADAs responded to CAR T-cell reinfusion, suggesting the potential role of these ADAs in CAR T-cell persistence or activity. Humanized or fully human constructs may be a way to overcome the negative impact of immunogenicity in CAR T-cell outcomes. Preliminary data from several phase 1 and 2 trials have shown encouraging clinical efficacy with high ORRs and deep and durable responses.22,23 Moreover, in 1 academic study evaluating ARI-002h, a human ized BCMA CAR T, patients with an ongoing response and the absence of significant toxicity to the first infusion were eligible for a reinfusion. Sixteen out of 30 patients received a second infusion, and 5 patients showed an improved response, suggesting an adequate expansion of the CAR T-cell product.24,25 Alternatively, the use of non–scFv-based CARs, such as engineered binding scaffolds, receptors, or natural ligands, might additionally serve to mitigate immunogenic reactions.26 Higher levels of memory T-cell phenotypes have been associ ated with better clinical outcomes, and several strategies, such as CD4/CD8 in a 1:1 ratio, may yield a higher proportion of these populations in the final product. An interesting approach was evaluated in the phase 1 CRB-402 study. In this trial, ide-cel was cultured with a PI3K inhibitor (bb007), resulting in a different CAR T-cell product (bb21217) enriched with T cells having a memorylike phenotype and a superior proliferative capacity upon adop tive transfer. Overall, 72 patients with a median of 6 prior lines were infused. The ORR was 69%, with 36% of the patients achiev ing a CR or better. The median DoR was 23.8 months and was not reached by patients in CR. Importantly, the persistence of CAR T cells was prolonged, with detectable CAR T cells in some patients up to 24 months.27 One limitation of targeted immunotherapies is the potential heterogeneity of antigen expression among tumor cells and the loss or downregulation of anti gen after expo sure.18 Simultaneously targeting different antigens could overcome this barrier, and several trials are evaluating this possibility. GC012F is a dual BCMA/CD19-targeted CAR T manufactured in 24 to 36 hours on the FASTCAR® platform. Sixteen patients with RRMM and a median of 5 prior lines of therapy were infused with GC012F (dose range: 100 000-300 000 CAR T cells per kilogram). The ORR was 94%, with 56.3% of patients achieving CR or better. The median DoR was not reached at 7.3 months of median follow-up.28 Likewise, other targets are being explored (Table 4). GPR5D (G protein–coupled receptor 5 family D)-directed CAR T cells are Dr Prakash Singh Shekhawat CAR T-cell therapy in multiple myeloma | 185 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 been reported after anti-BCMA treatment, with the potential to limit BCMA CAR T-cell efficacy.15-17 Additionally, strategies to shorten vein-to-vein time are being developed, such as platforms for rapid CAR T-cell manufactur ing or uni ver sal off-the-shelf allo ge neic CAR. ALLO-715 is an allogeneic BCMA-targeting CAR T cell. It is manufactured with a knockout of the T-cell receptor alpha constant and CD52 to minimize the risk of graft-versus-host disease and to allow pro found T-cell depletion using an anti-CD52 antibody to improve engraftment of the CAR T cells, respectively.18 The UNIVERSAL phase 1 study tested ALLO-715 in 43 RRMM patients using esca lating doses and different lympho-depleting regimens. The best