Oncogenic CSF3R mutations in chronic neutrophilic leukemia
and atypical CML.
N Engl J Med. 2013 May 9; 368:1781
Speaker: CR 呂學儒醫師
Moderator: VS 蕭樑材醫師
Definition of CNL and atypical CML
Leukocytosis and hypercellularity of BM, predominantly of granulocytic cell
Absence Ph chromosome , absence of rearrangements of PDGFR A/B and FGFR1
CNL: neutrophils in both periphreal and BM blood (segmented neutrophils and band forms > 80% of WBC)
Atypical CML : granulocytic dysplasia, increased neutrophil precursors in both peripheral and BM blood
(typically, ≥10% of white cells)
Hematology Am Soc Hematol Educ Program. 2011;2011:250-6
Clinical, hematological and cytogenetic characteristics of aCML
• Background:
– an infrequent chronic MPN
– leukocytosis, absence of Ph/BCR-ABL, marked myeloid dysplasia
– immature granulocytes > 15%, some with absolute monocytosis, D/D
CMML
• Result:
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median age: 65 years
55% had moderate anemia and 36% had thrombocytopenia
Most with marked dysplasia, particularly in the granulocytic lineage
Cytogenetic data: trisomy 8, 5q-, 13q-, 17p-, 12q-, and 11q-,
t(6;8)(p23;q22)
– median survival: 14 months
Ann Oncol. 2000;11:441
Histopathology of peripheral blood and BM of CNL
N Engl J Med. 2013;368:1781
G-CSF and its receptor in myeloid malignancy
• Granulocyte colony-stimulating factor(G-CSF/CSF3):
– major regulator of neutrophil production
– granulopoiesis during infections and enhances multiple neutrophil functions
– inducing proliferation and survival of myeloid progenitor cells
• The receptor for CSF3 (CSF3R)
– 17 exons and its protein 813 amino acids
– Activation with Jak/STAT, Ras/Raf/MAP kinase, and PKB/Akt pathways
• Colony-stimulating factor 3 receptor gene (CSF3R)
– mapping to chromosome 1p
– provides the proliferative and survival signal for granulocytes
– contributes to their differentiation and function
• CSF3R mutations:
– severe congenital neutropenia, secondary developed at AML (1%)
Blood. 2010;115:5131
Leukemia 2013. doi: 10.1038
Use of Hematopoietic Growth Factors in the Survival and Differentiation of
Hematopoietic Cells
NEJM 2013;368:1131
Hematopoietic Growth Factor Signaling
NEJM 2006;354:2034
Sequential gain of mutations in severe congenital neutropenia
(SCN) progressing to acute myeloid leukemia (AML)
Blood. 2012;119:5071
Model for Activation and Signaling of CSF3R Mutations
Two different classes of CSF3R mutations
N Engl J Med. 2013;368:1781
The mutations in CSF3R are a defining molecular abnormality of CNL/atypical CML
Testing for CSF3R mutations could aid in the diagnosis of these diseases
5 patients: both membrane proximal and
truncation mutations
AML(SCN progression): Q741X mutation
ETP-T-ALL: one of T618I
CSF3R deep sequencing
CNL, CSF3R (S783fs mutation)
Dependence on SRC Family-TNK2 or JAK Kinases
(Truncation mutations)
CNL, CSF3R (S783fs mutation) for 66 kinase inhibitor
Hypersensitive to dasatinib
Insensitive to JAK kinase inhibitors
IC50: 50% inhibitory concentration; SFK: SRC family kinase; TNK2: tyrosine
kinase nonreceptor 2
small interfering RNAs (siRNAs)
silencing of TNK2 and FGR (an SFK)
inhibited by dasatinib
Dependence on Src Family-TNK2 or JAK Kinases
(membrane proximal mutations)
CLL, CSF3R (T618I mutation) for 66 kinase inhibitor
Insensitive to dasatinib
Sensitive to JAK kinase inhibitors
Two different classes of CSF3R mutations
• Truncation mutations(S783fs)
– Dysregulation of SRC family–TNK2 kinases
– sensitivity to dasatinib but not to JAK kinase
inhibitors
• Membrane proximal mutations(T618I)
– Dysregulation of JAK family kinases
– sensitivity to JAK kinase inhibitors but not to
dasatinib
Distinct signaling-pathway dysregulation
To test the relative transforming capacity
Expressing wild-type CSF3R, membrane proximal mutations, or truncation mutations
Infected with murine retrovirus
Over a 10-day period
CSF3R mutations were capable to induce transformation
Membrane proximal mutations faster
The potential signaling
differences between the two classes of CSF3R
• Immunoblot analysis for JAK–STAT
phosphorylation
• T618I mutant induced high levels
of STAT3 -JAK2 phosphorylation;
S783fs mutant was low
• Two classes of CSF3R mutations
have different transformation
potential and downstream
signaling consequences
Use of tyrosine kinase inhibitors
the sensitivities of CSF3R, mice
Clinical efficacy of Ruxolitinib in a patient with
CSF3R T618I
CNL vs.atypical CML
• CNL and atypical CML
– separate neoplasms by the WHO
– But challenging for clinicians and hematopathologists
• The categorization relies on arbitrary thresholds
– Total WBC ( ≥25,000 for CNL; ≥13,000 for atypical CML)
– immature granulocytes (<10% for CNL; ≥10% for atypical CML)
– the presence or absence of dysgranulopoiesis (absent in CNL and
characteristic of atypical CML)
• CSF3R mutations :
– a biologically unifying feature of CNL and atypical CML
– the molecular classification of MPD and MDS/MPD
• Sequenced CSF3R in 54 cases
– CNL (n=35) or atypical CML(n=19)
– WHO defined: 12 patients diagnosed CNL; 5 monoclonal gammopathy
(MG)-associated CNL; 9 aCML
• 14 CSF3R mutations detected in 13 patients
– CSF3R T618I is the most mutation: 10/13
– CSF3R T618I frequency: 83% (10/12) in WHO-defined CNL
– CSF3R mutations not in aCML or MG-associated CNL
• CSF3RT618I also absent in PMF(n=76) and CMML(n=94)
• CSF3RT618I
– highly sensitive and specific molecular marker for CNL
– should be incorporated into current diagnostic criteria
Leukemia 2013. doi: 10.1038
Leukemia 2013. doi: 10.1038
Genetically Informed Therapy in Leukemia
Jerald Radich, M.D.
Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance
• Julia E. Maxson
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A TK mutation which play a major role in myeloid cancer
Identified a novel mutations in CSF3R
Tested in vitro with kinase inhibitors
Different types sensitivity to different therapeutic agents
Treated a patient with dramatic improved in WBC, Neutrophil, and PLT
• Therapeutic benefit in these rare disorders
– power of genetic screening to uncover new potential drug targets
• This is how we will beat cancer, one gene, one disease at a
time.
NEJM 2013;368:1838
Take home message
• CSF3R mutations: identified in >50% CNL/aCML
– Consider as a diagnostic criteria
• The oncogenic CSF3R mutations
– Truncation mutations or membrane proximal mutations
– sensitivity to inhibitors of SRC family–TNK2 and JAK kinases
Thank you for your attention