ALBERT

Description: Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY. Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones. Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and

Description: Abstract 1595 Background: Immunohistochemistry for Cyclin D1 (CCND1) expression is routine in cases suggestive of mantle cell lymphoma (MCL). Most MCL are t(11;14)/IGH-CCND1-positive by FISH. PCR based detection of the fusion transcript is hampered by widespread breakpoints. Only few data is available on quantitative real-time PCR (RQ-PCR) for CCND1 expression measurement. Aims: To assess CCND1 mRNA expression and correlate it with t(11;14) in mature B-cell neoplasms. Methods: We established a RQ-PCR assay for CCND1 mRNA measurement and investigated 451 cases: 142 MCL (in all cases IGH-CCND1 confirmed by FISH), 76 chronic lymphocytic leukemia (43 typical CLL, 33 CLL/PL), 20 hairy cell leukemia (HCL), 13 hairy cell leukemia-variant (HCL-v), 20 splenic marginal zone lymphoma (SMZL), 91 other mature B-cell neoplasms. CCND1 background expression was assessed in 29 pts with other hematological neoplasms and 60 healthy individuals. FISH and/or chromosome banding analysis for the t(11;14) was available in 364 pts. Bone marrow (BM, n=267) or peripheral blood (PB; n=184) samples were analyzed by cytomorphology, multiparameter flow cytometry (MFC), FISH, and RQ-PCR. CCND1 mRNA expression was given by RQ-PCR in comparison to ABL1 mRNA expression (%CCND1/ABL1). Limited dilution of high expressers into cDNA of healthy controls revealed a sensitivity of the assay of up to 0.1 %. Results: IGH-CCND1 translocation carriers had higher %CCND1/ABL1 than those without which hold true in the total cohort (mean±SD, 420.4±740.3 vs 17.8±128.3; p

Description: Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 24 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Median age was 59 (range, 37–70) years. The series included 8 females and 16 males. Prior to allo-SCT, all patients were heavily pretreated with a median of 6 chemotherapy cycles including autologous and allo-SCT in all patients. Also, prior to lenalidomide, salvage treatment included donor lymphocyte infusions (DLI) in 18 pts,, thalidomide in 11 pts and bortezomib in 13 pts. Lenalidomide was given at 15mg (n=4) or 25 mg (n=20) orally once daily on day 1–21 every 28 days and in 20 patients in combination with dexamethason.. No prophylactic anticoagulation was used. The median number of completed cycles was 5 (range 2–17). Myelotoxicity according NCI criteria was the primarily and major encountered side effect (leukopenia: 4% grade 4, 21% grade 3, 17% grade 2, thrombopenia: 17% grade 3, 29% grade 2) and led to dose reduction in 54% of the patients. Infectious complications were observed in 50%. Non-hematological toxicity consisted of cramps (n=9), fatique (n=5) and constipation (n=2). Thrombembolic complications (cerebral infarction) were observed in one patient, who received concomitant corticosteroid treatment for acute graft-vs.-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 3 patients (one grade 2, and 2 grade 1), with one case occurring shortly after an additional DLI.. Objective remission was achieved in 66% of the patients (CR: 8%, VGPR: 8%, PR: 50%) and stable disease (SD) in 13% of the patients, while in 21% progressive disease was noted. Prior treatment with thalidomide or with bortezomib did not influenced the rate of CR/PR. Surprisingly, patients with del 13q14 achieved a higher CR/PR rate than those without del 13q14 (p=0.02). The median time to progression was 9.7 months (95% CI: 7.5–11.9) and the median overall survival was 19.9 months (95% CI: 17.3–22.5). Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in the majority of patients. The optimal dose of lenalidomide after allo-SCT has to be investigated.

