1. health and fitness
2. disease
3. cancer

Abstrakts til frie foredrag lørdag d 16/3 i forbindelse
med DHS årsmøde 2013
Indholdsfortegnelse
Session 1 09.00-10.30 ................................................................................................................................... 2
Camilla Nielsen.......................................................................................................................................... 2
Mads Emil Bjørn ........................................................................................................................................ 4
Nana Brochmann ....................................................................................................................................... 6
Cecilie Utke Rank ...................................................................................................................................... 8
Lene Sofie Granfeldt Østgård ................................................................................................................... 10
Anne Mette Larsen ................................................................................................................................... 12
Sarah Farmer ............................................................................................................................................ 15
Sara Bohnstedt Hansen............................................................................................................................. 17
Session 2 11.00-12.20 .................................................................................................................................. 19
Anders Møller .......................................................................................................................................... 19
Kristian Thidemann Andersen .................................................................................................................. 21
Ditte Reker ............................................................................................................................................... 23
Simon Husby ........................................................................................................................................... 25
Karen Juul Mylam .................................................................................................................................... 27
Sif Gudbrandsdottir .................................................................................................................................. 29
Peter Brændstrup ...................................................................................................................................... 31
Bo Kok Mortensen ................................................................................................................................... 32
1
Session 1 09.00-10.30
Camilla Nielsen
Rs10974944 germline polymorphism, JAK2 V617F somatic mutation, haematological
parameters, and morbidity in 49,488 individuals from the general population
Camilla Nielsen,1,3,4 Henrik S. Birgens,2,4 Børge G. Nordestgaard,1,3,4 and Stig E.
Bojesen1,3,4
1
Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital; 2Department of
3
Haematology, Herlev Hospital, Copenhagen University Hospital; The Copenhagen General Study
4
Population, Herlev Hospital, Copenhagen University Hospital; and Faculty of Health Sciences, University of
Copenhagen, Denmark.
Correspondence: Stig E. Bojesen, Department of Clinical Biochemistry, 54M1, Herlev
Hospital, Copenhagen University Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark.
Phone: +45 3868 3843. Fax: +45 3868 3311. E-mail: [email protected].
Abstract
The rs10974944 germline polymorphism might predispose to the development of the
JAK2 V617F mutation. The JAK2 V617F is a somatic mutation resulting in autonomous
hyperproliferation of myeloid cells and present in patients with myeloproliferative cancer
(polycythaemia vera, essential thrombocytosis, and primary myelofibrosis). The
association of the rs10974944 germline polymorphism and the JAK2 somatic mutation with
haematological parameters and morbidity in the general population is however unknown.
In 49,488 individuals from the Danish general population (The Copenhagen General
Population Study), followed for up to 6 years, we examined the association between
rs10974944 genotype, JAK2 mutation status, and haematological phenotype.
Furthermore, we measured risk of any cancer, haematological cancer, myeloproliferative
cancer, ischemic heart disease, and venous thromboembolism as a function of
rs10974944 genotype as well as JAK2 mutation status. Finally, we evaluated the
2
diagnostic value of the test for the JAK2 V617F somatic mutation with respect to
myeloproliferative cancer in relevant subgroups of the general population.
The JAK2 V617F somatic mutation was present in 0.1% (n=68), increasing across
rs10974944 genotypes. JAK2 somatic mutation positives vs. negatives had higher
erythrocyte (p=2.3*10-5), leukocyte (p=3.5*10-9), and thrombocyte (p=1.8*10-16) counts,
and they had 2.5, 28, 97, and 3-fold risks of incident cancer, incident haematological
cancer, incident myeloproliferative cancer, and prevalent venous thromboembolism,
respectively. In combination with erythrocyte volume fraction >50%, the test for the JAK2
mutation status detected myeloproliferative cancer with 100% sensitivity and 100%
specificity. These results suggest a sequential model for myeloproliferative cancer where
the rs10974944 genotype predisposes to development of the JAK2 V617F somatic
mutation resulting in hypercellularity of all three myeloid lineages, but only in clinical
disease in a subset of these individuals. Finally, the combination of conventional
haematological parameters and test for the JAK2 V617F somatic mutation, detects
myeloproliferative cancer.
3
Mads Emil Bjørn
Circulating YKL-40 in Myelofibrosis and Related Neoplasms
Authors : Mads Emil Bjørn, Morten Krogh Jensen, Christen Lykkegaard Julia Johansson,
Hans C. Hasselbalch.
Background : Circulating YKL-40 has been suggested as a novel biomarker of disease
activity in patients with various chronic inflammatory diseases and several types of
cancer. Furthermore,
YKL-40 has been reported to reflect cardiovascular disease burden in patients with
ischemic heart disease . Myelofibrosis is the advanced burnt-out stage of early stage
disease (essential thrombocythemia and polycythemia vera) within the spectrum of chronic
myeloproliferative neoplasms. Endothelial proliferation with neovascularisation is another
important bone marrow stroma alteration in myelofibrosis. These neoplasms are also
featured by a chronic inflammatory state being most pronounced in myelofibrosis. Being
featured by chronic inflammation and bone marrow angiogenesis we hypothezised that
circulating YKL-40 might reflect these disease processes and – accordingly – potentially
serve as a novel disease marker for myelofibrosis .
Patients : We included 15 ET-patients (6 male/9 female, average age 54,9), 17 PCVpatients (11 male/6 female, average age 61,9), 17 PMF-patients (9 male/8 female,
average age 61,1) and 30 healthy controls (15 male/15 female, average age 57,4).
Results : Levels of YKL-40 were significantly elevated in PMF vs. control subjects, PMF
levels median 43 [26,5 - 154,5] vs controls median 28 [16,5 - 49,75], p = 0,033 . The
same pattern was not seen in ET or PV. Circulating YKL-40 levels were increasing from
ET-patients through PV-patients with the highest levels being recorded in patients with
myelofibrosis.
Conclusions : Our findings of highly elevated YKL-40 in myelofibrosis and the steady
increase from ET over PV may reflect the integrated impact of disease processes in
MPNs, including clonal evolution, bone marrow fibrosis/angiogenesis, concurrent chronic
inflammation and accelerating atherosclerosis. Studies on the impact of anti-inflammatory
and antiproliferative treatment (interferon-alpha2, JAK-inhibition, HDACi and statins) upon
circulating YKL-40 and YKL-40 distribution in bone marrows from MPN-patients are being
planned.
