Abstracts til frie foredrag lørdag d 14/3 i forbindelse med DHS
årsmøde 2015
Indholdsfortegnelse
Session 1, kl. 09.00-10.15
Marie Toft-Petersen
Fazila Asmar
Lene Jepsen
Derya Aslan
Helene Nielsen
side 2
side 4
side 5
side 6
side 7
Session 2, kl. 10.45-12.00
Sara Farmer
Fie Juhl Vojdeman
Tarec Christoffer El-Galaly
Jens Helby Petersen
Christian Brieghel
side 8
side 9
side 10
side 11
side 12
1
Session 1, kl. 09.00-10.15
Marie Toft-Petersen
CD34+CD38-hMICL+ cells from MDS patients are malignant and possess stem cell
properties when evaluated in the long-term colony initiating cell assayMarie ToftPetersen1, Line Nederby1, Eigil Kjeldsen1, Gitte Birk Kerndrup2, Peter Hokland1 and
Anne Stidsholt Roug1
1 Department of Hematology, Aarhus University Hospital, Denmark 2 Department of
Pathology, Aarhus University Hospital, Denmark
Introduction
The human Myeloid Inhibitory C-type lectin-like receptor (hMICL) has been proposed as a
marker of the leukemic stem cell (LSC) as it is present on CD34+CD38- cells in the
majority of AML samples, while absent on the CD34+CD38- compartment in normal bone
marrow. We have studied the expression of hMICL on the CD34+CD38- stem cell
compartment from 19 untreated MDS patients and found it aberrantly expressed in 84%
(16/19) of cases. In consistency with previous reports, hMICL was not found on
CD34+CD38- cells in any of the 11 normal control bone marrows examined (0.0%).
Patients with large fractions of hMICL positive CD34+CD38- cells showed a significantly
shorter AML-free survival (ASH14 abstract #3235).
To functionally assess the stem cell potential of the CD34+CD38-hMICL+/- subsets we
examined 4 selected MDS patients with known cytogenetic aberrancies and 2 normal
controls in long-term colony initiating cell assays (LTC-IC).
Materials and methods
FACS-sorted CD34+CD38-hMICL+/- cells were co-cultured on irradiated M2-10B4 stromal
feeder cells with weekly half-change of medium. After 6 weeks, cells were transferred to
MethoCult (StemCell Technologies) for an additional 2 weeks. Colony forming cells
(CFCs) were scored, individually picked and cytospin-slides were analyzed by FISH.
Results and perspectives
The majority of the CFCs from MDS patients (both hMICL+/- subsets) were smaller than
CFCs from normal controls and were almost exclusively of non-erythroid origin. In all
samples analyzed by FISH, CFCs from hMICL+ subsets were 100% malignant.
Interestingly, for MDS patient no. 4, hMICL-negative CFCs were a mixture of small
colonies and morphologically normal CFCs, which was in accordance with the FISHresults (Table 1).
In conclusion, we showed that CD34+C38-hMICL+ cells possess functional stem cell
properties when evaluated in LTC-IC. Aberrant expression of hMICL on CD34+CD38stem cells from MDS patients might serve as a potential marker of LSCs in this
heterogeneous group of diseases.
2
Table 1. Results from LTC-IC and subsequent FISH analyses
Karyotype
Sorted subsets
Seeded in LTCIC
19
MDS 1
CD34+CD38-hMICL+
48,XY,+1,del(1)(p11),+8
CD34+CD38-hMICLCD34+CD38-hMICL+
MDS 2
MDS 3
MDS 4
500
123
46,XY ,del(5q)
Complex karyotype with del(7q) +
del(5q)
CD34+CD38-hMICL-
500
CD34+CD38-hMICL+
500
CD34+CD38-hMICL-
500
CD34+CD38-hMICL+
146
CD34+CD38-hMICL-
500
47, XY, +8
* Scattered cells ** FISH-negative CFCs were morphologically normal
3
Fazila Asmar
MSH3 loss-of-function mutations in familial hematological cancer
1
1
1
Fazila Asmar , Lasse Sommer Kristensen , Sara Hansen , Morten Munk
1
2
3
3
Johansen ,Christian Bjørn Poulsen Guangliang Yin , Wenjun Cai , Mette Klarskov
4
1
Andersen , Kirsten Grønbæk .