Description: We characterized the mutational status of the FLT3 tyrosine kinase domain (FLT3-TLD) in 3082 patients with newly diagnosed AML. FLT3-TKD mutations were detected in 147 of 3082 (4.8%) patients. Similar to the FLT3 juxtamembrane domain mutations (FLT3-LM), there was a high correlation of FLT3-TKD mutations with normal karyotype (88 of 1472; 6.0%). FLT3-TKD mutations were most frequent in the AML FAB subtypes M5b (15 of 114; 13.2%), M3v (6 of 51; 11.8%), and M4 (39 of 484; 8.1%). Similar to FLT3-LM, the FLT3-TKD mutations show elevated peripheral leukocytes compared with FLT3wt AML. FLT3-TKD had a high incidence in cases with NPM1 mutations (23 of 262; 8.8%), CEBPA mutations (6 of 76; 7.9%), and NRAS mutations (6 of 78; 7.7%). FLT3-TKD in combination with FLT3-LM (17 of 594 patients; 2.9%) and KITD816 (1 of 44; 2.3%) was rare. Unlike the FLT3-LM, which are associated with inferior survival, prognosis was not influenced by FLT3-TKD in the total cohort of 1720 cases, where follow-up data were available (97 FLT3-TKD; 1623 FLT3-WT). In t(15;17)/PML-RARA with FLT3-TKD mutations, in FLT3-LM/TKD double-mutated, and in MLL-PTD/TKD double-mutated cases prognosis was unfavorably influenced by FLT3-TKD mutations. In contrast, we found an additional favorable impact of FLT3-TKD on EFS in prognostically favorable AML with NPM1- or CEBPA mutations.

Description: Introduction: In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), relapse after allogeneic stem cell transplantation (SCT) occurs with frequencies of 30–80% in dependence on risk profiles. Selected patients are offered a 2. allo-SCT. As the number of studies is limited, we here performed retrospective analysis of 20 consecutive MDS and AML pts who received at least 2 allografts from related/unrelated donors from 2000–2007. Patients: There were 4 children and 16 adults who received ≥2 allo-SCTs (10 males, 10 females; 2–63 years, median 38 yy). 14 pts had de novo AML, 3 pts s-AML, 2 MDS RAEB-1; one child had juvenile myelomonocytic leukemia (JMML). 19 pts received two allo-SCTs-18 due to another relapse after the 1. allo-SCT, one due to secondary graft failure after 1. SCT. One pt with MDS had 3 allo-SCTs due to relapses. 13/20 pts received treatment before the 2. allo-SCT, but only 6/20 achieved complete remission (CR). 10/20 pts had unfavorable cytogenetics. The median interval between the 1. and 2. allo-SCTs was 20 months (range 2–47 mo). Median disease free survival (DFS) after the 1. SCT was 16 months (range 3–45 mo). Characterization of 2. allo-SCTs: 4/20 second allo-SCTs were performed from related donors (3 HLA-identical, one haploidentical). 16 SCTs were performed from unrelated donors (HLA matched: n= 8, -mismatched: n=8). In 10 cases, the 2. allo-SCTs was performed from different, in 10 cases from the same donor. Reduced intensity conditioning was applied to 16 pts, myeloablative to 4 pts. Antithymocyte globuline was administered to 12/20 pts. One pt received bone marrow, 19 pts peripheral blood stem cells. Immunosuppression was performed in most pts by cyclosporine A plus short-course MTX or mycophenolate mofetil. Outcomes: Leukocyte engraftment was achieved in 16/20 cases with a median interval of 13 days (r. 10–24 d). 3 pts died prior to engraftment. One pt had secondary graft failure being followed by a 2. allo-SCT. CR was achieved by 16/20 pts. At the time of this report, 5/20 pts are still alive after a median follow-up of 15 months (r. 2–36 mo), three of those in CR with follow-ups of 7, 22, and 36 months. 9/20 pts developed relapse (45%), of whom 2 received donor lymphocytes. Median DFS after the 2. SCT was 7 months (range

