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Bone marrow (BM) derived adult multipotent mesenchymal stromal cells (MMSCs) and fibroblast colony-forming units (CFU-Fs) of 20 patients with acute myeloid leukemia (AML) and 15 patients with acute lymphoblastic leukemia (ALL) before and during 1 year after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) were studied. The growth characteristics of MMSCs of all patients before allo-HSCT were not altered; however, relative expression level (REL) of some genes in MMSCs, but not in CFU-Fs, from AML and ALL patients significantly changed. After allo-HSCT, CFU-F concentration and MMSC production were significantly decreased for 1 year; REL of several genes in MMSCs and CFU-F-derived colonies were also significantly downregulated. Thus, chemotherapy that was used for induction of remission did not impair the function of stromal precursors, but gene expression levels were altered. Allo-HSCT conditioning regimens significantly damaged MMSCs and CFU-Fs, and the effect lasted for at least 1 year.

Objective Dendritic cells (DC) play a key role in initiation of immune responses. In vitro modified acute myeloid leukemia (AML) blasts acquire certain specific features of DC and are suggested as a potential source of anti-leukemia vaccines. AML-DC have been characterized in terms of costimulatory molecule expression and cytokine production. In contrast, migratory capacity of AML-DC, which is a major attribute of DC required for their in vivo function, remains unknown. Here we present data on adhesion properties and profile of integrin expression of AML-DC. Materials and methods Blasts from nine patients were used to generate AML-DC by calcium ionophore treatment. Adhesion of AML-DC to the major components of the extracellular matrix and the profile of integrin expression was studied using flow cytometry. Results Similar to their normal counterparts, calcium ionophore-induced AML-DC acquired the ability to bind to fibronectin and in 4 of 7 studied cases to bind to denatured collagen. Adhesion to native collagen remained unchanged during DC-type differentiation of AML blasts. AML-DC and DC obtained from monocytes of healthy donors expressed CD49d, CD49e, α v β 3 , and α v β 5 . However, AML-DC from 3 of 8 patients down-regulated CD49d, which plays an important role in cell-to-cell and cell-to-matrix interactions and normally is coexpressed with CD83. Conclusion The results provide further evidence that AML blasts can be induced to display functional properties characteristic for DC and may prove useful for in vivo delivery and presentation of tumor antigens to the immune system. Abnormal CD49d expression and variability in AML-DC adhesion to denatured collagen indicate that motility of AML-DC from individual patients may vary, and a customized approach is essential for evaluating leukemic cell feasibility for vaccine design.

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is characterized by low response rate to standard therapy and relapses in the course of treatment. CHOP-like therapy shows 70-80 % of overall response rate, though 2-year progression-free survival is observed only in 30-40 % of patients. Older patients are more frequently diagnosed with AITL than younger subjects. Usually the median age of those patients is 60 years. A search for new approaches to treatment of AITL has an important medical and social aspect. Aim: The aim of our study is to define rational approaches to treatment of younger patients with AITL, to assess the overall and progression-free survival depending on the type of therapy. Patients and methods: 45 patients with AITL were treated between 2002 and 2016 in our center. The diagnosis was verified according to WHO classification. We analyzed clinical outcomes in 17 (38%) patients younger than 55 years treated by different protocols. Results: Of 17 patients there were 9 men and 8 women with a median age of 41 (range, 29-55) years. 14 of 17 patients had Ann Arbor stage IV. Bone marrow involvement was detected in 7 of 17 cases by histological tests. Lung involvement was observed in 10 cases. B symptoms and elevated LDH level were present in all patients. Despite the younger age of the patients, 12 of 17 were classified as having an intermediate-high (n=5) and high (n=7) risk IPI score. There were 5 of 17 patients, who received CHOP-like therapy; other 12 patients were treated by longed therapy. There were 4 of 5 patients after CHOP-like therapy, who showed the progression from 1 to 4 (median 2) months, one achieved partial remission. There were 12 of 17 patients who were treated by the longed therapy according to RALL-2009 protocol (ClinicalTrials.gov public site; NCT01193933), successfully used for treatment of acute lymphoblastic leukemia in Russia. The program is based on non-intensive but uninterrupted treatment. After induction + consolidation phases (150 days) patients received maintenance therapy by interferon-A and thalidomide over 2 years. All 12 patients achieved complete remission, none of them showed disease progression. Two patients received autologous stem cell transplant (Auto-SCT) as first line consolidation treatment, conditioning regimen was CEAM (CCNU, etoposide, Ara-C, melphalan). One patient died after Auto-SCT from infection complications without disease progression. 11 (92%) of 12 patients treated by RALL-2009 protocol are alive, follow-up survival varies from 3 to 73 (median 19) months. A univariate analysis of overall and progression-free survival curves showed that longed therapy is more effective than CHOP-like therapy (Fig.1). Conclusion: Though, the study is small, the results show that higher efficiency of RALL-2009 protocol than CHOP-like therapy in younger patients withAITL. It is particularly important for working patients. The principle of non-intensive but uninterrupted usage cytostatic drugs and longed maintenance therapy by immunomodulators allows to avoid the early treatment failures and provides optimistic long-term results. Figure 1 (A) Overall and (B) progression-free survival of younger patients with AITL depending on the type of therapy. Figure 1. (A) Overall and (B) progression-free survival of younger patients with AITL depending on the type of therapy. Disclosures No relevant conflicts of interest to declare.