1. Department of Hematology, Rigshospitalet,
Copenhagen, Denmark.
2. Department of Hematology Roskilde, Denmark.
3. BGI-Tech,
BGI-Shenzhen, Shenzhen, 518083, China.
4. Department of Clinical Genetics,
Rigshospitalet, Copenhagen, Denmark.
Introduction: Mutations in epigenetic regulators and splicing factors have been identified
across a wide spectrum of hematological cancers. In some cases, the mutations identified
in hematopoietic tumors are also present in the hematopoietic stem cells from the same
patients. Thus, a shared genetic predisposition may contribute to a range of hematopoietic
malignancies. To better understand the origin of hematological cancers, we are in the
process of analyzing epi-/genetic aberrations that may give rise to both lymphoid and
myeloid cancers, through the analysis of families and patients with cancers of both
lymphoid and myeloid lineages.
Results: In one of these families, three of the four siblings had developed different
hematological malignancies: acute myeloid leukemia (AML), chronic lymphocytic leukemia
(CLL), and chronic myeloproliferative syndrome (CMPD). After informed consent, these
family members were subjected to whole exome sequencing. The three siblings carried a
germline heterozygous splice site mutation of the DNA mismatch repair gene, MutS
homolog 3 (MSH3), likely to cause a truncation of the C-terminal domain that interacts with
MSH2. Both allelic variants are transcribed at mRNA level. We did not detect increased
microsatellite instability (MSI) based on evaluation of high resolution melting analysis for
two MSI markers (BAT25 and BAT26).
Discussion: Aberrations of MSH3 are rarely reported in hematological cancers. However,
loss of expression of MSH3 at the mRNA level is reported in some hematological
malignancies and MSH3 mutations have been detected in Sezary Syndrome (SS) patients.
Intriguingly, a recent study showed that MSH3 co-precipitated with oxidative derivatives of
methylated cytosine, suggesting that MSH3 may be involved in the process of active DNA
demethylation. Thus, we are in the process of investigating the role of MSH3 in active DNA
demethylation in hematological stem cells. We are currently also screening a large panel
of sporadic cases of hematological malignancies for MSH3 mutations.
4
Lene Jepsen
The experience of outpatient management by intensively treated acute leukemia
patients
Jepsen LØ1, Høybye MT2, Hansen DG3, Friis LS4
1Research Unit of Hematology, Odense University Hospital, Denmark 2Interdisciplinary
Research Unit, Elective Surgery Center, Silkeborg Regional Hospital, Denmark 3National
Research Center of Cancer Rehabilitation, Research Unit of General Practice, University
of Southern Denmark 4Department of Hematology, National University Hospital, Denmark
Outpatient management of intensively treated patients with acute leukemia (AL) is
performed in the Home Unit (HU) at the Department of Hematology, Odense University
Hospital. The knowledge about how this type of outpatient management is experienced by
patients and relatives is sparse. This qualitative study aims to explore how outpatient
management may offer an opportunity to meet well-known physical and psychosocial
challenges in AL treatment and what new challenges it may produce.
This ongoing study based on qualitative methods combines’ participant observation in the
HU, individual interviews three times with each patient (at the beginning in HU, ending HUmanagement and 6 months after the end of treatment) as well as focus group interviews
with their relatives. Initially, twenty-two patients will be interviewed in the HU followed by
interviews in their homes. The results will be analyzed in an everyday life relational
perspective.
Preliminary analysis of the first interviews with 22 patients suggests that the patients value
the possibility to stay at home instead of being in hospital in the observation period
between chemotherapy cycles. They value having significantly more time with their
families, even though special precautions must be adapted to counter the risk of infection.
Furthermore, the analysis suggests that time spent with fellow patients in similar
circumstances in the HU during follow-up visits every second day is an important and
highly valued part of their social life. They prioritize such interaction to individual physical
training during waiting time in the HU.
