Abstracts til frie foredrag lørdag d 15/3 i forbindelse
med DHS årsmøde 2014
Indholdsfortegnelse
Session 1, kl. 09.00-10.20
Charlotte Madsen
Christian Eskelund
Ina Hahn
Andreas Glenthøj
Bo Mortensen
Marietta Nygaard
Marie Warny
Mette Smith
2
3
4
5
6
7
8
9
Session 2, kl. 11.00-12.20
Michael Andersen
Sarah Farmer
Ulrik Ralfkjær
Lasse Kristensen
Simon Husby
Kim Theilgaard-Mönch
Andreas Ørskov
Thomas Kristensen
10
11
12
13
14
15
16
17
1
Session 1 09.00-10.20
Charlotte Madsen
In Transformed Indolent Lymphomas from the Rituximab Era the Efficacy of Upfront
Autologous Stem Cell Transplantation Depends on When Transformation Occurs in
the Course of the Disease.
C. Madsen1, M. B. Pedersen1, K. Bendix2, M. B. Møller3, P. Johansen4, B. A. Jensen5, P.
Jensen6, L. Munksgaard5, P. D. Brown7, E. K. Segel1, F. A. d'Amore1.
1
Department of Haematology, Aarhus University Hospital, Aarhus, Denmark, 2Department of
Pathology, Aarhus University Hospital, Aarhus, Denmark, 3Department of Pathology, Odense
University Hospital, Odense, Denmark, 4Department of Pathology, Aalborg University Hospital,
Aalborg, Denmark, 5Department of Haematology, Odense University Hospital, Odense, Denmark,
6
Department of Haematology, Aalborg University Hospital, Aalborg, Denmark, 7Department of
Haematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
Introduction: When indolent lymphomas (IL) transform to an aggressive histology (TRIL)
the result is often a rapid clinical course, treatment refractoriness and shortened survival.
Although rituximab-containing chemotherapy regimens (R-chemo) has generally become
standard-of-care in CD20-positive TRIL, no therapeutic gold standard has yet been
established. The purpose of this study was to determine whether the outcome of TRIL
patients improved if they, at the time of transformation, after R-chemo, also received
upfront autologous stem-cell transplantation (ASCT). We also analyzed whether pretransformation features and prior rituximab therapy affected outcome at transformation.
Patients and methods: Ninety-five patients (≤ 68 years) with histologically confirmed
TRIL were included. Five-year overall (OS) and progression-free survival (PFS) were
calculated and selected parameters tested in a multivariate analysis. All analyses were
conducted on three cohorts: (1) whole cohort (‘all TRIL’), (2) patients with co-existing
evidence of both indolent and aggressive histology at diagnosis (‘discordant/composite
TRIL’) and (3) patients with indolent lymphomas transformed over time (‘sequential TRIL’).
Results: Sixty-five TRIL patients (67%) received ASCT consolidation and 31 (33%) did
not. Within the ‘all TRIL’ cohort, the 5-year OS and PFS for R-chemo+ASCT vs. R-chemo
alone, were 65% vs. 48% (p=0.11) and 57% vs. 30% (p=0.02), respectively. At specific
sub-cohort level, the ‘discordant/composite TRIL’ showed no impact of R-chemo+ASCT on
neither OS (80% vs. 67%; p=0.51) nor PFS (75% vs. 61%; p=0.35). Conversely, Rchemo+ASCT improved the outcome of ‘sequential TRIL’ (OS 57% vs. 36%; p=0.09; PFS
47% vs. 6%; p=0.003), regardless of prior rituximab therapy. However, the beneficial effect
of ASCT was significantly higher in patients who had not received rituximab at IL stage.
Conclusions: In the rituximab era, upfront ASCT improved outcome in sequential, but not
composite/discordant TRIL. Prospective trials elucidating the role of upfront ASCT in TRIL
are warranted.
2
Christian Eskelund
Måling af piperacillin koncentration i plasma åbner mulighed for individualiseret behandling
af hæmatologiske patienter
Christian Winther Eskelund1, Ingolf Mølle1, Birgitte Brock2, Tore Forsingdal Hardlei2 og Ole
Halfdan Larsen1,2
Hæmatologisk Afdeling1 og Klinisk Biokemisk Afdeling2, Aarhus Universitetshospital
Piperacillin med tazobactam anvendes til empirisk intravenøs behandling af neutropen feber hos
patienter med maligne hæmatologiske sygdomme. Kendskabet til piperacillins farmakokinetik hos
disse patienter er begrænset. Formålet med dette studie var derfor at undersøge koncentrationen af
piperacillin i plasma fra hæmatologiske patienter både ved bolus dosering samt kontinuerlig
behandling. Syv patienter blev fulgt med 2 daglige blodprøver under behandlingens første 5 dage.
Patienterne fik initielt bolus dosering af 4g piperacillin/0,5g tazobactam intravenøst hver 8. time.