Blinatumomab is a bispecific anti-CD3/CD19 monoclonal antibody. It showed efficacy as a single agent in the treatment of relapsed/refractory Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). T-regulatory (T-reg) and T-helper cells can inhibit effector T-cells used by blinatumomab. Tyrosine-kinase inhibitors (TKI) are the basic treatment of the Ph+ ALL. To improve the long-term results of the relapsed Ph+ ALL treatment we combined blinatumomab with TKIs dasatinib or nilotinib or ponatinib. The aim of this study was to evaluate clinical efficacy of blinatumomab + TKI combination in relapsed Ph+ ALL patients and to study T-cell and NK subpopulations kinetics in R/R-ALL patients during the treatment. From Oct 2015 to June 2016 we treated 6 relapsed Ph+ ALL patients (5 overt hematological relapses and 1 cytogenetic relapse). Blinatumomab was administered in the dose 28 mcg/day by continuous IV infusion 28 days (4 week cycle) with 2 weeks intervals (up to 4 cycles). The dose of blinatumomab in the 1st week of the 1st cycle was 9 mcg/day. Dasatinib 140 mg/day PO started on 1 week and administered daily continuously. Nilotinib 400 mg bid PO was administered in the case of dasatinib toxicity. Ponatinib 45 mg /day PO was administered in the case of T315I ABL kinase domaine mutation. Lymphocytes subpopulations of the peripheral blood (T-helper CD3+/CD4+/CD8-, T-cytotoxic CD3+/CD4-/CD8+, T-reg CD3+/CD4+/CD25+, NK CD3-/CD56+) were studied by flow cytometry on the 1st day of every week during every blinatumomab cycle. Two pts received all 4 cycles of blinatomomab + TKI dasatinib (two MolCR after 2nd cycle). Two pts received 2 cycles of blinatumomab + TKI dasatinib (two MolCR) and are continuing treatment. 1 pt with T315I mutation received 1 cycle of blinatumomab + TKI ponatinib (MolCR). 1 pt progressed during 1st cycle of blinatumomab + dasatinib which were discontinued. AlloBMT was performed in 2 pts in MolCR and 3 pts in MolCR are awaiting alloBMT. All 6 pts are alive. The pt who progressed on the 1-st cycle of blinatumomab + dasatinib was later diagnosed with T315I mutation and CR was obtained on ponatinib + dexamethasone + vincristine. In 1 pt the dasatinib-related pleural effusions and lung infiltration observed which fully regressed after 2 weeks of dasatinib interruption. Dasatinib in that pt was later replaced with nilotinib during 4th cycle and so on. Hypogammaglobulinemia was common and corrected with intravenous human normal immunoglobulin replacement. No neurological toxicity observed. As a result, 5 MolCR were obtained in 6 pts who received blinatumomab + TKI except in 1 pt with T315I mutation who progressed on blinatumomab + dasatinib. In all pts T-helper population was about low limit of normal range or below it (Fig. 1). In 4 of 5 responded pts the restoration of cytotoxic T-cell subpopulation was observed (Fig. 2). In 4 of 5 responded pts the 2nd week T-reg dropping occurred (Fig. 3). In all of responded pts NK population returned to normal range. The blinatumomab + TKI combination is effective in relapsed Ph+ ALL. The MolCR obtaining is possible without glucocorticoid treatment. The combination has acceptable toxicity. The T-reg subpopulation depletion on 2 week and depleted pool of T-helper are correlated with MolCR obtaining during blinatumomab + TKI treatment. The T cell cytotoxic and NK lymphocyte subpopulation restoring possibly reflects normal hemopoiesis establishing in MolCR settings in effectively treated pts. Figure 1 T-helper subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Figure 1. T-helper subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Figure 2 T-cytotoxic subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Figure 2. T-cytotoxic subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Figure 3 T-regulatory subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Figure 3. T-regulatory subpopulation kinetics (Pt - patient; C - cycle; W - week; LLNR - lower limit of normal range). Disclosures No relevant conflicts of interest to declare.