This study provides valuable insight into the experience of AL patients under outpatient
management and how this affects and shapes the everyday life of themselves and their
relatives. This may help us understand the need for rehabilitation that arises from patients
in the outpatient management setting and how future initiatives and interventions in
outpatient management may be implemented in everyday practice.
5
Derya Aslan
How do spliceosome mutations affect microRNA expression in myelodysplastic
syndrome?
Aslan D1, Treppendahl MB1, Sjø LD2, Nygaard MK3, Severinsen MT3, Grønbæk K1, and
Kristensen LS1
1Department of Hematology, Rigshospitalet, Copenhagen Denmark.
2Department of Pathology, Rigshospitalet, Copenhagen Denmark.
3Department of Hematology, Aalborg University Hospital, Aalborg, Denmark.
Introduction: Mutations in spliceosome genes are frequently observed in myelodysplastic
syndrome (MDS). However, data concerning the effects of spliceosome mutations on the
pathogenesis of MDS are still very limited. MicroRNAs (miRs) are short non-coding RNA
sequences, which regulate genes at the post-transcriptional level. miRs are important
regulators of cell proliferation, differentiation, and apoptosis, and have been implicated in
most human cancers. Some miRs are directly processed by the spliceosome and are,
therefore, likely to be deregulated in MDS patients harboring a spliceosome mutation. This
project aims to investigate if spliceosome mutations affect the expression of miRs, and to
establish their roles in MDS pathogenesis.
Methods: 48 MDS patients were included in the study (23 RARS and 25 RA). All patients
were screened for spliceosome mutations using high-resolution melting (HRM) and
denaturing gradient gel electrophoresis (DGGE). miR expression analyses have been
initiated using reverse transcription quantitative PCR. For this purpose a custom 96-well
plate was designed containing miRs selected after an extensive literature search. The
miRs showing the largest differences in expression among samples with and without
spliceosome mutations will be investigated further in cell lines. Genes that are posttranscriptionally regulated by the deregulated miRs will be identified and their potential
roles in the pathogenesis of MDS will be investigated.
Results: All RARS patients had a spliceosome mutation, while this was only the case for
58% of the RA patients. All mutations were mutually exclusive. Overall, 35% of patients
had a mutation in SF3B1, 14.5% in SRSF2, and 8.3% in U2AF35. All patients were
screened for mutations using both HRM and DGGE, which gave 100% concordant results.
We are currently processing miR expression data, which will be presented at the meeting.
Perspectives: Deregulated miRs may be drug targets and/or have potential as prognostic
biomarkers in MDS.
6
Helene Myrtue Nielsen
DNA methylation profiling of sorted cells from myelofibrosis patients reveals
aberrant epigenetic regulation of immune pathways and identifies early MPN driver
genes
1,2
1,3
1
1
Helene M. Nielsen , Christen L. Andersen , Lasse S. Kristensen , Fazila Asmar ,
4
4
5
3,4
Torben A. Kruse , Mads Thomassen , Thomas S. Larsen , Vibe Skov , Caroline
6
2
1
3
7
Riley , Lise Lotte Hansen , Ole W. Bjerrum , Hans Hasselbalch , Vasu Punj , and
1
Kirsten Grønbæk
1Dept. of Hematology, Rigshospitalet, Denmark, 2Institut of Biomedicine, Aarhus
University, Denmark, 3Dept. of Hematology, Roskilde Hospital, Denmark, 4Dept. of
Clinical Genetics, Odense University Hospital, Denmark, 5Dept. of Hematology, Odense
University Hospital, Denmark, 6Dept. of Hematology, Herlev Hospital, Denmark, 7USC
Epigenome Center, California, USA
Myelofibrosis (MF) belongs to the heterogeneous group of myeloproliferative neoplasms
(MPN) together with essential thrombocytosis (ET) and polycythemia vera (PV). We
performed DNA methylation profiling of sorted cells from MF patients to unravel pathways
contributing to disease phenotype. As an aberrant DNA methylation pattern may be an
early event in tumorigenesis and may be crucial for progression of the malignant clone
from ET over PV to MF, we further aimed to identify methylated candidate driver genes.