Hos 6 patienter var der i observationsperioden rutinemæssigt skift til kontinuerlig infusion over 24
timer (12g/1,5g). Blodprøver blev taget gennem centralt venekateter. Piperacillin koncentrationen
blev kvantificeret ved en UHPLC metode (Agilent 1290). De inkludererede patienter var alle mænd
med maligne hæmatologiske sygdomme og neutropeni på grund af kemoterapi. Ved
behandlingsstart var temperaturen 38.4°C (38.2-38.7) (median (interkvartil interval)) og TOKS
score 3 (1-4). Der blev observeret markante interindividuelle forskelle i median piperacillin peakværdi: 171 µg/mL (107-190), halveringstid: 35 min (29-42) samt niveau ved steady state under
kontinuerlig behandling: 18 µg/mL (13-21). Der var ikke tegn til signifikant ændring over tid
(ANOVA p-værdier > 0.34). Interindividuelle forskelle kunne kun delvis forklares ved forskelle i
kropsvægt (R2 0.18-0.30). Nyrefunktionen var normal hos de undersøgte og bidrog ikke til de
observerede forskelle (alle R2<0.18). Dette studie viser at måling af piperacillin kan anvendes til
monitorering af behandlingen hos hæmatologiske patienter. Den nye metode påpeger markante
interindividuelle forskelle og åbner mulighed for individualiseret behandling med optimeret effekt
og bivirkningsprofil. Den fulde potentielle kliniske værdi heraf bør afklares i et større klinisk
randomiseret studie.
3
Ina Hahn
Prevalence of venous thromboembolism in patients with Diffuse large B-cell
lymphoma
Ina Hornemann Hahn1; Jakob Madsen1; Marianne Tang Severinsen1
1Department
of Haematology, Aalborg University Hospital, Aalborg
Introduction:
Venous thromboembolism (VTE) is a critical complication in patients with cancer but the
information on VTE among patients with diffuse large B-cell lymphoma(DLBCL) is sparse.
We aimed to evaluate the incidence of VTE in patients with DLBCL and to evaluate the
quality of registry data on VTE among DLBCL patients.
Methods:
Patients with a first time diagnose of DLBCL at the Department of Haematology Aalborg
University Hospital from January 1, 2007 to December 31, 2013 were included in the
study. Participants were followed from date of DLBCL and two years further. We reviewed
the entire medical records for all DLBCL patients and data on confirmed VTE events were
retracted. Also VTE risk factors, medication, platelet count, haemoglobin and leukocyte
count at date of VTE were noted.
We retracted register data on VTE discharge diagnosis from Patients Administrative
System from the participants and compared this with information from medical records on
VTE events.
Results:
A total of 296 patients had an incident diagnosis of DLBCL during the study period. We
identified 32 (10.8%) with a VTE event within a two-year follow-up period of which three
PEs were found on routine CT scan.
In the discharge register 20 of the 296 patients had a diagnosis of VTE during follow-up of
which 17 were confirmed. The positive predictive value of a VTE discharge diagnosis was
85%. Only 17 of the 32 confirmed VTE cases were registered therefore the sensitivity of a
VTE discharge diagnosis was 53%.
Conclusions:
10,8 % DLBCL patients had a VTE event during two years of follow-up. Register data on
VTE discharge diagnosis is valid among patients with DLBCL with a positive predictive
value of 85%. The sensitivity of a VTE discharge diagnosis among DLCBL patients is only
53%.
4
Andreas Glenthøj
PLASMA PROHNPS ARE SPECIFIC EARLY MARKERS OF NEUTROPHIL
ENGRAFTMENT IN PATIENTS UNDERGOING HIGH-DOSE CHEMOTHERAPY,
AUTOLOGOUS OR ALLOGENEIC STEM CELL TRANSPLANTATION
Andreas Glenthøj, Frederik Emmertsen, Jette Sønderskov, Peter Kampmann, Henrik Sengeløv,
Niels Borregaard.
Department of Hematology, Rigshospitalet, Copenhagen, Denmark
1.0
2.5
0.8
2.0
0.6
1.5
0.4
1.0
0.2
0.5
0.0
PMN
proHNPs
PMNs 109/L
Relative to max
Background: Neutropenia is invariably a consequence of myeloablative conditioning before
hematopoietic stem cell transplantation (SCT) as well as high-dose chemotherapy. Several plasma
proteins have been investigated as early markers of bone marrow recovery, but these lack
specificity which renders them clinically less useful.
Human neutrophil peptides (HNPs) are antimicrobial peptides exclusively synthesized by neutrophil
precursors. Most is synthesized by myelocytes, where HNPs remain unprocessed as proHNPs and
are secreted in large amounts to the bone marrow plasma, and from here to blood.
Aims: We hypothesized that proHNPs may serve as early and specific markers of granulopoiesis in
patients undergoing hematopoietic SCT or high-dose chemotherapy.
Methods: Plasma samples were obtained daily from patients undergoing allogeneic SCT (n=11),
autologous SCT (n=16), or high-dose chemotherapy (n=14). Furthermore, plasma samples were
taken from patients with AML prior to chemotherapy (n=19) and healthy controls (n=39).
ProHNPs, MPO, NGAL, and lysozyme were measured by ELISA.
Results: Neutrophils disappeared rapidly from circulation after myeloablative conditioning or highdose chemotherapy. Of the investigated proteins, only proHNPs consistently disappeared from
circulation along with neutrophils. Likewise, only proHNPs invariably preceded the rise in
neutrophils.