4
5
Nana Brochmann
Livskvalitet hos patienter med kronisk myeloproliferativ neoplasi
Nana Brochmann1, Christen Lykkegaard Andersen1,2, Ann-Dorthe Olsen Zwisler3, Hans
Carl Hasselbalch1
1
Hæmatologisk afdeling Roskilde Sygehus, 2Hæmatologisk afdeling Rigshospitalet,
3
Kardiologisk afdeling Roskilde Sygehus
Introduktion
Patienter med kroniske myeloproliferative neoplasier (MPNs) er ofte tynget af
hypermetabole symptomer, komplikationer og varierende komorbiditetsbyrde.
Ingen internationale livskvalitetsundersøgelser har fulgt patienternes livskvalitet
systematisk fra diagnose og gennem sygdomsforløbet. I Danmark er ikke udført
livskvalitetsundersøgelse på patientgruppen.
En naturlig konsekvens af øget sygdomsindsigt, nye behandlingsmuligheder og forlænget
levetid er systematisk monitorering af patienternes livskvalitet, så patient - reported
outcomes kan indgå i den samlede vurdering af behandlingseffekt og bivirkningsprofil for
herved at kvalitetssikre behandling, forebyggelse og rehabilitering.
I hæmatologisk ambulatorium Roskilde Sygehus udføres aktuelt en prospektiv
undersøgelse af symptomer og helbredsrelateret livskvalitet.
Materiale
I perioden 1-4-2012 – 22-2-2013 er inkluderet 112 patienter fordelt på 24 patienter med ET
(alder 46-76 år), 38 med PV (47-76 år), 22 med MF (66-87 år) og 28 med CML (36-93 år).
9 patienter har takket nej til deltagelse.
I alt forventes ca.150 patienter inkluderet.
Metode
Spørgeskemaerne EORTC QLQ C30, MPN SAF og SF36 anvendes. Spørgsmålene
besvares hver måned vha. et nyudviklet internetbaseret system eller på papir efter
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patientønske. Sammenhæng mellem forskellige aspekter af livskvalitet - total
livskvalitetsscore og symptomer - og sygdomsvarighed, sygdomsaktivitet, komplikationer,
medicinsk behandling og bivirkninger undersøges. Systemet kræver ikke installation, og
det kan tilgås fra alle computerplatforme. Patienterne registreres til automatisk at få
tilsendt sms og/eller mail, når det er tid til at udfylde spørgeskemaer. Årsager til eventuel
nedsat livskvalitet f.eks. gennem et behandlingsforløb kan analyseres ved multivariat
analyse af variable i systemet (subjektive og objektive data). Systemet implementeres ifm.
undersøgelsen i hæmatologisk ambulatorium Roskilde Sygehus til fremtidigt brug som led
i vanlig behandling.
98 patienter har valgt at besvare spørgeskemaer via internettet og 14 patienter på papir.
Konklusion
I hæmatologisk ambulatorium Roskilde Sygehus gennemføres en undersøgelse af
livskvalitet blandt patienter med MPNs. Samtidig med undersøgelsen implementeres et nyt
internetbaseret system, som giver mulighed for besvarelse af livskvalitetsspørgeskemaer.
Således kan patient – reported outcomes indgå i den samlede vurdering – og
kvalitetssikring af behandling, forebyggelse og rehabilitering.
7
Cecilie Utke Rank
Low burden JAK2 V617F in Patients with Essential Thrombocythemia, Polycythemia
Vera, and Primary Myelofibrosis. Clinical and Biochemical Phenotype with Particular
Focus upon the Impact of Therapy.
Cecilie Utke Rank1, Thomas Stauffer Larsen2, Lasse Kjær3, Morten Krogh Jensen3, Karin
de Stricker4, Niels Pallisgaard5, Ole Weis Bjerrum6, Maj Westman7, Inge Helleberg8,
Mikael Frederiksen9, Mette Weidinger Nordmann10 Maja Irene Dam Andersen11, Torben
Mourits-Andersen12, Torben Plesner13 , Hans Carl Hasselbalch1
1 Department of Hematology, Roskilde Hospital, University of Copenhagen; 2 Department of Hematology X,
Odense University Hospital, University of Southern Denmark; 3 Department of Hematology, Herlev Hospital,
University of Copenhagen; 4 Department of Pathology, Odense Hospital, University of Southern Denmark; 5
Department of Clinical Biochemistry, Vejle Hospital, University of Southern Denmark; 6 Department of
Hematology L, Rigshospitalet, University of Copenhagen; 7 Department of Clinical Genetics, Rigshospitalet,
University of Copenhagen; 8 Department of Hematology, Aalborg University Hospital; 9 Department of
Hematology, Haderslev Hospital; 10 Department of Pathology, Næstved Hospital; 11 Department of
Hematology, Viborg Hospital, 12 Department of Hematology , Sydvestjysk Hospital, 13 Department of
Medicine, Hospital of Lillebælt.
Abstract
Background: The Ph-chromosome-negative chronic myeloproliferative neoplasms
(MPNs) ET etc. are clonal hematopoietic disorders arising due to an acquired genetic
defect in the pluripotent stem cell. The JAK2V617F-mutation is detected in more than 98%
of PV patients and in about 50% of patients with ET and PMF. The MPNs have
overlapping clinical features but exhibit different phenotypes, which may be determined by
the JAK2V617F-allele-burden together with physiological and genetic modifiers. Most
recently, chronic inflammation has been proposed to be the common denominator for
clonal evolution, premature atherosclerosis, thrombosis, and second cancer. Aims: 1. To
describe the clinical and biochemical phenotype of a large Danish cohort of 540 MPNpatients with low allele burden JAK2V617F≤10%, of which 235 patients presented lowburden at the time of diagnosis. 2. To describe the impact of therapeutic intervention with
IFN-alpha2. Results: 17/235 (7,2%) patients presented with thrombosis at the time of
8
diagnosis. Of these 6 had suffered previous thrombosis. In total 63/235 (26,8%) had
experienced thrombosis prior to diagnosis. The number of patients presenting with
thrombosis was 3 in patients receiving aspirin only, 3 in patients receiving a statin only,
and 2 in those patients receiving both aspirin and a statin. A proportion of the patients had
co-morbidities such as cardiovascular diseases, COLD, autoimmune and chronic
inflammatory disease, diabetes, and second cancer. A low-burden JAK2V617F was
achieved by treatment with IFN-alpha2 in 42/59 patients. In none IFN-alpha2 was
associated with a complete molecular remission. Conclusion: An analysis of the largest
reported cohort of low-burden JAK2V617F-positive MPN-patients has shown this cohort to
be associated with a diagnosis of ET, female dominance, a low MPN-disease-burden, low
rate of thrombosis, and a low co-morbidity-burden. Long-term IFN-alpha2 treatment was
associated with low-burden JAK2V617F in a large proportion of the patients. The cohort
constitutes a unique platform for prospective studies on the impact of IFN-alpha2 on
disease evolution, the JAK2V617F-allele-burden, thrombotic complications, and the
potential of IFN-alpha2 to induce minimal residual disease. A complete data-analysis will
be presented at the meeting.