Cells were sorted in CD34+ cells, granulocytes and mononuclear cells from 16 MF patients
and three healthy age-matched controls, and analysed for differential methylated regions
using the Infinium HumanMethylation 450K BeadChip. Candidate genes were validated by
pyrosequencing in a second cohort (n=30). To identify potential driver genes the DNA
methylation status of candidate genes were likewise analysed in 60 ET/PV patients. In MF
CD34+ cells 1628 CpG sites were differential methylated compared to 519 and 213
differential methylated CpG sites observed in MF granulocytes and MF mononuclear cells,
respectively (Δβ = 0.2, adjusted p-value < 0.05, T-test). Differentially methylated genes
were mainly involved in cancer and embryogenic pathways together with inflammatory and
immunological diseases. The DNA methylation level of the promoter region of TRIM59,
ZNF577, and LEP could differentiate between the three disease entities ET, PV, and MF
and controls. In conclusion genome-wide DNA methylation profiling of sorted MF blood
cells provided an insight into which pathways that contribute to MF pathogenesis and our
data suggests that it may be possible to select a panel of genes that can discriminate early
MPN from benign, reactive myeloproliferation.
7
Session 2, kl. 10.45-12.00
Sara Farmer
Knoglemineral indhold hos patienter med ET og PV
Sarah Farmer og Henrik Frederiksen
Hæmatologisk afdeling, Odense Universitets Hospital
Baggrund
En række systemiske inflammatoriske sygdomme associeres med øget forekomst af
osteoporose og frakturer på baggrund af nedsat knoglemineral indhold (BMD).
Inflammation er efterhånden ved at være accepteret som en del af patofysiologien hos de
kronisk myeloproliferative neoplasier (CMPN). Vi har tidligere vist, at patienter med
essentiel trombocytose (ET) og polycytæmia vera (PV) har øget risiko for frakturer
sammenlignet med baggrundsbefolkningen. Årsagen hertil er ikke kendt eller beskrevet i
kliniske studier.
Formål
At evaluere knoglemineralindholdet hos CMPN patienter i en klinisk
tværsnitsundersøgelse.
Metoder
Patienter med diagnoserne ET og PV fra hæmatologisk afdeling, OUH blev inkluderet.
BMD blev målt med duel energy X-ray absorption (DXA) i hofte og columna. Resultaterne
blev sammenlignet med raske personer fra en dansk knogle database matchet på alder,
køn, højde og vægt 1:1 ratio.
Resultater
47 patienter blev inkluderet. Gennemsnitsalderen var 56,2. Resultaterne af BMD målt i
hofte og columna er vist i figuren, og det fremgår at resultaterne er sammenlignelige.
Konklusion
Dette studie har målt BMD hos CMPN patienter og sammenlignet resultaterne med raske
kontroller. Dette studie kan derfor ikke forklare den øgede risiko for frakturer blandt CMPN
patienter. Resultaterne bør understøttes med longitudinelle målinger eller suppleres med
mere avancerede knogleskanninger eller knogleomsætningsmarkører.
Reference cohorte
1.4
CMPN cohort
.4
.6
.6
.8
.8
1
1
1.2
1.2
Reference cohort
aBMD hip
aBMD L1-L4
8
CMPN patients
Fie Juhl Vojdeman
HOVON68: LYMPHOPENIA IN THE FIRST YEAR AFTER TREATMENT IS RELATED
TO SHORTER OVERALL SURVIVAL IN CLL PATIENTS AGED 65+ WHO HAVE
SURVIVED MORE THAN 48 MONTHS.
Authors: Fie Juhl Vojdeman and Christian H. Geisler on behalf of the HOVON68 study
group.
Affiliations: Hæmatologisk klinik, Rigshospitalet, Denmark.
Background:
In the HOVON68 trial patients aged 65+ did not benefit from the addition of low-dose
alemtuzumab to fludarabine and cyclophosphamide (FCA) and significantly more
opportunistic infections were observed in the alemtuzumab treated patients (Geisler et al.