Introduction It is postulated that the improvement in the overall treatment outcome in adult Ph-negative ALL came from the implementation of more aggressive pediatric-like protocols and higher portion of allogeneic HSCT. Here we report the results of the adult (15-55 yy) Ph-negative ALL protocol based on the opposite approaches: less intensive but non-interruptive treatment with low numbers of allo-HSCT. The study is registered on the ClinicalTrials.gov public site; NCT01193933. Patients and Methods The ALL-2009 is based on: (1) the replacement of prednisolone (Pdn) 60 mg/m2 with dexamethazone (Dexa) 10 mg/m2 if blast cells are >25% in b/m after prephase (7d); (2) de-intensified but non-interruptive 5 months induction/consolidation treatment (5 wks prd/dexa with 3 instead of 4 dauno/vncr pulses, 4 weeks of 6MP with 5 L-asp, 2 instead of 4 ARA-C blocks, 1 instead of 2 Cph injections during induction; induction-like 3 consolidations for 3wks, 2wks, 4wks-continuously without intervals), followed by (3) 2 late (at 6 mo) intensifications- with 1 day HD MTX and with 1 d HD ARA-C, both with L-asp and 3 ds dexa and (4) 2-yrs continuous 6MP/MTX maintenance with doses modification according to myelosuppression with monthly 3-days dexa/vncr/L-asp pulses (∑ L-asp = 590.000 IU/m2). The protocol was identical for all risk groups. Allo-HSCT was indicated only for extremely high-risk BCP-ALL (t(4;11),L>100). No central MRD monitoring was performed. Since Apr 2009 till June 2015 20 centers had recruited 168 BCP-Ph-negative ALL pts with a median age 28 years (15-54), 84f/84 m. Full cytogenetics was available in 67,3% (n=113), 43,4% of them (n=49) had normal karyotype (NK), 10% (n=9%) had no mitosis, 47,6% (n=54) - different abnormalities (hypoploid-1, hyperploid-12, t(11q23)/MLL-8, del11q23-2, t(1;19)-2, t(12;21)-1;others-28). 26,7% of pts (n=45) were in the standard risk (SR) group (WBC 30; EGIL BI, LDH > 2N; late CR; t(4;11)-positive), 28 patients (n=16,8%) were not qualified by the risk. The analysis was performed in June 2015. 158 pts were available for analysis. Results CR rate in 158 available for analysis pts was 87,7% (n=139), induction death occurred in 9,1% (n=14), resistance was registered in 3,2% (n=5). The majority of CR pts (87,8%) achieved it after prephase (12,2%, n=17) and the 1st phase of induction (75,6%, n=105). Late responders constituted 12,2% (n=17). Allogeneic BMT was performed only in 9 of 144 patients who survived induction (6,2%). Totally 31 pts (22,3%) had relapsed. At 60 mo OS for the whole group constituted - 50%, DFS - 51.3%. In a univariate analysis among various risk factors (age <> 30y, initial risk group, WBC, LDH, immunophenotype, late response >35d, PRD resistance) age (>30 y) became statistically significant for OS, DFS and relapse probability (RP) (pic.1), abnormal karyotype - for DFS (30% vs 68%, p=0,04) and RP (42% vs 19%, p= 0,04). In a multivariate analysis no common risk factors were significant. Conclusions Our data demonstrate that the proposed treatment approach is rather effective. We believe that constant non-interruptive treatment without intensive highly myelosuppressive consolidation courses and high portion of allogeneic HSCT may become an alternative and reproducible approach in adult Ph-negative ALL, though we have to stress that it should be very strict compliance of the pts to the protocol. All pts, mostly from the region hospitals who refused prolonged and constant treatment (~5%), relapsed. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: The number of inhibitor hemophilia patients in Russia is presently about 200 patients (4% of all hemophilia patients). Current medical community is moving towards increasing the efficacy and safety of treatment, as well as the standardization of services and improvement of standards of care in rare diseases area. Patient registries and databases are key tools for the improvement of patient care, healthcare resources planning as well as social, economical and quality of life outcomes. Due to the lack of data on clinical course of disease, treatment approaches and outcomes, National Hematology Society initiated continuously running prospective, multi-center, open-label, non-interventional, observational Registry of INhibitor Hemophilia patients (RING) in July 2014. Aims: to document the natural history of hemophilia A or B disease in subjects with any type of inhibitors and to