In patients undergoing allogeneic SCT, proHNPs rose five to six days before neutrophil counts.
Patients undergoing autologous SCT were treated with G-CSF to speed up neutrophil recovery and
in these, proHNPs rose approximately two days before neutrophils were detected. As for patients
receiving induction or consolidation chemotherapy, proHNPs predated neutrophils by two to four
days.
Patients with AML had significantly lower levels of plasma proHNPs than healthy controls,
indicating that proHNPs are not produced by leukemic blasts.
Conclusion: ProHNPs are markers of normal granulopoiesis. Clinically, plasma proHNP could be
an important tool to distinguish late take from primary graft failure in hematopoietic SCT patients,
since measurement of proHNPs in plasma allows early detection of donor cell engraftment.
0.0
0
5
10
15
20
25
Day
Figure 1: ProHNPs and polymorphonuclear neutrophils (PMNs) in peripheral blood of one
patient undergoing allogeneic SCT. Patient is representative of the group. Day 0 marks the
day of SCT.
5
Bo Mortensen
Similar long term overall survival after allogeneic HCT for severe aplastic anemia in patients
transplanted with a related donor compared to an unrelated donor
Mortensen BK, Jacobsen N, Heilmann C, Sengeløv H.
Department of Hematology, National University Hospital, Rigshospitalet, Copenhagen, Denmark
Treatment of severe aplastic anemia (SAA) is based on immunosuppressive therapy (IST)
and allogeneic hematopoietic cell transplantation (HCT). For patients younger than 55
years with an HLA-identical sibling donor, allogeneic HCT is the treatment of choice. For
patients younger than 55 years who lack an HLA-identical sibling donor, IST is the
treatment of choice and allogeneic HCT is recommended after failure to respond to IST.
We retrospectively analysed the outcome of all patients treated with allogeneic HCT for
SAA at Rigshospitalet between 1977 and 2013 and compared the outcome of allogeneic
HCT for patients transplanted with a related donor vs. an unrelated donor.
The median age at HCT was 16 years (0-61). The donor was an HLA-identical sibling in 51
(53 %), syngen in 2 (2 %), related HLA mismatched in 4 (4 %), HLA matched unrelated in
29 (30 %), and HLA mismatched unrelated in 10 (11 %). Forty-five (47 %) patients were
transplanted between year 1977 and 2000, and 51 (53 %) were transplanted after year
2000.
Overall survival (OS) in patients transplanted before 2000 was 58 %, whereas survival
after 2000 increased to 92% (P<0.0001), median observation times were 15,0 years (0-33)
and 5,4 years (0-13), respectively. In patients transplanted after year 2000, OS was 89%
both after transplantation with a related donor or an unrelated donor (figure 1) (Fig 1).
Median observation times were 6,5 years (0-13) and 4,1 years (0-11), respectively.
In this study we observe a marked improvement in survival among SAA patients receiving
an allogeneic HCT since 2000. Interestingly, we find similar long term OS in patients
transplanted with a related donor as compared to an unrelated donor. Our study supports,
that allogeneic HCT with an unrelated donor should be considered as first line treatment in
selected patients with SAA lacking an HLA-identical sibling donor.
Figure 1: Overall survival in patients transplanted for SAA after year 2000 with a related or
an unrelated donor:
6
Marietta Nygaard
Close Monitoring of Cyclosporine A Concentration as a Means of Preventing Acute
Graft-versus-Host Disease in Allogeneic Stem Cell Recipients
Nygaard M, Hovgaard D, Schjødt I, Smedegaard Andersen N, Vindeløv L and Sengeløv H
Department of Hematology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark
Introduction
The whole blood concentration of cyclosporine A (CsA) after allogeneic stem cell transplantation is
important in prevention of acute graft versus host disease (aGVHD). Low concentrations may
cause aGVHD and high concentrations toxicity or relapse. The target concentration and method of
monitoring and administering CsA varies between transplant centers. As one of few centers we
measure CsA daily and furthermore administer CsA orally for as long as possible.
Methods
We retrospectively analyzed data from 48 adult patients, who consecutively underwent
myeloablative allogeneic stem cell transplantation in the years 2010 and 2011 at Rigshospitalet.
Transplant indications were AML (n=18), ALL (n=14), MDS (n=3), SAA (n=6), MM (n=2) and other
(n=5). Whole blood concentration of CsA was measured once daily. The median CsA
concentration in weeks 1 to 4 was calculated, and patients were divided into two equally sized
groups according to the median CsA concentration. Renal impairment was defined as doubling of
serum creatinine during the first 28 days after transplantation.
Results
Assessing the cumulative incidence of aGVHD with death as competing event, the proportion of
patients with aGVHD grade II-IV was higher in those with CsA levels below the median in week 2
after transplantation (p=0.012, figure 1). CsA levels were not correlated with excess risk of renal
impairment or death from relapse.
21 patients (43%) received CsA orally for the entire transplantation period. There was no difference
in occurrence of aGVHD between groups on intravenous and oral CsA (p=0,2).