Description: In chromic myeloproliferative diseases (CMPD) CML can be identified by the presence of a BCR-ABL fusion gene. The genetic spectrum of the BCR-ABL negative disorders is diverse. The most frequent aberrations especially in PV, ET, and CIMF is the JAK2V617F mutation. Initially these two aberrations were considered to occur mutually exclusive and define different diseases. However, recently 3 reports documented coincidences of the JAK2V617F and BCR-ABL in individual cases. For further clarification we analyzed a cohort of 2317 cases which were screened for BCR-ABL and JAK2 in parallel due to suspected CMPD. Within this cohort 1249 (53.9%) were BCR-ABL-/JAK2V617F-, 119 (5.1%) were BCR-ABL+/JAK2V617F-, and 945 were BCR-ABL-/JAK2V617F+ (40.8%). Double positivity for BCR-ABL and JAK2V617F was detected in 4 cases (0.17%). Real-time PCR for BCR-ABL expression and the V617F was performed for all available timepoints during follow-up. In 2 cases only one timepoint was available and 2 pts were followed for 1 year and 7 months, respectively. After retrospective analysis of the clinical data different patterns of the mutations were detected: 1 pt: A 56-year old male patient was diagnosed with CML in chronic phase in 9/2004. Imatinib treatment led to a major molecular response, but in 10/2006 a marked thrombocytosis led to the diagnosis of a JAK2V617F positive CMPD. Retrospective real-time quantification revealed that the JAK2V617F stayed at the same level as was detected at diagnosis of CML and during one year of follow up whereas the %BCR-ABL/ABL decreased during treatment with imatinib over three log ranges. This indicated that the two mutations occurred in separate clones. 2 pt: A 45 year old male was diagnosed with CML in chronic phase in 9/2006. After 7 months of imatinib treatment RQ-PCR showed decrease of %BCR-ABL/ABL to 0.225 but JAK2V617F remained at a 30% level. This case resembled the first case and suggests different clones - one with BCR-ABL fusiongene and one with JAK2V617F. 3 pt: A 75-year old female, was diagnosed with BCR-ABL negative CMPD in 11/2000. Cytoreduction by hydroxurea (HU) was successful from 2004–2005, but in May 2006 the disease showed signs of acceleration. Molecular diagnostics showed a JAK2V617F and for the first time a BCR-ABL fusion gene, both with expression levels typical for untreated cases. Imatinib was not tolerated, and HU was started again. Over 7 months %BCR-ABL/ABL and %JAK2V617F/ABL remained at the same high level suggesting that one clone harbored both mutations. 4 pt: A 53 years old female was diagnosed with CMPD. 21 years later in 6/2005 molecular diagnostics was performed upon increasing thrombocytosis and showed BCR-ABL positivity and a homozygous JAK2V617F. Both mutations had high expression levels which persisted after 12 months of imatinib treatment. As in the third case the homozygous JAK2 mutational status and the high BCR-ABL expression suggested coexistence of both mutations in the same clone and resistance to imatinib. In conclusion, these four new cases support the idea that BCR-ABL and JAK2 mutations can occur in same patients in the same clone or in different clones and at different time points. It may be speculated that in these cases there is an unknown common antecedent defect.