clinical results were obtained with 320 × 106 cells in combina tion with fludarabine, cyclophosphamide, and anti-CD52 anti body (ALLO-647). In this cohort, 24 patients were infused. The ORR was 71%, and the median DoR was 8.3 months.19 Potential immunogenicity and short persistence are the main challenges when using allogenic CAR T cells, and other cellular platforms are under investigation, such as natural killer CAR or γδ T cells.18 Treating patients in earlier lines of therapy with less refrac tory disease may help overcome some of the aforementioned problems. Therefore, different studies are indeed evaluating the feasibility and efficacy of this therapy in the context of early relapse or frontline disease. In cohort A from the phase 2 CARTITUDE-2 study, patients with 1 to 3 prior lines and lenalidomiderefractory disease were included and treated with cilta-cel. The median number of prior lines was 2 (range, 1-3). Interestingly, 40% of the patients were already triple-class refractory. Nineteen out of 20 patients responded (95%), and 85% achieved at least CR. The median PFS has not yet been reached, with a PFS at 12 months of 84% (95% CI, 59.1-94.7). Safety was comparable to that of the CARTITUDE-1 trial without new safety signals.20 Likewise, in cohort B of the same study, patients with early relapse (within the first 12 months) after frontline therapy with a proteasome inhibitor and immunomodulatory drugs were included. A total of 19 patients were infused. The ORR was 95%, and 79% of patients achieved at least CR, with a 12-month PFS of 84% (95% CI, 57.9-94.5) and a safety profile comparable to that of the CARTITUDE-1 trial.21 Altogether, these results suggest that CAR T-cell therapy in earlier lines of therapy is safe and may yield high and deep responses in different unmet-need populations. Still, its efficacy needs to be confirmed in the context of large phase 3 studies both in early relapse (KarMMa-3, NCT03651128; CARTITUDE-4, NCT04181827) and the frontline setting (BMTCTN1902, NCT05032820; CARTITUDE-5, NCT04923893; CARTITUDE-6, NCT04181827) (Table 3). Dr Prakash Singh Shekhawat NDMM high-risk NDMM NDMM not intended for ASCT NDMM-TE high-risk NDMM TE NDMM high-risk (R-ISS 3) Multicohort Multicohort 1-3 PL + Len R ≥3 PL RRMM 2-4 prior lines Len-ref & antiCD38 exposed 1-3 prior lines Len refractory NDMM postASCT FUMANBA-2 (NCT05181501) NCT04287660 CARTITUDE-5 NCT04923893 NCT04935580 CARTITUDE-6 NCT05257083 KarMMa-4 NCT04196491 CARTITUDE-2 NCT04133636 KarMMa-2 NCT03601078 KarMMa-7 NCT04855136 KarMMa-3 NCT03651128 CARTITUDE-4 NCT04181827 BMTCTN1902 NCT05032820 Ide-cel + Len maintenance A: PVd or DPd B: cilta-cel A: DVd/DPd/ EloPd/Ixa-Rd/Kd B: Ide-cel (A): Ide-cel + iberdomide maintenance (B): Ide-cel + gamma secretase inhibitor 2a + b: R-ISS 3 + early relapse 2c: suboptimal response to ASCT Cohort A: 1-3 prior lines Len-ref Cohort B: early relapse after frontline treatment Cohort C: relapse after BCMA Cohort F: NDMM after front line Cohort E: NDMM-Tx not planned (high-risk) Standard induction×3 cycles + Ide-cel A: DVRD×4 + ASCT +2×DVRD + Len B: DVRD×6 + cilta-cel + Len (2y) 2 cycles induction + CAR T + Len maintenance A: VRD×8 + Rd B: VRD (6+2) + cilta-cel Ide-cel Cilta-cel Ide-cel Ide-cel Murine Llama Murine Murine Murine Llama Cilta-cel Ide-cel Murine Ide-cel Llama Human GC012F Cilta-cel Llama Cilta-cel NA NA BiRD + BCMA CAR T cell Human CT103A VRD/PAD/PCD×3-ASCT (if eligible) >> CT103A ScFv