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Lene Sofie Granfeldt Østgård
Validation of the Danish National Acute Leukemia Registry: A Hematological Data
Resource
Lene Sofie Granfeldt Østgård1,2, Jan Maxwell Nørgaard1, Marianne Tang Severinsen3,
Lone Friis4, Morten Krogh Jensen5, Ove Juul Nielsen6 og Mette Nørgaard2
1
2
Affiliations: Department of Hematology, Aarhus University Hospital , Department of Clinical Epidemiology ,
Aarhus University Hospital, Department of Hematology, Aalborg University Hospital3, Department of
Hematology, Odense University Hospital4, Department of Hematology, Herlev Hospital5, Department of
6
hematology, Rigshospitalet, Copenhagen
Abstract:
Background: Danish health care, disease, and population registries are generally known
for high completeness and high validity. The Danish National Acute Leukemia Registry
(DNLR) has a documented coverage above 98.5%. Less is known about the validity of the
recorded variables.
Objective: We describe the history, structure, present coverage, and data quality of the
DNLR in relation to acute myeloid leukemia (AML). Furthermore, we report the incidence
of this severe blood cancer in Danish patients and compare these with incidences based
on other AML registries.
Patients and methods: By the end of December 2011, the DNLR (established January
2000) included detailed data on a large, well-defined, non-selected population of 2665
AML patients. We investigated the validity of 30 central variables in 260 randomly selected
patients, corresponding to 10% of all patients registered in the DNLR. We used medical
records as gold standard.
Results: The completeness of the registry was 99.6% using Danish National Registry of
Patients (DNRP) as reference. The positive predictive values (PPVs) of the variables
ranged from 89.4% to 100%. The completeness of individual variables varied between
60.7% and 100%. One out of eight departments of hematology had delayed registration of
treatment data, and two departments of follow up data. When stratifying by time of
registration (before versus after Jan 2006) we found higher PPVs and a lower frequency of
missing data in the most recent period.
Conclusions With few exceptions, both PPVs and completeness of registered data were
high. The quality of data improved during the registration period. In conclusion, the DNLR
10
can be a valuable epidemiological tool for research in relation to the prognosis of AML,
though effort should be made to reduce the proportion of missing data.
11
Anne Mette Larsen
High Syndecan-1 Levels in Acute Myeloid Leukemia Are Associated with Bleeding,
Thrombocytopathy, Endothelial Disruption and Leukocytosis
Anne Mette Larsen1, Eva Birgitte Leinøe2, Pär I. Johansson3,4, Henrik Birgens1 and Sisse
R. Ostrowski3
1
Department of Hematology, Herlev University Hospital, Herlev, Denmark
2
Thrombosis and Hemostasis Unit, Department of Hematology, Copenhagen University Hospital,
Rigshospitalet, Copenhagen, Denmark
3
Section for Transfusion Medicine, Capital Region Blood Bank, Copenhagen University Hospital,
Rigshospitalet, Copenhagen, Denmark
4
Department of Surgery, Division of Acute Care Surgery, Center for Translational Injury Research, CeTIR,
University of Texas Medical School at Houston, TX, USA
Background: The risk of hemorrhage in acute myeloid leukemia (AML) is influenced by a
multitude of factors. In this study we investigated whether hemorrhage in AML patients is
associated with endothelial disruption, potentially caused by thrombocytopenia, platelet
dysfunction and leukocytosis. In general, dysfunction of the inner lining of the vessel wall,
the endothelium, contributes significantly to capillary leakage and bleeding and to the
pathophysiology of systemic critical illness like sepsis and trauma. Trauma patients with
excessive degradation of the endothelial glycocalyx, have increased coagulopathy,
bleeding and mortality. In AML patients, bleeding may also in part be attributed to direct
activation and dysfunction of the endothelium, since leukemic blast cells and
thrombocytopenia both activate or disrupts the endothelium.
Design and Methods: All study patients were participants in a previous prospective study
of functional platelet defects and hemorrhage. Patients were diagnosed with AML
according to the WHO-classification, and enrolled in the study at the time of diagnosis
before onset of treatment. In the present study, 49 patients with available serum or plasma
samples from the original cohort were included. We measured soluble markers of
glycocalyx degradation and endothelial cell activation and damage (syndecan-1, Inter
Cellular Adhesion Molecule-1 (sICAM-1), sE-selectin, thrombomodulin (sTM)), natural
12
anticoagulation (protein C), platelet activation (sCD40L) and cell turn-over (histone
complexed DNA (hcDNA)) along with previously collected data on bleeding status and
platelet activation markers (CD62P, CD63, PAC-1) before and after thrombin receptor
agonist peptide (TRAP) stimulation. Hemorrhage was evaluated according to the Common
Toxicity Criteria on a 0-4 scale and performed by the same physician in 47 patients.
Results: Median age was 67 years (IQR: 51-74) and 41% (n=20) were female. Twenty
three patients (47%) had no bleeding (grade 0), whereas 22 (45%) and 4 (8%) patients
experienced bleeding according to grade 1 and 2 classification respectively. No patients
suffered from grade 3 or 4 bleeding.
Patients with bleeding (grade 1 and 2) had, as expected, lower platelet count (median: 22
(IQR: 17-45) vs. 92 (38-153) mia/L) but also had higher circulating levels of syndecan-1
(46 (30-102) vs. 27 (21-57) ng/mL) and sICAM-1 (384 (293-553) vs. 264 (193-376) ng/mL)
indicating increased endothelial glycocalyx degradation and endothelial cell activation.
Also, bleeding patients had lower TRAP-stimulated CD62P and CD63 platelet expression,
indicating impaired platelet function. Leukocyte count (blast count) correlated positively
with syndecan-1, sE-selectin and sTM, suggesting that the degree of glycocalyx
degradation and endothelial cell activation and damage was related to the number of
circulating blasts.