Blood 2014). We hypothesize that lymphopenia is predictive of survival (OS) and that late
excess mortality in the 65+ patients is related to opportunistic infections.
Methods:
Fischer’s exact and Chi squared test for categorical data and survival analyses with log
rank test were performed in SAS statistical software 9.3 and Graph Pad Prism 5.0.
Results:
There were 69 patients aged 65+ in the HOVON68 study with 25 patients still alive after 48
months. Six deaths only in FCA patients of the 25 patients were registered and the causes
of death were progression, infection, or other, including cardiovascular death.
Lymphocyte counts in the first year after treatment were available for 50 of 69 and 23 of 25
patients. Six out of 6 who died with OS>48 months experienced lymphopenia in the first
year after treatment (lymphopenia FU1) as opposed to only 7 of 17 of the ones still alive
(p=0.019). In survival analysis, patients with lymphopenia FU1 had a tendency of shorter
OS (log rank p=0.0681) irrespective of treatment arm (figure 1).
Patients still alive after 48 months experienced more opportunistic infections in the FCA
arm compared to the FC arm (0/13 vs. 4/12, p=0.04) and a significantly higher number of
deaths due to opportunistic infection (p=0.03) with only a trend for shorter survival in the
FCA arm (p=0.059).
Conclusion:
Lymphopenia in the first year after treatment in the HOVON68 protocol is associated with
a late excess mortality in elderly patients who have survived more than 48 months
irrespective of treatment arm and opportunistic infections are related to the excess
mortality in the elderly.
9
Tarec Christoffer El-Galaly
Surveillance imaging for diffuse large B-Cell lymphoma in complete remission is not
associated with better survival – A Danish-Swedish observational study
Tarec Christoffer El-Galaly,1 Lasse Hjort Jakobsen,1,2 Martin Hutchings,3 Peter de Nully
Brown,3 Herman Nilsson-Ehle,4 Elisabeth Székely,5 Viktoria Hjalmar,6 Karen Juul Mylam,7
Hans Erik Johnsen,1 Martin Bøgsted,1,8 and Mats Jerkeman5
1Department of Hematology, Aalborg University Hospital; 2Department of Mathematical
Science, Aalborg University; 3Department of Hematology, Rigshospitalet; 4Department of
Hematology, Sahlgrenska University Hospital;5Lund University Hospital; 6Karolinska
Institutet, Stockholm; 7Department of Hematology, Odense University Hospital; 8 Unit of
Clinical Biostatistics, Aalborg University
Background: Routine surveillance imaging plays a limited role in detecting recurrent
diffuse large B-cell lymphoma (DLBCL) and beneficial effects have never been shown in
properly designed studies.
Methods: Patients enrolled in the Danish (LYFO) and Swedish population based
lymphoma registries were included by following criteria: a) newly diagnosed DLBCL in the
period 2007-2012, b) age 18-65 years, and c) CR after 1st line treatment with RCHOP/CHOEP. Follow-up (FU) for Swedish patients includes symptom assessment,
clinical examinations, and blood tests with three months intervals for two years, and with
longer intervals later in follow-up. Imaging is only performed in response to suspected
relapse. FU for Danish patients is equivalent, but includes additional routine surveillance
imaging.
Results: A total of 525 Danish and 696 Swedish patients were included. Danish and
Swedish patients had similar male:female ratio, median age, and proportion of IPI high risk
disease (IPI>2). The 3-yr OS for the entire patient cohort was 92% (95%CI 90-93) with no
difference between Danish and Swedish patients (P=0.5, log-rank). Age >60 years (HR
2.3), elevated LDH (HR 2.3), B-symptoms (HR 1.6) and ECOG performance ≥2 (HR 1.8)
were associated with inferior post-remission OS. An imaging-based national FU strategy
had no impact on the post-remission OS for patients grouped according to the IPI scores
(P=0.2 for IPI≤2 and P=0.8 for IPI>2). The cumulative two-year progression rate was 6%
(95%CI 4-9) for patients with IPI≤2 vs. 21% (95%CI 13-28) for patients with IPI>2.