origin CAR name Study design 2 3 randomized 3 Randomized 2 2 2 1 3 randomized 1/2 3 randomized 3 1 Phase 40 419 381 181 181 157 13 750 20 650 20 20 n BCMA BCMA BCMA BCMA BCMA BCMA BCMA BCMA BCMACD19 BCMA BCMA BCMA Antigen Recruiting Recruiting Recruiting Active Not recruiting Recruiting 150-450×106 150-450×106 450×106 0.75×106/kg 450×106 ORR ORR PFS PFS Autologous Autologous Autologous Autologous Autologous ORR Recruiting 0.75×106/kg Autologous MRD negativity at 1 year Not yet enrolling 0.75×106/kg Autologous Recruiting 3×105/kg Active Not recruiting Recruiting Recruiting 0.75×106/kg 450×106 PFS Safety ORR, PFS, MRD PFS Not yet enrolling 1×106/kg 2-3×107/kg Status Dose Safety Autologous Autologous Autologous ORR at 4 weeks MRD negativity 2-year PFS Autologous Autologous End point Cell source ASCT, autologous stem cell transplantation; BiRD, bortezomib, claritromicine, lenalidomide, dexamethasone; DPd, daratumumab, pomalidomide, dexamethasone; DVd, daratumumab, bortezomib, dexamethasone; EloPd, elotuzumab, pomalidomide, dexamethasone; IxaRd, izaxomib, lenalidomide, dexamethasone; Kd, carfilzomib, dexamethasone; Len, lenalidomide; Len-ref, lenalidomide refractory; NA, not available; NDMM, newly diagnosed multiple myeloma; NDMM-TE, newly diagnosed transplant eligible; PAD, bortezomib, adryamicine, dexamethasone; PCD, bortezomib, cyclophosphamide, dexamethasone; PVd, pomalidomide, bortezomib, dexamethasone; Rd, lenalidomide-dexamethasone; Tx, transplant. Study population Trial Table 3. Summary of ongoing studies evaluating CAR T cell therapy in earlier lines of therapy or in combinations with standard of care treatment Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 186 | Hematology 2022 | ASH Education Program CAR T-cell therapy in multiple myeloma | 187 Peter MacCallum Cancer Centre, Australia Zhejiang University Tongji Hospital Memorial Sloan Kettering Cancer Center NCT01716364 NCT05016778 NCT05219721 NCT04555551 MCARH109 CAR-GPR5D GPR5D-CAR T LeY NA CAR2 antiCD38 A2 NKG2D-CAR UCARTCS1 NA κ.CARTs CART-138 Human Human NA NA NA NA Human NA NA 1 1 1 1 1 1 17 18 15 6 NA 72 12 18 1 1 42 1 5 7 1/2 Murine 10 38 n 1 1 Murine Murine 1 Phase Murine ScFv origin GPRC5d GPRC5d GPRC5d Lewis Y CD44v6 (+HSVTK suicide gene) CD38 NKG2D ligands SLAMF7 (TALEN-targeted gene editing TCR and SLAMF7) SLAMF7 κ light chain CD138 CD19 SLAMF7 Antigen 4-1BB 4-1BB NA CD28 CD28 NA DAP10 4-1BB CD28 or 4-1BB/ CD3z + inducible caspase 9 (IC9) cell suicide CD28 4-1BB 4-1BB NR Costimulatory domain Autologous Autologous Autologous Autologous Autologous Autologous Autologous Allogeneic Autologous Autologous Autologous Autologous Autologous Cell source NA, not available; NR, not reached; PD, progressive disease; PR, partial response; SD, stable disease; VGPR, very good partial response. EU Horizon 2020 Program Cellectis S.A. NCT04142619 EU-CART National Cancer Institute NCT03958656 Sorrento Therapeutics Baylor College of Medicine NCT00881920 NCT03464916 Chinese PLA General Hospital NCT01886976 Celyad University of Pennsylvania NCT02135406 NCT02203825 CARAMBA European Union & CARAMBA NCT04499339 CTL019 CAR name Sponsor Trial Table 4. Clinical trials in multiple myeloma evaluating non–BCMA-directed CAR T-cell constructs 1.1-6×108 Lentiviral NA NA NA NA 1.0, 3.0, or 6.0×106/kg 0.5, 1.0, and 2.0×106/kg 25, 50, 150, 450×106 Retroviral Retroviral NA NA Lentiviral ORR 69% CR 25% NA NA NA NA NA NA NA NA 4 SD 1-3×107 NA 0.3-12.0×106 