Patients with endothelial glycocalyx degradation, evidenced by syndecan-1 level ≥ median,
had more bleeding (16 vs. 9 patients) and reduced TRAP-stimulated CD62P expression
despite comparable platelet counts. These patients were older (70 (63-78) vs. 58 (47-71)
years), displayed signs of profound endothelial cell activation and damage, evidenced by
higher levels of ICAM-1 (361 (300-502) vs. 255 (198-389) ng/mL) and sTM (4.1 (3.5-5.4)
vs. 1.8 (1.5-2.5) ng/mL), and had higher leukocyte count (25 (3-89) vs. 5 (2-18) mia/L) (all
p < 0.05).
Conclusions: Patients with glycocalyx degradation, indicated by high syndecan-1 levels,
had more bleeding and impaired platelet function, regardless of platelet counts.
Furthermore, patients with high levels of syndecan-1 were older, displayed signs of
profound endothelial activation and damage, and had higher leukocyte counts.
The bleeding tendency in AML patients is influenced by a multitude of factors. This is
supported by the results of the present study demonstrating associations between
bleeding, endothelial perturbation, platelet dysfunction and leukocytosis.
13
We suggest that platelet dysfunction and leukocytosis in AML causes endothelial
perturbation.
Studies are needed to evaluate the functional significance of endothelial biomarkers in
AML, so that future interventions designed to stabilize the endothelium and prevent
endothelial degradation, may reduce the risk of fatal hemorrhage in AML patients.
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Sarah Farmer
Chronic Myeloproliferative Neoplasms and Risk of Osteoporosis.
A Nationwide Population-based Cohort Study
Sarah Farmer, MD1, Erzsebet Horvath-Puho MSc 2, Hanne Vestergaard, MD, PhD1,
Henrik Toft Sørensen, MD, PhD2, Pernille Hermann, MD, PhD3, Henrik Frederiksen, MD,
PhD1,2
1. Department of Hematology, Odense University Hospital, Odense, Denmark
2. Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark
3. Department of Endocrinology, Odense University Hospital, Odense, Denmark
Background: Essential thrombocytemia (ET), polycythemia vera (PV), and chronic
myeloid
leukemia
(CML),
are
chronic
myeloproliferative
neoplasms
(CMPNs),
characterized by accelerated proliferation of hematopoietic tissue. Systemic mastocytosis,
also a CMPN and has been associated with increased risk of osteoporosis. However, to
our knowledge, no data is available on the risk of osteoporosis among patients with
classical CMPNs.
Method: We conducted a Danish population-based cohort study of the risk of osteoporosis
among patients with ET, PV, and CML using data from the Danish health care system.
ET, PV, and CML patients were identified from Danish National Registry of Patients
(DNRP), and linked to the Danish Civil Registration System (CRS) in the study period 1
January 1980 to 31 December 2010. Patients with a first-ever CMPN diagnosis in the
DNRP were identified by means of their ICD-8 diagnosis code until 1994 and ICD-10
diagnosis code thereafter. We established three distinct cohorts of ET, PV, and CML
patients. For each CMPN patient, 50 general population comparison cohort members
without CMPN were identified in the CRS matched on age, sex, and calendar year,
creating three comparison cohorts. Follow-up started 1-year from the date of diagnosis for
CMPN patients. The comparison cohort members were assigned the same index date as
their index CMPN case.
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A diagnosis of proximal femoral fracture was used an indicator of osteoporosis, since this
fracture type invariably leads to hospitalization, and therefore would be registered in the
DNRP throughout our observation period. The CMPN and comparison cohorts were
followed until a diagnosis of femoral fracture, emigration, death, or 31 December 2010,
whichever came first. Patients and comparison cohort members with a previous diagnosis
of osteoporosis or osteoporotic fractures were excluded.
The Kaplan-Meier method was used to estimate the cumulative rate of fractures. Cox
regression was used to estimate hazard ratios (HRs) as a measure of relative risk of
femoral fracture for each CMPN cohort compared to the comparison cohort, adjusted for
comorbidity.
Results: We identified 7,595 MPN patients (1,864 with ET; 4,418 with PV; and 1,313 with
CML) and 338,974 comparison cohort members. Fracture rates are shown in table 1.
The cumulative rate of proximal femoral fractures was higher among CMPN patients than
among comparison cohort members, as depicted in the figure.
CMPN cohort: prox.
femur fract. rate
(per 1000PYRs)
Parameter (95% CI)
ET
6.6 (CI: 5.2 - 8.3)
PV
9.9 (CI: 8.8 - 11.0)
CML
8.2 (CI: 6.0 - 10.7)
Comparison cohort:
Prox. femur fract. rate
(per 1000PYRs)
(95% CI)
HR (95% CI)
5.3 (CI: 5.1 - 5.4)
1.19 (0.94 - 1.51)
6.2 (CI: 6.1 - 6.3)
1.82 (1.62 - 2.04)
4.4 (CI: 4.3 - 4.6)
2.67 (1.97 - 3.62)
Conclusion: CMPN patients are at higher risk of osteoporotic fractures than the general
population.
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Sara Bohnstedt Hansen
Findes der en fælles oprindelse for myeloid og lymfoid cancer?
Sara Bohnstedt Hansen et al. Epi-/Genom laboratoriet, Hæmatologisk klinik,
Rigshospitalet.
Indledning:
Den aktuelle klinisk-patologiske klassificering af hæmatologiske maligniteter er baseret på
den antagelse, at hæmatologiske kræftformer opstår ved klonal ekspansion af en bestemt
celle på et bestemt udviklingstrin i den normale differentiering af blodets celler.
Imidlertid er mutationer i bl.a. epigenetiske regulatorer (fx TET2) blevet identificeret på
tværs af de klassiske sygdomsenheder, og mutationer, som identificeres i tumorer med
moden fænotype, kan i nogle tilfælde også findes i de bloddannende stamceller hos de
samme patienter.
Vores hypotese er således, at nogle myeloide og lymfoide cancerformer opstår som følge
af den samme primære genetiske defekt.
Mål/ Metoder:
-
Et populationsbaseret epidemiologisk studie med henblik på incidensen af:
1) patienter med samtidig myeloid og lymfoid diagnose, start dato 0101-2000.
2) patienter med terapirelateret MDS/AML (tMDS/AML) efter kemoterapeutisk
behandling for malign lymfoid sygdom, start dato 0101-1990.
Hertil anvendes søgning i Patobank.
-
Indsamling af data på familier hvori medlemmer har hæmatologiske kræftformer af
både lymfoid og myeloid oprindelse.
Her har vi brug for hjælp fra vore gode DHS-kolleger!
-
Et laboratorie studie, hvor vi vil undersøge patientprøver fra såvel familierne som de
enkelte patienter med både myeloid og lymfoid cancer for mutationer ved exon/
eller whole genome sekventering (ES/WGS).