describe long-term treatment related outcomes in routine clinical practice. Methods: Data of all inhibitor patients are collected by physicians from patients medical records documenting the routine patient's examination and reflecting the medical practice at the particular treatment center. All data are entered into e-database using on-line access secured by the Operator. Results: In total 183 patients (101 adult and 82 children) from 47 centers were included in the Registry by June 2015. The majority of patients (90%) have severe hemophilia. The age of inhibitors diagnostics was: ≤10 years - 83%, 11-20 years - 3%, 21- 50 years - 5%, ≥51 years - 3% of patients. Chronic viral infections are diagnosed in 55 (30%) patients: 1 - HIV, 5- HBV and 49 - HCV. In 28% of patients annual bleeding rate (ABR) are ≥ 16, in 42% - 6-15, in 21% - ≤ 5. Approximately 25% of patients have zero joint ABR. Life threatening bleedings were registered in 25 patients (14%). Only 15 (8%) patients undergo immune tolerance induction (ITI); treatment with bypassing agents is administered in 151 (82%) patients. Due to the fact that activated prothrombin complex concentrate (aPCC) is not supplied by the State guarantees program 83% of patients receive rFVIIa (on-demand treatment - 50% and prophylaxis - 33%) and only 17% of patients have an access to aPCC treatment. The current data show that 34 (27%) patients with rFVIIa treatment need ≥3 injections to stop a bleeding episode thus they are identified as non-responders. Conclusion: The clear view of current treatment situation based on available Registry data demonstrates the necessity of administering alternative treatment approaches for non-responding to rFVIIa inhibitor patients, which would optimize resources of the State guarantees program. Disclosures No relevant conflicts of interest to declare.

Background: FL is one of the most frequent types of lymphomas: it consists about 22% of non-Hodgkin lymphomas in Russia. 20 % of pts have an aggressive tumor after standard therapy. In FL pts with unfavorable prognosis (FLIPI-1 III-IV, fast growth of tumor mass, B-symptoms, third cytological grade of tumor, bulky, absence of tumor response after first line R-CHOP-21 therapy) autoSCT allows to increase the overall (OS) and progression-free survival (PFS). Aim: To estimate an efficacy of HDT (high dose therapy) with following autoSCT in front-line therapy of FL pts with factors of poor prognosis. Patients and Methods: Since 2000 to 2015 years in National Research Center for Hematology were treated 39 FL pts with poor prognosis. All pts received R-CHOP-21 induction therapy. In 24 patients (62%) who hadn't partial response or poor prognosis patients with partial response after 4-6 courses autoSCT was performed. This group consists of 17 males and 7 females with median age 46 years old (31-68). Majority of pts (23/24) had IV stage of disease according to Ann Arbor. In 16/24 (66%) cases pts had bulky disease. 3/24 pts had phenomenon of leukaemization. Basing on FLIPI-1 criteria all pts were divided into 3 groups: low risk - 5/24, intermediate - 3/24, high - 16/24. B-symptoms were in majority of cases (18/24). 18/24 pts were diagnosed with I-II cytological grade of FL, 6/24 - III A/B. Basing on tumor growth pattern there was following ratio: nodular tumor growth revealed in 6/24 pts, nodular-diffuse tumor growth - in 15/24, diffuse tumor growth - in 3/24. Immunochemical analysis of serum and urine proteins was performed in 20/24 cases, among them increased level of serum β2-microglobulin was diagnosed in 10/20 pts. Increased activity of lactate dehydrogenase (LDH) was revealed in 16/24 pts. Bone marrow involvement at the time of disease onset was diagnosed in 18/24 pts. Results: After induction R-CHOP-21 chemotherapy pts underwent HDT which included two courses R-DHAP, following high-dose cyclophosphamide with rituximab, then high dose etoposide with rituximab. As condition regimen R-BEAM was performed. All 24 FL pts, who underwent autoSCT, continue to be in complete remission. Median follow-up was 31 months (7-178). Conclusions: Preliminary results of the first prospective study of intensive therapy of FL in Russia demonstrate that autoSCT in front line therapy provides to achieve a complete remission in pts with poor prognosis and allows to increase an overall and disease-free survival. Disclosures No relevant conflicts of interest to declare.