2.0×108 SD: 4; PD: 1 VGPR: 6; PR: 2; PD: 2 NR Efficacy Retroviral NA NA Retroviral 0.44-1.51×107 NA Sleeping beauty Retroviral Dose Transfer method Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 Dr Prakash Singh Shekhawat also under investigation. In a first-in-human study of MCARH109, 17 patients were infused. Four doses were tested (25, 50, 150, and 450×106 CAR+T cells). Fifty-nine percent of the patients had prior BCMA therapy, including 47% with prior CAR T-cell treat ment. The ORR was 60% and 80% in patients with prior BCMA therapy. Data are still preliminary since the median follow-up was only 18 weeks.29 In addi tion, dif fer ent strat e gies aiming to improve CAR potency and sensitivity are under evaluation, and the possibil ities are endless. Therefore, the fine-tuning of CAR density in the T cell,30 the co-infusion of CAR and chimeric costimulatory receptors,31 the use of HLA-independent T-cell receptors instead of CAR,32 armored CAR T, or a combination with gamma-secretase inhibitors to increase BCMA density in the tumor cell are potential venues to improve.33 Clinical data, however, are still scanty (Figure 2). Conclusions In conclusion, BCMA-directed CAR T-cell therapies have dem onstrated impressive clinical results in the context of advanced triple-class exposed RRMM, leading to the approval of 2 CARs. New modalities are in development to overcome some of the current limitations of CAR T-cell therapy, aiming to improve out comes, shorten vein-to-vein time, and reduce toxicity. The fur ther use of BCMA-directed CAR T-cell treatment in earlier lines of therapy is sure to improve outcomes and, hopefully, find the way to curing MM patients. Conflict-of-interest disclosure Paula Rodriguez-Otero: honoraria: Abbvie, Amgen, Bristol Myers Squibb/Celgene, GSK, Janssen, Kite Pharma, Oncopeptides, Pfizer, Sanofi; consultancy: Celgene, GSK, Janssen, Pfizer. Jesús F. San-Miguel: honoraria: Abbvie, Amgen, Bristol Myers Squibb, Celgene, Janssen, MSD, Novartis, Sanofi, Roche, Takeda. 188 | Hematology 2022 | ASH Education Program Off-label drug use Paula Rodriguez-Otero: nothing to disclose. Jesús F. San-Miguel: nothing to disclose. Correspondence Paula Rodríguez-Otero, Clinica Universidad de Navarra, Av de PIO XII, 36, 31008 Pamplona, Spain; e-mail: paurodriguez@unav.es. References 1. Fonseca R, Abouzaid S, Bonafede M, et al. Trends in overall survival and costs of multiple myeloma, 2000-2014. Leukemia. 2017;31(9):1915-1921. 2. Walker BA, Mavrommatis K, Wardell CP, et al. A high-risk, double-hit, group of newly diagnosed myeloma identified by genomic analysis. Leukemia. 2019;33(1):159-170. 3. Usmani SZ, Rodriguez-Otero P, Bhutani M, Mateos MV, Miguel JS. Defining and treating high-risk multiple myeloma. Leukemia. 2015;29(11):2119-2125. 4. Garcés JJ, Bretones G, Burgos L, et al. Circulating tumor cells for compre hensive and multiregional non-invasive genetic characterization of multiple myeloma. Leukemia. 2020;34(11):3007-3018. 5. Paiva B, Puig N, Cedena MT, et al. Measurable residual disease by next-gen eration flow cytometry in multiple myeloma. J Clin Oncol Off J Am Soc Clin Oncol. 2020;38(8):784-792. 6. Gandhi UH, Cornell RF, Lakshman A, et al. Outcomes of patients with mul tiple myeloma refractory to CD38-targeted monoclonal antibody therapy. Leukemia. 2019;33(9):2266-2275. 