Conclusion
Close monitoring of the CsA concentration in the early transplant period is important in maintaining
CsA within target concentration and thus preventing aGVHD. Target concentration of CsA should
possibly be adjusted to higher levels than we observed in this series of patients. It seems safe to
administer CsA orally with regards to aGHVD.
Figure 1
Cumulative incidence of aGVHD (1) and death (2) for groups with high and low CsA.
7
Marie Warny
G6PD deficiency in the Danish immigrant population
Marie Warny, Jesper Petersen, Tobias Wirenfeldt Clausen and Henrik Birgens
Hæmatologisk afdeling, Herlev Hospital
Abstract
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common occurring enzyme
deficiency, with a high prevalence in areas historically exposed to malaria. In this study
1508 Danish immigrants (307 males and 1201 females) were tested using quantitative and
molecular methods, to determine the prevalence of G6PD deficiency in the Danish
immigrant population. We found the allelic frequency to be 2,7% in the female population.
Furthermore, a map of the mutation pattern in the Danish immigrant population was made,
202A
showing a high prevalence of the G6PD Avariant in African immigrants, a high
prevalence of the G6PD Mediterranean variant in immigrants coming from the
Mediterranean and western Asia countries and a high variant heterogeneity in the
populations of the South East Asia-Pacific. The Danish immigrant population from the
abovementioned areas, is characterized by a high prevalence of thalassaemia trait, and
thus microcytosis and low mean corpuscular haemoglobin (MCH). The lowered mean
haemoglobin content of the red blood cells, cause deviations in the G6PD activity when
measured as activity per g of Hgb. Since this study included many patients with various
thalassemic syndromes it gave us an opportunity to evaluate the interaction between a
thalassemia gene and the G6PD gene on the clinical phenotype especially in
heterozygous females. We found some overlap between patients without G6PD deficiency
and heterozygous females, but the positive predictive value of a sample with low or
intermediate activity of G6PD was highly influenced by the MCH level. Measuring MCH is
a helpful tool to evaluate whether a female with seemingly normal G6PD activity is at risk
of being heterozygous G6PD deficient or not.
Figure: It is obvious that there is no diagnostic trouble concerning males, since their G6PD
activities is split in two, those with normal activity and those with abnormal activity. Three
G6PD hemizygous males with near-normal activity (Δ), all had blood transfusion prior to
the quantitative assay. Females are more difficult to classify, in that there is no clear
distinction between the three activity levels. This is consistent with the problem in
diagnosing heterozygous females, because they present a broad spectrum of activities.
8
Mette Smith
Red cell mass measurement in the diagnostic of PV and ET anno 2013
Mette Aaby Smith(1), Anders Møjbæk(1), Allan Johansen(2), Hanne Vestergaard(3)
1: stud med, SDU, 2: ledende overlæge, Nuklearmedicinsk afdeling, OUH, 3: konst. ledende overlæge, PhD,
Hæmatologisk afdeling, OUH
Introduction and background: Essential thrombocytosis (ET) and polycythemia vera (PV) belong
to the Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPN).
After the discovery of the JAK2 mutation, WHO in 2008 changed the diagnostic criteria for
PV and ET, omitting the measurement of red cell mass (RCM) and replacing it with haemoglobin
measurements.
Patients with early PV may be difficult to discriminate from patients with ET. The aim of this
study was to clarify to what extent EV determination contributes to the differential diagnosis
between PV and ET and to the diagnosis of PV.
Methods and results: From 2007 to 2012, a total of 95 patients were referred from the Department
of Haematology X, Odense University Hospital, for RCM measurement. Of these, 79 patients were
included in the study and assigned to one of two groups, depending on whether they presented with
thrombocytosis (study population) or with a normal number of thrombocytes (the rest).
In the study population, 21 patients had normal haemoglobin and haematocrit values. Twelve
of these had increased RCM, and they were diagnosed with PV. Nine of these patients did not get
their final diagnosis till after the RCM measurement.
Conclusion: RCM measurement contributes to the differential diagnosis between PV and ET, and it
is necessary to include it for patients presenting with isolated thrombocytosis.
9
Session 2 11.00-12.20
Michael Andersen
Korrelation mellem hypogammaglobulinæmi og cytogenetiske aberrationer og
paraklinisk karakteristik af en CLL kohorte fra en enkelt institution.
Michael A Andersen, Ole W Bjerrum, Mette Klarskov Andersen og Christian Geisler
Hæmatologisk Klinik, Rigshospitalet
Karakterisering af hypogammaglobulinæmi, cytogenetiske aberrationer og IGVH
mutationsstatus kan hjælpe til forståelse af patogenese, sygdomsprogression og
immundefekt for B-CLL. Forekomsten af lave immunglobulinniveauer er ikke undersøgt i
forhold til de cytogenetiske abnormiteter. Det primære formål med studiet var at
undersøge sammenhænge mellem Ig A, G og M, IGVH og cytogenetiske aberrationer. Det
sekundære formål var at korrelere parametre i nyligt diagnosticerede CLL-patienter for at
illustrere sammenhænge i sygdomsbiologi.
Metode
Journalen fra 97 konsekutive patienter over 3 år, identificeret ved DRG kode, blev
gennemgået for at registrere de kliniske, patologiske og cytogenetiske karakteristika
retrospektivt.
