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Billedjagt
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 6 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. 9 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. 14 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. 15 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. 16 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. 18 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