Resultater: (Indtil videre…)
-
1) 60 patienter med samtidig myeloid og lymfoid sygdom, heraf er 26 nulevende.
17
-
2) 109 patienter med tMDS/AML. Tiden fra lymfoid til myeloid diagnose var median
5,7 år (0,5- 23,3 år).
-
Sammen med vore kolleger fra Dansk Hæmatologisk Selskab, har vi indtil videre
identificeret 6 familier.
-
Vi har undersøgt en familie for mutationer i TET2, hvilket ikke fandtes, hvorfor vi går
videre med (ES/WGS).
Perspektiver:
Identifikationen af sådanne tilgrundliggende mutationer vil kunne være potentielle mål for
ny behandling, og vil kunne være af væsentlig betydning for anvendelsen af visse
behandlingsmodaliteter, så som autolog knoglemarvstransplantation.
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Session 2 11.00-12.20
Anders Møller
Diagnostisk og terapeutisk lumbalpunktur udført sikkert og effektivt med en tynd
stump nål
Anders Møller, Hvidovre Hospital, Anæstesiologisk afdeling & Rigshospitalet,
Hæmatologisk afdeling. Arash Afshari, Geneva Hospital, Department of Paediatric and
Neonatal Intensive Care & Rigshospitalet, Anæstesiologisk afdeling. Ole Weis Bjerrum,
Rigshospitalet, Hæmatologisk afdeling
Introduktion
Postdurapunkturhovedpine (PDPH) er en bivirkning til lumbalpunktur (LP). Risiko herfor
reduceres ved brug af en tynd eller stump kanylespids [1], men studier tyder på at brug af
stumpe nåle ikke er implementeret uden for det anæstesiologiske speciale [2]. Formålet
med undersøgelsen var at beskrive praksis blandt danske hæmatologer, samt at måle
tappetiden af spinalvæske med en tynd stump kanyle, anvendt til spinal anæstesi, for at
vise om LP hermed også er anvendelig i diagnostisk øjemed.
Materiale og metode
1. del: Et elektronisk spørgeskema om udførelse af LP blev sendt til alle hæmatologiske
afdelinger i Danmark. Link: https://survey.mamut.com/s?s=25876.
2. del: Tappetiden af LP udført med en stump 27 gauge (0,4mm) kanyle måltes på
patienter i siddende stilling. Seks glas á 1mL spinalvæske blev udtaget per LP.
Resultater
1.del: Elleve afdelinger deltog. Kun tre
afdelinger (27%) angav at anvende stumpe
nåle rutinemæssigt. Fem afdelinger (45%)
bruger nåle af stor kaliber, 18-20 gauge (1,20,9mm), mens de resterende seks (55%)
19
Figur 1. Anvendt nålestørrelse og nåletype til
lumbalpunktur (n=11), gauge (G)
bruger nåle af mindre kaliber, ≥22 gauge (≤0,7mm), figur 1.
Foruden læger, angav tre (27%) at have lægestuderende ansat til at udføre LP. På tre
afdelinger (27%) anvendes lokalbedø-velse rutinemæssigt. Otte afdelinger (73%)
anvender rygleje rutinemæssigt efter LP, median varighed 60 min (interval 30-120 min).
På alle afdelinger gennemføres ryg-leje efter intrathekal kemoterapi, median varighed 60
min (interval 30-120 min). På fem afdelinger (45%) opsamles fem prøveglas, tre (27%)
opsamler to glas. På de resterende tre afdelinger tages hhv. et, tre og fire glas fra.
2. del: tappetiden for LP blev målt på 64 patienter på én afdeling. Den mediane tappetid
var 11 min 59 sek med 27 gauge nål (sv.t. 30mL/time) Fyldning af 1-5 prøveglas med 1
mL spinalvæske vil således forventes at vare 2-10minutter.
Diskussion
Udførelse af LP varierer indenfor specialet, både mht lejring, nåletype og sengeleje. Det er
ikke et mål i sig selv at have ensrettede procedurer, men generelt kan undlades sengeleje
efter diagnostisk LP og der bør med aktuelle evidens anbefales brug af stumpe nåle for at
mindske risiko for PDPH. Anvendelse af stumpe nåle er for nylig vist at være forbundet
med en besparelse på $26 per patient, når både omkostninger ved procedure og behandling af PDPH tages i betragtning [3]. Det forudsætter en oplæring af personale. Tappetiden
ved opsamling af prøver kan være relativt lang. Antal prøveglas er på nogle afdelinger
derfor reduceret rutinemæssigt.
1.
Arendt K, Demaerschalk BM, Wingerchuk DM et al. Atraumatic lumbar puncture needles: after all
these years, are we still missing the point? Neurologist 2009;15:17-20.
2.
Stendell L, Fomsgaard JS, Olsen KS. There is room for improvement in the prevention and
treatment of headache after lumbar puncture. Dan Med J 2012;59:A4483.
3.
Tung CE, So YT, Lansberg MG. Cost comparison between the atraumatic and cutting lumbar
puncture needles. Neurology 2012;78:109-13.
20
Kristian Thidemann Andersen
MMY2083 (ACVDL): An Investigator Initiated Phase 2 Trial for First-Line Treatment
of Previously Untreated Patients with Multiple Myeloma.
Kristian Thidemann Andersen (1), Maja Hinge(1), Paw Holdgaard (2), Tina Jensen (3), Henrik Jørgensen (2),
Gitte Kerndrup (4), Thomas Lund (1), Holger Møller (5), Niels Pallisgaard (6), Erik Segel (7), Lone
Østergaard (3) and Torben Plesner (1).
Departments of Hematology (1), Nuclear Medicine (2), Radiology (3), Biochemistry (6) and Pathology (4),
Vejle Hospital, and Departments of Hematology (7) and Biochemistry (5), Aarhus University Hospital.
Aim: To improve the quality of response and obtain healing of osteolytic lesions in
previously untreated patients with Multiple Myeloma (MM).
Method: Previously untreated patients with MM were treated with Adriamycin,
Cyclophosphamide, Bortezomib, Dexamethasone and Lenalidomide in a CHOP-like 21days schedule with G-CSF support. Patients eligible for HDT received 4 courses of
ACVDL followed by HDT. Patients ineligible for HDT received 8 courses of ACVDL.