Resultater
En reduktion i mindst en af immunglobulinklasserne blev observeret i 57 %. IgA var lavt
hos 23 % af patienterne, 17 % havde lavt IgG og 52 % af patienterne havde lavt IgM
niveau. Blandt de 75 % af patienterne med mindst en anomali havde 48 % 13q deletion,
17 % havde trisomi 12, 7,7 % havde 17p deletion, 6,6 % havde 11 q deletion og 6,6 %
havde 14q32 translokation.
Patienter med trisomi 12 viste signifikant forhøjet IgA- og IgG-niveau. Patienter med 14q32
translokationer havde signifikant lavere IgA og IgG. Statistisk signifikante korrelationer blev
demonstreret mellem stadier og rutine parakliniske parametre.
Konklusioner
1. Ved trisomi 12 var IgM og IgA signifikant forhøjet. Patienter med 14q32 translokation
havde signifikant nedsat IgA og IgG. Det indikerer, at genetiske forhold på kromosom
henholdsvist 12 og 14 kan have betydning for regulering af immunglobulin. 14q32
translokation involverer IGH genet, hvilket kan have betydning herfor.
2. Statistisk signifikante sammenhænge kunne demonstreres for en række rutine
blodprøver, hvilket illustrerer CLLs biologi. Princippet kan bruges som en indikator for at
dokumentere anvendelse af en repræsentativ CLL population i behandlings- eller studie
sammenhæng.
10
Sarah Farmer
Bone manifestations in patients with myelofibrosis - A cross-sectional study using DXA and
HR-pQCT.
Sarah Farmer1, Pernille Hermann2, Vikram Shanbhogue2, Stinus Hansen2, Hanne Vestergaard1,
Claudia I Stahlberg3, Henrik Frederiksen1
1
Department of Hematology, 2Department of Endocrinology, 3Department of Pathology,
Odense University Hospital, Odense, Denmark
Objectives
In patients with myelofibrosis, osteosclerotic changes appear in the bone tissue as the result of
growing and thickening of the bone trabeculae. We conducted a cross-sectional study to evaluate
bone structure MF patients using conventional DXA and High-resolution peripheral quantitative CT
(HR-pQCT).
Materials and Methods
Patients who meet the diagnostic criteria of primary myelofibrosis or myelofibrosis secondary to
another CMPN according to WHO 2010-criteria with International Classification of Diseases, 10th
revision, from the department of haematology, Odense University Hospital, Denmark were
included.
Areal Bone mineral density was assessed by DXA (duel energy X-ray absorption), and 3D
assessment of Bone geometry, volumetric BMD, and micro-architecture were measured using HRpQCT
Data are compared with healthy volunteers matched on age, sex, and height, in a 1:1 ratio.
Blood samples were analyzes for PINP1 (procollagen type I N-terminal pro-peptide), a markers of
bone formation. Diagnostic bone marrow biopsies were reviewed by a hematopathologist to
confirm diagnosis, classification of fibrosis grade and presence of osteosclerotic changes.
Results
20 MF patients were included, 11 with grade 1-2 and 9 with grade 3. Mean age was 69.8 (95% CI:
66.3-73.4). BMD in patients were increased in spine 1.10 (0.93-1.08) vs. 0.93 (0.87-1.00), but not
of the hip 0.93 (0.85-1.02) vs. 0.90 (0.82-0.98) and HR-pQCT showed increased bone mass,
particular trabecular volumetric BMD, trabecular BV/TV, and trabecular number, although
statistical significance was not reached. PINP was increased in patients (fig 1).
Conclusion
This study demonstrated elevated level of PINP in MF patients indicating increased formation rate
of collagen 1 correlated to new bone formation. DXA indicated increased bone mass, and the HRpQCT could locate this predominately on trabecular site. Myelofibrosis is a rare disease and the
number of patients included was small.
MF patients
50
100
150
Healthy donors
0
Ulrik Ralfkjær
Level of PINP (ng/mL)
11
Ulrik Ralfkjær
The role of microRNA 203 in cutaneous T-cell lymphoma
[1,2]
[2]
[3]
[4]
[5]
[5]
Ralfkiær U, Kopp KL, Gjerdrum LM, Marstrand T, Ahler CB, Glue C,
[4]
[6]
[7]
[3]
[2]
[2]
Røpke M, Gniadecki R, Skov L, Ralfkiær E, Woetman, A, Ødum N and
[1]
Grønbæk K
[1]
[2]
Rigshospitalet, dept. of Hematology, University of Copenhagen, dept. of International Health,
[3]
[4]
[5]
Immunology and Microbiology, Rigshospitalet, dept. of Pathology, LEO Pharma, Exiqon,
[6]
[7]
Bispebjerg Hospital, dept. of Dermatology and Gentofte Hospital, dept. of DermatoAllergology
Cutaneous T-cell lymphoma (CTCL) is a rare disease which originates from malignant
transformation of skin homing memory T-cells. A major challenge in CTCL is early
diagnosis, since the early phases often resemble benign skin disorders (BSD) both
clinically and histologically. As a result many patients are followed for years with repeated
examinations by morphology, pheno-typing and geno-typing before a definite diagnosis
and relevant therapy can be initiated. MicroRNAs (miRs) constitute a novel class of noncoding RNA that are expressed in a cell and tissue specific manner in both normal and
malignant tissues. In this study we have focused on miR expression in CTCL vs. benign
diseases.