Description: Introduction Acute myeloid leukemia (AML) is a heterogenous hematological malignancy driven by leukemia stem cells (LSCs) (Lapidot et al, 1994). LSCs resistant against conventional chemotherapy represent the major cause of relapse. Elderly or unfit AML patients not eligible for intensive chemotherapy are treated in a palliative setting with hypomethylating agents (HMA) or low dose Ara-C, but responses are modest and not durable. The reason for the low efficacy of HMA treatment is their insufficient action on the disease initiating- and -maintaining LSC population (Craddock et al, 2013). We recently demonstrated that CD34+ AML cells (progenitors and LSC) consistently express the tumor necrosis factor family ligand CD70 as well as its receptor CD27 and that cell-autonomous CD70/CD27-signaling propagates the disease (Riether et al. 2017). The aim of the current study was to evaluate whether resistance to HMA treatment can be overcome by combining HMA with an anti-CD70 monoclonal antibody treatment. Experimental design The effect of HMA treatment on the expression of CD70 on primary human CD34+CD38- AML LSCs was determined in vitro cultures and in patients treated with HMA in vivo. The therapeutic potential of targeting CD70-expressing LSCs in presence and absence of HMA was assessed using the anti-CD70 ADCC-optimized monoclonal antibody (mAb), cusatuzumab, and an effector-dead anti-CD70 mAb in colony formation and re-plating assays as well as patient-derived xenograft models (Silence et al, 2014). The clinical relevance of the findings was determined in a clinical phase 1 trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (AZA, NCT03030612). Four different dose levels of cusatuzumab (1, 3, 10 and 20 mg/kg Q2W) were studied; AZA was administered at 75 mg/m² for 7 days every 28 days. Results We found that resistance of AML LSCs to HMA treatment is mediated by the up-regulation of the CD70. The up-regulation of CD70 triggered cell-autonomous CD70/CD27 signaling on AML LSCs. Based on these findings we hypothesized that the upregulation of CD70 by HMA may render LSCs more susceptible to CD70-targeting interventions. Targeting CD70-expressing LSCs by a blocking anti-CD70 mAb and the anti-CD70 mAb cusatuzumab, which blocks CD70/CD27-signaling and additionally mediates ADCC and CDC, eradicated LSCs in colony and re-plating assays in vitro and in xenotransplantation experiments in vivo. HMA in combination with blocking αCD70 mAb synergistically reduced LSC numbers in vivo and this was even more efficient when ADCC-enhanced αCD70 mAb cusatuzumab was added in the presence of NK cells. In order to test the hypothesis that targeting CD70 in combination with HMA eliminates LSCs in AML patients, we initiated a phase 1 dose-escalation trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with azacitidine. No dose-limiting toxicities (DLT) were observed in the dose-escalation phase 1 trial and responses were observed across the dose levels (1-20 mg/kg). A single dose of cusatuzumab reduced bone marrow blasts in just two weeks in all patients on average by 32%. Cusatuzumab monotherapy significantly reduced LSC numbers and frequencies in all patients analyzed in the bone marrow as assessed in limiting dilution colony assays. Single cell sequencing analysis revealed that cusatuzumab induced gene signatures related to myeloid differentiation and apoptosis in LSCs. In combination with azacitidine, cusatuzumab induced CR/CRi in 10 out of 12 patients. Responses were observed at all dose levels of cusatuzumab and median time to response was 3.3 months. Conclusions Blocking CD70/CD27-signaling and targeting CD70-expressing LSCs by the ADCC-optimized mAb, cusatuzumab, eliminated LSCs in vitro and in xenotransplantation experiments. In a phase 1 study promising activity of cusatuzumab in combination with HMA was observed in AML patients, in which translational data indicate that cusatuzumab selectively eliminates CD70-expressing LSCs. Disclosures Van Rompaey: argenx: Employment, Equity Ownership, Patents & Royalties. Moshir:Argenx: Employment, Equity Ownership. Delahaye:argenx: Employment, Equity Ownership. Gandini:Argenx: Employment, Equity Ownership. Erzeel:argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Fung:argenx: Consultancy, Equity Ownership. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties.