Response was assed after 4 courses of ACVDL (interim) and at the end of induction
therapy (EOT). Patients that are not in molecular CR at EOT will be offered consolidation
therapy with sc Bortezomib once weekly for five 5-weeks cycles (4 weeks on, 1 week off
therapy). The first patient was enrolled in November 2011, and the inclusion period for a
total of 35 patients is expected to be 2½ years. The duration of the induction treatment is
approximately 28-30 weeks followed by 4 years of follow-up. Patients eligible for
consolidation therapy will receive 25 weeks of treatment during the follow-up period.
Response assessment is by IMWG criteria supplemented with Heavy-Lite® chain
measurements, PCR using patient-specific primers and PET-CT scans. Osteolytic bone
lesions are examined by consecutive conventional X-ray of the skeleton, CT-scan of the
axial skeleton, DEXA-scan and SPECT-CT scan supplemented with biochemical markers
of bone turn-over (serum CTX, bone-specific alkaline phosphatise, and P1NP).
Neurotoxicity and QoL questionnaires are included in the study
Results: 20 patients have been screened and 18 out of the target population of 35
patients have been enrolled in the study, 14 in the HDT group, 4 in the non-HDT group. 14
patients have passed interim assessment and 11 patients have had EOT assessment.
21
Two patients have been withdrawn from the study. One due to lack of response (SD by
IMWG, PD by PET-CT) and one due to severe depression caused by the sudden death of
his spouse. The response according to IMWG at interim assessment was 4 SD, 5 PR, 1
VGPR, 1 CR and 3 sCR. At EOT the responses were 6 PR, 1 VGPR and 4 sCR.
13 patients had pathological bone lesions by PET-CT at the time of screening, 5 were
PET-CT negative.
Out of the 13 PET positive patients eight could be evaluated for “PET-response” at this
time point. Five became PET negative (3 PR and 2 sCR by IMWG), one had attenuated
PET signal (PR by IMWG), one was unchanged (PR by IMWG), and one had progression
(non-secretory MM).
22 SAEs have been reported including 5 considered related to study medication. There
have been no SUSARs and no treatment-related deaths All of the patients enrolled in the
study are alive.
Interpretation: The skewed balance of patients enrolled in this study with a
preponderance of younger patients eligible for HDT reflects competition from a
simultaneous study, the NMSG18 study of MPT versus MPR, which has now been
concluded. In the future we expect to recruit more elderly patients that will be treated with
appropriate dose-modifications according to the protocol. These recommendations are
based on decades of experience with CHOP for elderly patients with high-grade NHL.
PET-CT may contribute with important information about the quality of remissions. The
study is progressing in accordance with the planned schedule.
22
Ditte Reker
MicroRNA Profiling Predicts Survival and Identifies a Novel Putative Onco-miR in
Diffuse Large B-Cell Lymphoma Treated with Immunochemotherapy
Ditte Reker, MSc1, Christoffer Hother, MD1, Konstantions Dimopoulos, MD1, Steen
Knudsen, PhD2, Thomas Jensen2, Michael B. Møller, MD, DMSc3, Peter De Nully Brown1,
Elisabeth Ralfkiaer, Prof., MD, DMSc4 and Kirsten Grønbæk, MD, DMSc1
1
2
Department of Hematology, Rigshospitalet, Copenhagen, Denmark; Medical Prognosis Institute, Hørsholm,
Denmark; 3Department of Pathology, Odense University Hospital, Odense, Denmark; 4Dept. of Pathology,
Rigshospitalet, Copenhagen, Denmark;
The introduction of Rituximab as supplement to chemotherapy has significantly improved
outcome in diffuse large B-cell lymphoma (DLBCL). Still, a fraction of patients are resistant
or relapse shortly after treatment, and for these patients the current alternative therapies
are often not curative. Hence, there is an urgent need to improve stratification of patients
for immunochemotherapy, and to develop novel therapies to improve outcome in DLBCL.
Novel prognostic predictors have been proposed based on mRNA profiling and
immunohistochemistry, however, these have limited clinical applicability and prognostic
value, respectively. MicroRNAs (miRNAs) are particularly attractive for clinical
applications, as they are well conserved in formalin-fixed paraffin-embedded (FFPE)
tissue, and they have potential prognostic value, as they are differentially expressed in
human cancers. All in all, they show promising potential as clinical biomarkers in diagnosis
and prognosis, and as putative therapeutic targets.
We hypothesize that the outcome of immunochemotherapy in DLBCL patients can be
predicted from the expression levels of particular miRNAs, and by identifying these
miRNAs we hope to form a novel prognostic miRNA predictor for clinical use.
In this study we performed miRNA profiling by miRNA microarray on FFPE tissue samples
from 97 DLBCL patients, all uniformly treated with immunochemotherapy (R-CHOP, n =
80; R-CHOEP, n = 17). The miRNA profiles revealed a 17 miRNAs signature with
prognostic value, of which 14 were validated by real-time quantitative PCR. These are
currently being validated in an individual patient cohort.
The preliminary data show promising potential for this novel prognostic miRNA predictor.
Survival analyses, using Kaplan-Meier plots and ROC curves, show that the miRNA
23
predictor, both alone and in combination with the international prognostic index (IPI),
performs better than the IPI alone. Additionally, the miRNA predictor revealed a novel
putative onco-miR in DLBCL, miR1275 that might be a promising therapeutic target.
24
Simon Husby
miRNA expression profiling in two prospective clinical mantle cell lymphoma
cohorts
Simon Husby1*, Ulrik Ralfkiaer1*, Christopher Workman2, Christian Garde2, Sara Ek. Arne
Kolstad3, Mats Jerkeman4, Anna Laurell5, Riikka Räty6, Mats Ehinger5, Christer
Sundström7, , Marja-Liisa Karjalainen-Lindsberg6, Jan Delabie3, Elisabeth Ralfkiaer8, Erik
Clasen-Linde8, Christian H. Geisler1# and Kirsten Grønbæk1#.
On behalf of the Nordic mantle cell lymphoma study group.
* # These authors contributed equally to this work.
1
Department of Hematology, Rigshospitalet, Copenhagen, Denmark. 2Department of Systems Biology,
3
Technical University of Denmark, Lyngby, Denmark. Departments of Oncology,and Pathologyt,
4
Rikshospitalet Oslo, Norway. Department of Oncology, Skåne University Hospital, Lund, Sweden.
5
Department of Pathology, Skåne University Hospital, Lund, Sweden. 6Department of Hematology and
7
Pathology, Helsinki University Central Hospital, Helsinki, Finland. Department of Immunology, Genetics and
Pathology, Uppsala University Hospital, Uppsala, Sweden. 8Department of Pathology, Rigshospitalet,
Copenhagen, Denmark.