The first aim of this project has been to develop a diagnostic kit, which can assist in
establishing an early diagnosis. We used Exiqon miR microarrays to show that the
expression level of 3 miRs (203, 205 and 155) can discriminate CTCL from BSD with
>95% classification accuracy (Ralfkiaer U, Hagedorn PH et al., Blood. 2011 Nov 24). We
validated these findings in an independent cohort using a TaqMan qPCR based method
Marstrand et al., Leuk Lymphoma. 2013 Jul 29).
The second aim of this project has been to investigate the regulation and function of miR203 in CTCL. MiR-203 is critical for normal development of stratified epithelia and acts to
repress stemness and induce differentiation. Consistent with this function in normal cells,
miR-203 is down-regulated in many cancers and has been recognized as an important
tumor suppressor in both epithelial and hematological malignancies. We show that miR203 is down-regulated in CTCL compared to BSD and normal blood T-cells, and that a
probable cause of this down-regulation is epigenetic silencing by DNA promoter
methylation. We also show that induced miR-203 expression in transfected cells targets
known the oncogenes, p63, survivin and cREB. To search for new potential targets of miR203, miR-203 mimic and mock transfected cells were examined on Affymetrix gene
expression arrays. This resulted in the identification of a list of 19 significantly de-regulated
genes, including a previously unrecognized miR-203 target molecule involved in cytokine
induced JAK/STAT signaling, a pathways frequently aberrantly activated in CTCL.
In conclusion, we have identified a miR classifier that may aid early recognition of CTCL
and have added significant novel insight into the possible biological means of action of mir203 in CTCL pathogenesis.
12
Lasse Christensen
Investigation of allelic methylation patterns of the death-associated protein kinase
(DAPK) gene in diffuse large B-cell lymphoma (DLBCL) using a novel
pyrosequencing-based method.
Lasse Sommer Kristensen1, Marianne Bach Treppendahl1, Fazila Asmar1, Mia Seremet
Girkov1, Helene Myrtue Nielsen1,2, Tina Ellegaard Kjeldsen2, Elisabeth Ralfkiaer3, Lise
Lotte Hansen2 & Kirsten Grønbæk1
1
Department of Hematology, Rigshospitalet, Copenhagen, Denmark.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
3
Department of Pathology, Rigshospitalet, Copenhagen, Denmark.
2
Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of nonHodgkin’s lymphoma in the western world. Improvements in overall survival have been
observed with the introduction of rituximab in combination with cyclophosphamide,
doxorubicin, vincristine, and prednisone (R-CHOP), however, prognostic markers are still
needed. Methylation of the death-associated protein kinase (DAPK or DAPK1) gene is
likely to have prognostic value in DLBCL patients treated with R-CHOP.
Findings: Allele-specific methylation patterns of the DAPK promoter CpG island were
assessed using allelic MSP-pyrosequencing in a cohort of 106 DLBCL patients uniformly
treated with R-CHOP. Mutation status of the TP53 gene was assessed using denaturing
gradient gel electrophoresis (DGGE) and Sanger sequencing for 80 of the patients. DAPK
mono- and biallelic methylation was associated with shorter overall survival (p =0.029).
When only considering individuals heterozygous for the rs13300553 SNP a shorter overall
survival was associated with monoallelic methylation of the A-allele (p =0.005). Patients
carrying both DAPK methylation and a TP53 mutation did not have a statistically significant
difference in overall survival compared to patients carrying only one of these molecular
alterations (p =0.134).
Conclusions: This study confirms the potential of DAPK methylation as a prognostic
marker in DLBCL. Moreover, monoallelic methylation of the A-allele of the rs13300553
DAPK promoter CpG island SNP is associated with inferior overall survival as compared to
all other allelic DAPK methylation patterns.
13
Simon Husby
miRNA-18b overexpression identifies mantle cell lymphoma sub-group with poor survival
and improves MIPI-B prognostication
Simon Husby1*, Ulrik Ralfkiaer1*, Christian Garde2,9, Sara Ek5, Arne Kolstad3, Mats Jerkeman4,
Anna Laurell5, Riikka Räty6, Mats Ehinger5, Christer Sundström7, Marja-Liisa KarjalainenLindsberg6, Jan Delabie3, Elisabeth Ralfkiaer8, Erik Clasen-Linde8, Peter Brown1, Christopher T.
Workman2,9, Christian H. Geisler1# and Kirsten Grønbæk1#.
Purpose Mantle cell lymphoma (MCL) is an aggressive rare NHL subtype with a variable clinical
course. We assessed whether MCL miRNA expression could identify patients with poor survival
and improve current prognostication of MCL patients.