Description: Abstract 3496 Introduction: Allogeneic hematopoietic stem cell transplantation (allogeneic HSCT) has found entrance into treatment of patients (patients) with poor-risk T-cell malignancies, but post-transplant relapse rates of ~30% were reported (Marks et al. Blood 2009). Improvement of the pre-transplant remission status in relapsed/refractory disease might lead to better outcomes. Nelarabine - a novel purine antimetabolite - has been approved by the FDA in 2005 as salvage approach for patients with T-cell malignancies, but data on pre-transplant use are limited (Goekbuget et al. ASH Annual Meeting 2005). Patients/Methods: Aiming to estimate its clinical applicability and efficacy in the pre-transplant period, we evaluated its use in 8 patients (5 males, 3 females; 22–56 years) with precursor/mature T-cell neoplasms who had failed to ≥2 previous conventional/intensified chemotherapy regimens (including a history of autologous/allogeneic HSCT in 2 patients) and were consequently proceeded to allogeneic HSCT (in 6 cases the 1st, in 2 cases the 2nd allogeneic HSCT). Five had T-ALL (1 pre-T-, 3 cortical, 1 mature), 3 had mature T-cell lymphomas (angioimmunoblastic lymphoma, n=1; peripheral T-cell lymphoma not otherwise specified, n=2), all with bone marrow involvement. Patients entered conventional chemotherapy in 6 cases at the 1st manifestation of disease, in 2 cases at the 1st relapse. Following conventional chemotherapy, 5 patients developed an early relapse, while 2 patients were refractory to treatment; one patient was minimal residual disease (MRD) positive as assessed by multiparameter flow cytometry. Therefore, all 8 patients were offered an allogeneic HSCT (1st allogeneic HSCT: n=6; 2nd allogeneic HSCT: n=2) with pre-treatment with nelarabine. The median interval from the last chemotherapy to 1st nelarabine administration was 8 weeks (range, 1– 252 weeks). Nelarabine (1,500 mg/m2 i.v.) was administrated on days 1, 3, and 5 at a median of 11 weeks (range, 2–53 weeks) before allogeneic HSCT. Three patients received a 2nd cycle of nelarabine after a median of 4 weeks (range, 2–9 weeks) from the 1st. Six patients had unrelated (HLA-match, n=4; mismatch, n=2) and 2 patients matched related donors. Most patients received myeloablative conditioning (TBI, 12 Gy; cyclophosphamide 120 mg/kg; n=3; treosulfan 36 g/m2; etoposide 30 mg/kg; cyclophosphamide 120 mg/kg; n=4). In the 8th patient who had already received 2 courses of nelarabine, a 3rd nelarabine application was incorporated as pre-phase into reduced-intensity conditioning (nelarabine 2 × 1,500 mg/m2; fludarabine 90 mg/m2; TBI 8 Gy). Results: i) Pre-transplant application of nelarabine: Immediate neurotoxicity was observed in 1 patient (grand mal seizure day +3 after the last nelarabine administration). Grade II hematotoxicity was observed in 3/8 patients. There were no treatment-related deaths. Seven of 8 patients showed response to nelarabine (86%; CR, n=5; PR, n=2) after a median of 4 weeks (1 – 35 days) from the 1st cycle. One patient had stable disease (SD). ii) Peri-/post-transplant period: there were no uncommon adverse events. After HSCT, prolonged leukocytopenia (WBC 20 days) and thrombocytopenia (thrombocytes 30 days) were observed in 3 and 5 patients, respectively. At the 1st post-transplant bone marrow control, 3 patients with residual disease (PR or SD) prior to allogeneic HSCT achieved CR, and all other patients maintained CR. Acute (grade II) and chronic GvHD developed in 2/8 (25%) and in 2/6 (33%) patients, respectively; there was no case of grade III-IV acute GvHD. At a median follow up of 9 months (range, 1–24), 3 patients had died (38%; relapse, n=1, t-AML, n=1; transplant-related toxicity, n=1). All 5 patients being alive were in CR; 4 patients required no further treatment, and one patient received donor lymphocyte infusions (DLIs) due to mixed chimerism. Conclusions: Nelarabine improves pre-transplant remission in patients with relapsed/refractory T-cell neoplasms and seems to be well tolerated immediately before allogeneic HSCT even in heavily pre-treated patients. The compound might be used as pre-phase or be incorporated into conditioning for allogeneic HSCT which should be further evaluated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3393 Poster Board III-281 Background: HLA-mismatched