Abstract
Introduction: Mantle cell lymphoma (MCL) is an aggressive NHL subtype. Recent progress
including immunochemotherapy and autologous stem cell transplantation (ASCT) has
improved the survival. However, many patients still relapse, and the underlying
mechanisms are unknown. MicroRNAs (miRNAs) have been implicated in disease biology
and as prognosticators, but have not been tested in large prospective clinical cohorts.
Material and methods:
Patients: In the present study, we assessed the miRNA profile in diagnostic tumour
samples of 76 of the 160 patients of the Nordic MCL2 protocol, of whom material was
available in the biobank established in the 5 pathology reference centers of the Nordic
MCL2 collaboration. The patients were otherwise unselected. All patients received
induction therapy with rituximab (R) + cyclophosphamide, vincristine, doxorubicin,
25
prednisone (maxi-CHOP), alternating with R + high dose cytarabine followed by BEAM
and ASCT. The median follow-up was 6.4 years (range, 0.2 to 9.9 years).
Methods: RNA was purified from formalin-fixed paraffin embedded tissue and analysed by
miRNA microarray. Differentially expressed miRNAs (adjusted p-value < 0,05) were
subsequently re-analysed by quantitative (q)RT-PCR.
Results: Of the 76 patients studied, 29 have died, 25 (32,9%) of MCL. 17 miRNAs were
differentially expressed in relation to death from MCL or not. Six miRNAs (miRNA-4417, 3687, -144-3p, -486-5p, 18b-5p, -378d) were confirmed to be differentially expressed by
qRT-PCR. Moreover, five of these miRNAs (miRNA-4417, -3687, -144-3p, -486-5p, 18b5p) were also differentially expressed in patients with relapse of MCL. These findings are
currently being validated in 96 patients of the similarly treated 3. Nordic MCL (MCL3)
cohort, and results will be presented at the meeting. We also analyzed the expression
levels of the miR-29 family in the MCL3 cohort, and confirm down-regulation of miR-29a as
a prognostic marker in MCL.
Conclusion: Abberant microRNA expression may contribute to the mechanisms of therapy
resistance in MCL.
26
Karen Juul Mylam
Prognostic impact of clinician-based interpretation of FDG-PET/CT reports obtained
in patients with newly-diagnosed diffuse large B-cell lymphoma.
Karen Juul Mylam1, Tarec Christoffer El-Galaly2, Martin Hutchings3, Peter Brown3, Bodil
Himmelstrup4, Dorte Gillstrøm5, Ida Blok Sillesen4, Lars Munksgaard1, Bjarne Bach
Pedersen6, Ilse Christiansen2, Paw Jensen2, Lars Møller Pedersen4.
1
2
Department of Hematology, Odense University Hospital; Department of Hematology, Aalborg University
3
4
Hospital; Department of Hematology, Rigshospitalet, University of Copenhagen; Department of
Hematology, Roskilde Hospital, 5Department of Hematology, Aarhus University Hospital; 6Department of
Hematology, Viborg Hospital.
Purpose: The prognostic value of mid- and post-therapy 18-fluoro-deoxyglucose positron
emission tomography (FDG-PET/CT) in newly diagnosed diffuse large B-cell lymphoma
(DLBCL) has been extensively evaluated. Little is known about the value of PET reports
when they undergo a second interpretation by clinicians handling the treatment of DLBCL
patients in daily practice. The main aim of this study was to evaluate the prognostic value
of the clinician-based interpretation of PET/CT reports in newly diagnosed DLBCL at midtherapy (I-PET) and end-therapy (E-PET).
Patients/methods: 434 patients with DLBCL diagnosed between September 2005 and
December 2009 at eight Danish specialized centers of hematology were enrolled in this
study comprising a total of 617 PET reports. Each report was independently evaluated by
three expert hematologists. Reports were labeled positive or negative if all three
interpreters independently agreed. All others were considered indeterminate.
Results: The distribution of PET/CT report interpretation is shown in Table 1. The
progression free survival (PFS) and overall survival (OS) for the I-PET reports were not
significantly different between the indeterminate and negative results (p=0.6). However,
patients with an indeterminate result had a worse OS according to the E-PET reports
(p=0.006). Patients with a positive I-PET and/or E-PET report both had a significantly
lower PFS (p<0.0001) and OS (p<0.0001) compared to the two other groups.
27
Discussion: In this study, we found a high number of indeterminate evaluations according
to both I-PET and E-PET reports. There was no significant prognostic difference between
the negative and the indeterminate group of the I-PET. However, we observed a significant
difference in outcome between patients with a negative and indeterminate E-PET. Patients
with a positive E-PET and I-PET had a very poor prognosis. With the majority of study
reports being indeterminate due to disagreement between clinicians, this study shows the
importance of having a multidisciplinary setting for PET-based clinical decision making.
Indeterminate
Negative
Positive
I-PET (n=241)
59%(n=142)
30%(n=73)
11%(n=26)
E-PET (n=376)
49%(n=186)
41%(n=153)
10%(n=37)
Table 1: Distribution of 617 PET/CT reports by hematologist interpretation
28
Sif Gudbrandsdottir
Rituximab and Dexamethasone vs Dexamethasone Monotherapy in Newly
Diagnosed Patients with Primary Immune Thrombocytopenia
Sif Gudbrandsdottir1,2, Henrik Sverre Birgens3, Henrik Frederiksen4, Bjarne Anker Jensen3,
Morten Krogh Jensen3, Lars Kjeldsen5, Tobias Wirenfeldt Klausen3, Herdis Larsen6, Hans
Torben Mourits-Andersen7, Claus Henrik Nielsen2, Ove Juul Nielsen5, Torben Plesner8,
Stanislaw Pulczynski9, Inge Helleberg Rasmussen10, Dorthe Rønnov-Jessen6, Hans Carl
Hasselbalch1
1
Department of Hematology, Copenhagen University Hospital Roskilde; 2 Institute for Inflammation
Research, Department of Infectious Diseases and Rheumatology, Copenhagen University Hospital
Rigshospitalet; 3Department of Hematology, Copenhagen University Hospital Herlev; 4Department of
5
Hematology, Odense University Hospital; Department of Hematology, Copenhagen University Hospital
6
7
Rigshospitalet; Department of Internal Medicine, Hematology section, Viborg Hospital; Department of
Hematology and Infectious Diseases, Esbjerg Hospital; 8Department of Hematology, Vejle Hospital; 9
10
Department of Internal Medicine, Hematology section, Holstebro Hospital; Department of Hematology,
Aalborg Hospital, Aarhus University Hospital
Primary Immune Thrombocytopenia (ITP) is an autoimmune disease characterized by low
platelet counts and risk of bleeding. Recommended first line treatments include
glucocorticoids and IVIG, second line treatments include splenectomy, rituximab,
thrombopoiesis-stimulating agents and a range of chemotherapies. In this study we report
the results from the largest cohort to date of patients with newly diagnosed ITP
randomized to first line treatment with dexamethasone (DXM) alone or in combination with
rituximab (RTX + DXM). Eligible were patients with platelet counts ≤ 25 x109/L or ≤ 50
x109/L with bleeding symptoms. 133 patients were randomly assigned to either DXM
monotherapy 40 mg/day for 4 days (n=71) or in combination with RTX 375 mg/m2 weekly
for 4 weeks (n=62). Patients in both groups were allowed supplemental DXM every 1-4
week for up to 6 cycles. The median follow-up time was 922 days. Our primary endpoint,
sustained response (i.e. platelets ≥ 50 x109/L) at 6 months follow-up, was reached in 58 %
of patients in the RTX + DXM group vs 37 % in the DXM group (p = 0.02). We found
29
longer time-to-relapse (p = 0.03) [Figure 1] and longer time-to-rescue-treatment (p =
0.007) in the RTX + DXM group than in the DXM group. The treatments were well
tolerated, although there were more grade 3-4 adverse events reported in the RTX + DXM
group (p = 0.04). In conclusion, RTX + DXM proved more effective than DXM alone and
we propose that RTX should be considered early in the treatment course of ITP.