7. Munshi NC, Anderson LD, Shah N, et al. Idecabtagene vicleucel (ide-cel; bb2121), a BCMA-targeted CAR T-cell therapy, in patients with relapsed and refractory multiple myeloma (RRMM): initial KarMMa results [abstract]. J Clin Oncol. 2020;38(suppl 15). Abstract 8503. 8. Berdeja JG, Madduri D, Usmani SZ, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refrac tory mul ti ple mye loma (CARTITUDE-1): a phase 1b/2 open-label study. Lancet (London, England). 2021;398(10297):314324. 9. Munshi NC, Anderson LDJ, Shah N, et al. Idecabtagene vicleucel in relapsed and refractory multiple myeloma. N Engl J Med. 2021;384(8):705-716. 10. Raje NS, Siegel DS, Jagannath S, et al. Idecabtagene vicleucel (ide-cel, bb2121) in relapsed and refractory multiple myeloma: analyses of high-risk subgroups in the KarMMa study. Blood. 2020;136(suppl 1):37-38. Dr Prakash Singh Shekhawat Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 Figure 2. Possibilities for the future development of CAR T-cell therapy. Depicted are some of the novel new-generation CAR T constructs, including third-generation CARs incorporating 2 different costimulatory domains; fourth-generation CARs (also named “armored” CARs) incorporating transducible genes codifying for cytokines, antibodies, or receptors (among others); and novel target (non-BCMA) and dual targeting strategies, including tandem CAR and dual CAR. 28. Jiang H, Dong B, Gao L, et al. Clinical results of a multicenter study of the first-in-human dual BCMA and CD19 targeted novel platform fast CAR-T cell therapy for patients with relapsed/refractory multiple myeloma. Blood. 2020;136(suppl 1):25-26. 29. Mailankody S, Diamonte C, Fitzgerald L, et al. Phase I first-in-class trial of MCARH109, a G protein coupled receptor class C group 5 member D (GPRC5D) targeted CAR T cell therapy in patients with relapsed or refrac tory multiple myeloma. Blood. 2021;138(suppl 1):827. 30. Rodriguez-Marquez P, Calleja-Cervantes ME, Serrano G, et al. CAR density influences antitumoral efficacy of BCMA CAR-T cells and correlates with clinical outcome. Blood. 2021;138(suppl 1):735. 31. Katsarou A, Sjöstrand M, Naik J, et al. Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence. Sci Transl Med. 2021;13(623):eabh1962. 32. Mansilla-Soto J, Eyquem J, Haubner S, et al. HLA-inde pen dent T cell recep tors for targeting tumors with low anti gen den sity. Nat Med. 2022;28(2):345-352. 33. Cowan AJ, Pont M, Sather BD, et al. Safety and efficacy of fully human BCMA CAR T cells in combination with a gamma secretase inhibitor to increase BCMA surface expression in patients with relapsed or refractory multiple myeloma. Blood. 2021;138(suppl 1):551. 34. Anderson LD, Munshi NC, Shah N, et al. Idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy, in relapsed and refractory multiple myeloma: updated KarMMa results. J Clin Oncol. 2021;39(suppl 15):8016. 35. Oriol A, San-Miguel J, Kansagra A, et al. Idecabtagene vicleucel (IDE-CEL, BB2121), a BCMA-directed CAR T cell therapy, in patients with relapsed and refractory multiple myeloma: updated KarMMa results. EHA Library. 2021;324732. 36. Martin T, Usmani SZ, Berdeja JG, et al. Updated results from CARTITUDE-1: phase 1b/2Study of ciltacabtagene autoleucel, a B-Cell maturation antigendirected chimeric antigen receptor T cell therapy, in patients with relapsed/refractory multiple myeloma. Blood. 2021;138(suppl 1):549. 37. Mailankody S, Jakubowiak AJ, Htut M, et al. Orvacabtagene autoleucel (orva-cel), a B-cell maturation antigen (BCMA)-directed CAR T cell therapy for patients (pts) with relapsed/refractory multiple myeloma (RRMM): update of the phase 1/2 EVOLVE study (NCT03430011). J Clin Oncol. 