unrelated donors are increasingly becoming a graft source for both standard myeloablative (MAC) and reduced intensity conditioning (RIC) hematopoetic stem cell transplantation (HSCT). Patients and methods: We retrospectively analysed the effects of HLA-mismatching in 553 patients undergoing MAC (n = 342) or RIC (n = 211) unrelated donor HLA-matched (n = 289) or -mismatched (n = 264) HSCT with antithymocyte globulin as part of conditioning. Patient characteristics well matched between HLA-matched and –mismatched groups of MAC and RIC patients. Results: Median follow-up was 1946 days for MAC patients and 765 days for RIC patients. In MAC patients, there was no difference in the incidence of aGvHD, treatment related mortality (TRM) and overall survival (OS) between recipients of HLA-matched vs. –mismatched allografts. In RIC patients on the contrary, the incidence of aGvHD II-IV (46%, vs. 32.0% p = 0.05), III-IV (18%, % vs. 9.0 p = 0.07) and TRM (35% vs. 20%, p = 0.04) were higher and OS lower (31% vs. 50%, p = 0.001) for recipients of HLA-mismatched vs. –matched transplants. In the RIC patients, female donor gender (RR: 1.8; p = 0.02) and HLA-mismatch (RR: 1.8; p = 0.02) negatively influenced TRM while female donor gender (RR: 1.58, p = 0.03), HLA-mismatch (RR: 1.82, p = 0.003) and bad risk disease (RR: 2.14, p 〈 0.001) negatively impacted OS. Conclusion: Our analyses surprisingly reveal that the positive effect of ATG in HLA-mismatched transplants is only limited to patients undergoing standard myeloablative conditioning. HLA-mismatching and female donor gender negatively impact outcome of RIC allogeneic unrelated donor HSCT despite the use of ATG, highlighting the need for improved strategies. Acknowledgments We thank the staff of the BMT unit for providing outstanding care to our patients and the medical technicians for their excellent work in the BMT laboratory. Disclosures: Off Label Use: Antithymocyte globulin for prevention of severe GVHD.

Description: Abstract 4689 Introduction: Allogenic stem cell transplantation (allo SCT) offers a potential curative approach for many malignant and non malignant haematological diseases. Despite its therapeutic benefit, long term immunodeficiency, poor immune reconstitution and Graft vs. Host Disease (GvHD) can often be limiting drawbacks. Since the nineties, regulatory T cell subsets (Treg) have been described and several lines of evidence indicated their implication on GvHD occurrence and progression. We analysed the immune reconstitution of 184 patients who underwent allo SCT at our Transplant Center from 2007 till 2009. Patients, Materials and Methods: Differential lymphocyte subsets were analysed by flow cytometry. Antigens were stained by usage of the following mAb: CD3, CD4, CD8, CD19, HLA-DR, CD56/CD16, CD45RA, CD45RO, CD45, γδ TCR, CD25, and CD127. Tregs were evaluated on simultaneous expression of CD4/CD25hi/CD127low. Data were obtained in monthly intervals for the first six months and thereafter every six months for the next 3 years. Data were analysed for three different subgroups: Multiple Myeloma (MM: n=83), Myelofibrosis (PMF: n=22) and AML/MDS (n=51). Smaller number subgroups of patients with CML (n=11), NHL (n=10) and ALL (n=7) were included into the overall analysis but not evaluated separately. Results: The mean value of Treg cell number before allo SCT was 2,5% of the total leukocyte number in all patients. There was no significant difference in the Treg level in any of the three major groups (MM: 2,2%; PMF: 2,1% and AML/MDS: 2,03%). All patients exhibited a significant reduced number of Treg cells during the first 30 days after allo SCT (MM: 0,79%; p= 0,009; PMF: 0,41%; p= 0,01; MDS/AML: 0,6%; p=0,01). Between day 30 and 60 after allo SCT patients with MM had a transient Treg recovery to baseline level (2,4%) while Tregs of patients with PMF or MDS/AML remained significantly lower in comparison to baseline value (PMF: 0,72%, p=0,002 and MDS/AML 0,81%, p=0,01 respectively). One year after allo SCT a faster Treg recovery (1,3% and 1,8% respectively) was observed in MM and MDS/AML patients while patients with PMF still maintained a significant reduction (0,65%; p=0,01). Interestingly, in the second year after allo SCT, Treg cell levels were decreased in all investigated subgroups (MM: 1,1%, p=0,008; PMF: 0,7%, p=0,02 and MDS/AML: 0,7%, p