Patients and Methods Diagnostic MCL samples from 172 patients in the Nordic MCL2 and MCL3
clinical trials were retrieved. All patients had confirmed CyclinD1 overexpression and received
high dose immunochemotherapy followed by ASCT. The median follow-up was 6.4 years for the
MCL2 cohort and 3.7 years for the MCL3 cohort. Genome-wide miRNA microarray profiling was
performed in 74 patients of the screening cohort (MCL2). Differentially expressed miRNAs
(adjusted p-value < 0.05) were re-analyzed by qRT-PCR. Prognostic miRNAs were validated by
qRT-PCR in 94 patients of the validation cohort (MCL3).
Results In the screening cohort 17 miRNAs were differentially expressed by microarray analysis in
patients who died from MCL. Follow-up qRT-PCR analysis found that 3 of the 17 miRNAs (miR18b, miR-92a, miR378d) were differentially expressed in patients who died from MCL in the
validation cohort. On the basis of data from the screening cohort, feature selection and leave-oneout cross-validation, miR-18b was identified to hold the highest prognostic value. We generated a
new MIPI-B-miR prognosticator, by combining expression-levels of miR-18b with MIPI-B data,
which improved prognostication compared to the current gold standard with regards to causespecific survival, overall survival and progression-free survival.
Conclusion Overexpression of miR-18b identifies patients with poor prognosis in two large
prospective MCL cohorts and adds prognostic
information to the MIPI-B prognosticator.
14
Kim Theilgaard-Mönch
Comparing cancer vs. normal gene expression profiles identifies common
transcriptional cancer programs in AML patients.
Nicolas Rapin1,2,3,4, Frederik Otzen Bagger1,2,3,4, Johan Jendholm1,2,4, Helena MoraJensen5, Anders Krogh2,3, Alexander Kohlmann6, Christian Thiede7, Niels Borregaard5,
Lars Bullinger8, Ole Winther2,3,9, Bo T. Porse1,2,4,11, Kim Theilgaard-Mönch1,2,10,11
1
The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen,
Denmark; 2Biotech Research and Innovation Centre (BRIC), University of Copenhagen,
Copenhagen, Denmark; 3The Bioinformatics Centre, Department of Biology, Faculty of Natural
Sciences, University of Copenhagen; 4Danish Stem Cell Centre (DanStem) Faculty of Health
Sciences, University of Copenhagen, Denmark; 5The Granulocyte Research Laboratory,
Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Denmark; 6MLL Munich
Leukemia Laboratory, Munich, Germany; 7University Hospital Carl Gustav Carus, Medical Dept. 1,
University of Technics Dresden, Germany; 8Department of Internal Medicine III, University Hospital
of Ulm, Ulm, Germany; 9DTU Compute, Technical University of Denmark, Lyngby, Denmark: and
10
Dept. of Hematology, Skanes University Hospital, University of Lund, Sweden, 11These authors
contributed equally to this work
Gene expression profiling (GEP) has been used extensively to characterize cancer,
identify novel cancer entities and improve patient prognostification. However, it has largely
failed to identify transcriptional programs that differ between cancer and corresponding
normal cells, and are fundamental to disease etiology. Here we report a newly developed
method that facilitates the comparison of any cancer sample to its nearest normal cellular
counterpart and apply this method using acute myeloid leukemia (AML) as a model. First
we generated a gene expression-based landscape of the normal hematopoietic hierarchy
using expression profiles from highly purified normal hematopoietic stem/progenitor cells
(HSPCs) and their mature myeloid progeny, and mapped gene expression profiles of AML
patients to this landscape. This allowed us to identify the closest normal cell population for
individual AML samples and determine gene expression changes between cancer and
normal. We find the cancer vs. normal method (CvN-method) to be superior to
conventional methods in stratifying AML patients with aberrant-karyotype (AK-AML).
Moreover, the CvN-method uncovered a novel poor-outcome subtype of normal-karyotype
AML (NK-AML), which allowed for the generation of a highly prognostic survival signature.
Our analysis also unraveled common transcriptional programs that are associated with
elevated signaling activity, inflammatory response and hypoxia. Strikingly, these common
transcriptional programs are shared by most AML patients despite their otherwise
profound clinical and genetic heterogeneity. As these common transcriptional programs
represent disrupted core cellular functions including aberrant oncogenic pathway activity
some of them are highly relevant for targeting by antibody- and small molecule-based
therapies. Collectively, our study implicates that our CvN-method holds great potential as a
tool for prognostification as well as the identification of transcriptional cancer programs for
future targeted therapies.
15
Andreas Ørskov
Is there a rationale for combined targeting of PD-1 and DNA methylation in
myelodysplastic syndrome?
Ørskov AD, Treppendahl MB and Grønbæk K
Epi-/Genome Laboratory, Dept. of Hematology, Rigshospitalet, Copenhagen
Introduction: The programmed death-1 (PD-1) is a molecule that is involved in
immunosuppression. It is expressed on activated T-cells and inhibits T-cell function upon
binding to its ligand B7-H1. Numerous cancer studies indicate that the tumour
microenvironment manipulates the PD-1/B7-H1 pathway by upregulating B7-H1 on tumour
cells. The demethylating agent, azacytidine (AZA), is used as standard therapy for higher
risk MDS patients; however, the majority of the patients will lose their response to AZA
over time. It has recently been shown that the expression of PD-1 is regulated by DNA
methylation. Accordingly, we suggest that AZA, in addition to its beneficial function,
induces the PD-1 expression on T-cells in the MDS microenvironment thereby hampering
the immune response against the MDS blasts.