Conclusions: The vast majority of young DLBCL patients in CR stay in remission, and the
post-remission survival of Danish and Swedish DLBCL patients was identical despite the
use of surveillance imaging in Denmark, favoring a non-imaging based FU strategy for
DLBCL in 1st CR.
10
Jens Helby Petersen
High IgE levels are associated with low risk of chronic lymphocytic leukemia: a
prospective study of 37 747 individuals from the general population
Jens Helby Petersen.
Department of Clinical Biochemistry, Herlev Hospital, Copenhagen.
Background
Immunoglobulin E (IgE) is produced by plasma cells, often as part of an allergic immune
response. It is currently unknown whether plasma IgE levels are associated with risk of
cancer in individuals from the general population. We tested the hypothesis that high
levels of plasma total IgE are associated with overall risk of cancer and with risk of specific
cancers.
Materials and methods
Plasma total IgE was measured in 37 747 individuals from the general population, and the
participants were followed prospectively for up to 30 years. All statistical tests were twosided.
Results
During a mean follow-up of 7 years, a first cancer was diagnosed in 3454 participants. The
multivariable adjusted hazard ratio for a 10-fold higher level of IgE was 1.05 (95%
confidence interval [CI] 1.00 to 1.11;P=0.04) for any cancer, 0.44 (0.30 to 0.64;P=0.00002)
for chronic lymphocytic leukemia, 0.53 (0.33 to 0.84;P=0.007) for multiple myeloma, 1.54
(1.04 to 2.29;P=0.03) for non-Hodgkin lymphoma, 1.38 (1.04 to 1.84;P=0.03) for cancer of
the oral cavity and pharynx, and 1.12 (1.00 to 1.25;P=0.05) for lung cancer. The findings
for chronic lymphocytic leukemia and multiple myeloma were generally robust; however,
after correcting for 27 multiple comparisons only the finding for chronic lymphocytic
leukemia remained significant. The risk estimate for chronic lymphocytic leukemia
remained stable even when restricting the analysis to only include cases diagnosed more
than 10 years after blood sampling.
Conclusion
High levels of plasma total IgE were associated with low risk of chronic lymphocytic
leukemia and possibly of multiple myeloma, without convincing evidence for high risk of
any cancer type.
11
Christian Brieghel
Novel 31.2 kilobase 0 deletion in a Palestinian family with -thalassemia.
Christian Brieghel1, Henrik Birgens1, Henrik Frederiksen2, Jens M. Hertz3, Maria Steenhof3
and Jesper Petersen1
1Center
for Haemoglobinopathies, Department of Haematology, Copenhagen University
Hospital, Herlev.
2Department of Haematology, Odense University Hospital, Odense.
3Department of Clinical Genetics, Odense University Hospital, Odense.
Abstract
-Thalassemia is a common monogenic hereditary disorder mainly due to deletions in the
 gene cluster in which one (-/) or two (--/)  genes are lost. Such deletions are
designated + or 0, respectively. Clinically, the course of disease associated with + deletions or one 0 deletion is very mild and elusive,
while deletional HbH disease with compound heterozygosity for an 0 and an + deletion (-/-) causes more moderate microcytic hemolytic anemia and gives rise to the formation of
-globin tetramer. Homozygous 0 thalassemia (--/--) is incompatible with life or will lead to
intrauterine or neonatal death (HbBart’s Hydrops Foetalis) depending on whether the zeta
gene (HBZ) is involved or not.
We have located a previously unknown 0 deletion designated --DANE found in 3
generations of a Danish family of Palestinian origin. Six patients were heterozygous and 3
patients had deletional HbH disease due to compound heterozygosity with the common 3.7 deletion. Multiplex ligation-dependent probe amplification (MLPA) supplemented by
repeated polymerase chain reaction amplifications identified the 5’ and 3’ breakpoint in the
 globin gene cluster. This novel 31.2 kilobase deletion
(NG_000006.1:g.8800_40007del31208) leads to removal of the HBZ, HBA2 and HBA1
genes.
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