2020;38(15):8504. 38. Chen W, Fu C, Cai Z, et al. Sustainable efficacy and safety results from LUMMICAR study 1: a phase 1/2 study of fully human B-cell maturation antigen-specific CAR T cells (CT053) in Chinese subjects with relapsed and/or refractory multiple myeloma. Blood. 2021;138(suppl 1):2821. 39. Chunrui L, Wang D, Song Y, et al. A phase 1/2 study of a novel fully human B-cell maturation antigen-specific CAR T cells (CT103A) in patients with relapsed and/or refractory multiple myeloma. Blood. 2021;138(suppl 1): 547. 40. Gan An, et al. Blood. 2020;136 (suppl 1):29-30. 41. Costello CL, Cohen AD, Patel KK, et al. Phase 1/2 study of the safety and response of P-BCMA-101 CAR-T cells in patients with relapsed/refractory (r/r) multiple myeloma (MM) (PRIME) with novel therapeutic strategies. Blood. 2020;136(suppl 1):29-30. 42. Mailankody S, Matous JV, Liedtke M, et al. Universal: an allogeneic firstin-human study of the amt-BCMA ALLO-715 and the anti-CD52 ALLO647 in relapsed/refractory multiple myeloma. Blood. 2020;136(suppl 1): 24-25. © 2022 by The American Society of Hematology DOI 10.1182/hematology.2022000396 Dr Prakash Singh Shekhawat CAR T-cell therapy in multiple myeloma | 189 Downloaded from http://ashpublications.org/hematology/article-pdf/2022/1/180/2021675/180rodriguez-otero.pdf by guest on 09 December 2022 11. Jakubowiak A, Usmani SZ, Berdeja JG, et al. Efficacy and safety of ciltacabtagene autoleucel in patients with relapsed/refractory multiple myeloma: CARTITUDE-1 subgroup analysis. Blood. 2021;138(suppl 1):3938. 12. Shah N, Munshi NC, Berdeja JG, et al. Baseline cor re lates of com plete response to idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy in patients with relapsed and refractory multiple myeloma: subanalysis of the KarMMa trial. Blood. 2021;138(suppl 1):1739. 13. Cohen AD, Mateos M-V, Cohen YC, et al. Efficacy and safety of cilta-cel in patients with progressive MM after exposure to other BCMA-targeting agents [published online ahead of print 12 September 2022]. Blood. 14. Hansen DK, Sidana S, Peres L, et al. Idecabtagene vicleucel (Ide-cel) chi meric antigen receptor (CAR) T-cell therapy for relapsed/refractory multi ple myeloma (RRMM): real-world experience. J Clin Oncol. 2022;40(suppl 16):8042. 15. Da Vià MC, Dietrich O, Truger M, et al. Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma. Nat Med. 2021;27(4):616-619. 16. Samur MK, Fulciniti M, Aktas Samur A, et al. Biallelic loss of BCMA as a resis tance mechanism to CAR T cell therapy in a patient with multiple myeloma. Nat Commun. 2021;12(1):868. 17. Cohen AD, Garfall AL, Stadtmauer EA, et al. B cell maturation antigenspecific CAR T cells are clinically active in multiple myeloma. J Clin Investig. 2019;129(6):2210-2221. 18. Manier S, Ingegnere T, Escure G, et al. Current state and next-generation CAR-T cells in multiple myeloma. Blood Rev. 2022;54(suppl 1):100929. 19. Mailankody S, Liedtke M, Sidana S, et al. Universal updated phase 1 data validates the feasibility of allogeneic anti-BCMA ALLO-715 therapy for relapsed/refractory multiple myeloma. Blood. 2021;138(suppl 1):651. 20. Cohen YC, Cohen AD, Delforge M, et al. Efficacy and safety of ciltacabtagene autoleucel (Cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy, in

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