+
+
Methods: CD4 and CD8 T-cells from MDS patients sampled before and during AZA
treatment were isolated by magnetic cell sorting. PD-1 promoter methylation was analysed
by pyrosequencing. PD-1 expression was analysed by RT-qPCR and cell surface
expression will be analysed by flowcytometry. The expression of B7-H1 on MDS blasts
from the same patients will be analysed by flowcytometry.
Results: 20 MDS patients have currently been analysed. Our preliminary results
demonstrate an ongoing demethylation of the PD-1 promoter in the T-cells during
treatment with AZA in ~60% of the patients. The demethylation is accompanied by an
upregulation of the PD-1 mRNA expression. Data on cell surface expression of PD-1 and
B7-H1 will be presented at the meeting.
Discussion: The first and very preliminary results of this study suggest that PD-1 signalling
may be involved in MDS pathogenesis and in resistance to AZA. Based on these
preliminary data we hypothesize that the combination of AZA and PD-1/B7-H1 pathway
inhibitors may be efficient therapy in AZA refractory MDS.
16
Thomas Kristensen
Clinical relevance of sensitive and quantitative STAT3 mutation analysis using next
generation sequencing in T-cell large granular lymphocytic leukemia
Thomas Kristensen1, Martin Larsen2,3, Annika Rewes4, Henrik Frederiksen4. Mads
Thomassen2,3, Michael Boe Møller1
1
Department of Pathology, Odense University Hospital, 2Department of Clinical Genetics, Odense
University Hospital, 3Human Genetics, Clinical Institute, University of Southern Denmark,
4
Department of Hematology, Odense University Hospital, Odense, Denmark
Abstract: Diagnosis of T-cell large granular lymphocytic leukemia (T-LGL) is often
challenging because clinical and laboratory characteristics are overlapping with nonneoplastic conditions. Recently, mutation in the STAT3 gene has been identified as a
recurrent genetic abnormality in T-LGL. STAT3 mutation therefore represents a promising
marker in T-LGL diagnostics.
We therefore developed a new quantitative next generation sequencing (NGS) assay that
allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of
STAT3 mutation analysis as a diagnostic tool in T-LGL. Sixteen T-LGL patients were
included in the study.
A total of 15 mutations, including two new mutations (G618R and K658R), were detected
in 12 patients (75%) with mutation levels ranging from 2.5 – 45.6% mutation positive
alleles. NGS detected 50% more mutations than Sanger sequencing. Blood samples from
20 healthy blood donors all tested negative, thus demonstrating the specificity of the
assay. Also, the results indicated that mutation levels in blood and bone marrow are not
systematically different and that NGS-based STAT3 mutation analysis represents a
sensitive method for monitoring of residual disease as demonstrated in a patient receiving
pentostatin.
In conclusion, we demonstrate the clinical relevance of NGS-based STAT3 mutation
analysis which represents a sensitive and specific diagnostic marker in T-LGL that allows
assessment of molecular residual disease which may improve clinical decision making.
17
A common Nordic/Baltic treatment strategy for children and
adults (1- 45 years) with acute lymphoblastic leukaemia
Kjeld Schmiegelow, Department of Paediatrics & Adolescent Medicine, Rigshospitalet Copenhagen, kschmiegelow@rh.dk
Historically, adolescents and adults with acute lymphoblastic leukaemia have had a
significantly poor cure rate than young children with ALL. For both children and adults
treatment has primarily been based on clinical characteristics at diagnosis (age, tumour
burden, immune phenotype, cytogenetics) and time to obtained morphological remission.
With intensive chemotherapy, prophylactic and therapeutic cranial irradiation for high-risk
patients, and haematopoietic stem cell transplantation for the highest risk group, the eventfree survival obtained has been 80 % for children and approximately 40 % for patients 1845 years of age. Since July 2008 a common treatment protocol has been used for all
children with ALL (1-18 years) in Denmark, Norway, Sweden, Iceland, Finland, Estonia,
and Lithuania, and an increasing number of adult departments of hematology have joined
this strategy, which now includes all adults of 18-45 years in Denmark, Norway, Sweden,
Estonia, Lithuania, and Helsinki. Risk grouping (i.e. treatment intensity) is based on
cytogenetics and minimal residual disease in bone marrow after one and three months of
therapy. No patients receive cranial irradiation during first remission, and transplantation is
only offered to patients not being in remission at the end of induction therapy or who has a
tumour burden above 0,1 % in bone marrow after three months of therapy. With more than
1300 patients included (>200 above 18 years), the five year event-free survival for children
(1-18 years) is now 85 % and the event-free survival for adults is identical to that of
children, if survival analyses are stratified by risk group. Thus, the overall event-free
survival for adults is only slightly lower than that for children due to a higher frequency of
T-cell ALL, MLL rearrangements, and poorer MRD-response at end of induction therapy.
In addition to significant improvements in cure rates, the common Nordic/Baltic treatment
strategy is a unique platform for future research.
18