Figure 1. Time-to-relapse in patients
treated with RTX + DXM or DXM alone.
30
Peter Brændstrup
Staining of antigen-specific CD4+ T cells using MHC class II tetramers generated
according to a novel method of peptide-MHC class II monomer production and
purification
Brændstrup P1, 2, Justesen S1, Østerby T1, Mortensen B1, 2, Madsen MK1, Rasmussen M1,
Malone R3, Vindeløv L2, Stryhn A1, Buus S1
1
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Denmark
2
The Allogeneic Hematopoietic Cell Transplantation Laboratory, Department of Hematology, Rigshospitalet,
Copenhagen University Hospital, Denmark
3
Institut National de la Santé et de la Recherche Médicale, Unité 986, DeAR Lab Avenir, Hôpital Saint
Vincent de Paul, Paris, France.
CD4+ T cells recognize linear peptides bound in the peptide-binding cleft of MHC class II
molecules displayed on the surface of antigen-presenting cells. CD4+ T cells are key
players in adaptive immunity orchestrating both CD8+ T cells (e.g. activation,
differentiation, proliferation, maintenance) and B cells (e.g. antibody class switching, high
affinity antibody production).
The use of MHC class I tetramers to stain antigen-specific CD8+ T cells emerged in 1996
and has since transformed the field of cellular immunology, effectively becoming the
golden standard for direct enumeration, analysis and manipulation of CD8+ T cells. On the
other hand generation of functional peptide-MHC class II molecules has proven
challenging. It is fair to say that no consensus on how to generate MHC class II molecules
and/or tetramers with the purpose of staining CD4+ T cells has yet emerged.
We have developed an alternative approach to generate peptide-MHC class II tetramers.
Recombinant MHC class II alpha and beta chains were refolded in vitro in the presence of
peptides that had been extended by a hexa-histidine sequence. The resulting peptideMHC class II complexes could readily be purified and concentrated by immobilized metal
affinity chromatography, and subsequently tetramerized using fluorochrome-labeled
streptavidin. Here, we demonstrate that these MHC class II tetramers can be used to stain,
and even purify, antigen-specific, MHC class II-restricted CD4+ T lymphocytes.
31
Bo Kok Mortensen
Identification of Y-chromosomally encoded minor histocompatibility antigens using
a reverse immunology approach.
Bo Kok Mortensen, MD1, Peter Brændstrup, MD1, Malene Erup Larsen, Ph.d2, Mette
Voldby Larsen, Ph.d2, Ole Lund, Ph.d2, Michael Rasmussen, Msc3, Søren Buus, MD3,
Anette Stryhn, Ph.d3 and Lars Vindeløv, MD1
(1)Allogeneic Hematopoietic Cell Transplantation Laboratory, Rigshospitalet, Copenhagen Ø, Denmark,
(2)Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark,
Lyngby, Denmark, (3)Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen N,
Denmark
Introduction: In allogeneic hematopoietic cell transplantation (HCT), minor
histocompatibility antigens (mHags) are known to play an important role in generating
immune responses leading to graft-versus-leukaemia (GVL) effects and graft-versus-hostdisease (GVHD). mHags are results of polymorphisms in the recipients genome, which
cause expression of peptides that can be recognised by donor T-cells. Y-chromosomally
encoded proteins constitute a constant source of mHags relevant in allogeneic HCTs with
female donor and male recipient due to the disparities between these and their homologue
X-chromosomally encoded counterparts.
Methods: A panel containing 8-11 mer peptides encompassing multiple putative and
known mHags encoded by the Y-chromosome was designed using a bioinformatics
predictor of peptide-HLA binding, NetMHCpan. These peptides were synthesized and
used to screen for peptide-specific T-cell responses in peripheral blood mononuclear cells
(PBMCs) obtained post-HCT from male recipients of female donor grafts. Following in vitro
stimulation, PBMCs were analysed with an inteferon-γ ELISpot assay or intracellular
cytokine staining (ICS) for T-cell rectivity against these peptides. When a response was
found, the optimal epitope and the HLA-restriction was determined by staining with
peptide/HLA tetramers.
32
Results: In one male recipient of a female donor graft, a T-cell response was observed
with ELISpot assay against the peptide RESEEESVSL which is an already described
mHags restricted to HLA-B60. ICS and flow cytometry revealed that it was a CD8
responses. By tetramerstaining, the HLA-restriction was determined to be HLA-B*40:01,
which is a member of the previously designated HLA-B60 specificity. In another male
recipient of a female donor graft, a CD8+ T cell response was observed against the
peptide stretch YFYYNAFHWAI. Using tetramers, the HLA-restriction was determined to
be HLA-A*24:02 and the optimal epitope was YYNAFHWAI. CD8+ T cells specific for the
same H-Y mHag were subsequently found in two out of four additional HLA-A*24:02positive male recipients of female donor grafts
Conclusion: In conclusion, we have identified a new mHag and demonstrated the HLA
restriction elements at high resolution for an already described mHag. Furthermore we
have demonstrated the feasibility of a reverse immunology approach in mHag discovery.
33