Introduction. Nowadays high-dose post-transplantation cyclophosphamide (CY) replace standard immunosuppression (IST). Thereby, the investigation of T-cells reconstitution after post-transplant-CY doesn't reach appropriate level, and probably it's very different from what we see after standard IST. We studied the reconstitution of memory T-cells on day of engraftment (WBC>1000 cells\us) after allogenic hematopoietic stem cell transplantation (allo-HSCT) with post-transplant-CY and standard immunosuppression therapy. Patients and methods. During 2 years, 29 patients with different hematological malignancies were included in this study. Median of age was 36 years (24-60 years). 16 patients were males, 13 - females. 22 received RIC, 7 - myeloablative regime. Match unrelated donor (MUD) was in 17 cases, "Mismatch" MUD - 2, Match related donor (MRD) - 9, "Mismatch"MRD - 1. 4 patients were in relapse or disease progression. CY as alternative IST was administrated to 6 patients. Standard immunosuppression consisted of CSA, MMF or MTX at standard dose. Peripheral blood samples were collected in EDTA-tubes at day of engraftment after allo-HSCT (Me= 20 day (14-35)). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD4- APC-Cy7 antibody (Becton Dickinson, USA) and FSC/SSC were used for determine population of CD4+T-cells. Anti-CD45R0- FITC (Becton Dickinson, USA) antibody were used to determine memory T-cells as subpopulation of CD4+ T-cells. Due to cyto- and lymphopenia 1,000,000 events was analyzed. Results. Mann-Whitney U test was used to test for differences between memory T-cells (CD4+ CD45R0+) after post-transplant-CY alone and in a group with standard IST. The percent of memory T-cells in CD4+ cell population at day of engraftment after post-transplant CY alone was statistically higher (74,3% ± 5,1% ,p=0.048*) than in patients with standard immunosuppression (49,4%±6,7%). Conclusion. We may conclude that patients with post-transplant CY had a different "T cell reconstitution profile". Reported data show us that probably post-transplant CY spares memory T-cells in contrast with standard IST, and also probably that CY is more selectively immunosuppressor than "gold standard" (such as CSA, MMF and etc.) not only on effector T-cells population. Despite the fact that the analyzed group is small, obtained data is important and needs further investigation. Disclosures No relevant conflicts of interest to declare.

Long-term survival of AA patients as a result of effective immunosuppressive therapy (IST) is accompanied by an increased risk of MDS / AML development. Clonal evolution of AA in MDS / AML is detected in 2,3-10,6% of cases ( M.G.Afable et al, Hematology ASH Educ Program., 2011; p.90-95). From 1996 till 2015 385 pts were treated in National Research Center of Hematology, Moscow, Russia. Seventeen (4,4%) pts ( 8 men and 9 women; median age 23 years) with AA (6 severe and 11 moderate AA) were diagnosed with clonal evolution to MDS, AML and CMML. At the moment of AA diagnostics all those 17 pts had normal cytogenetics and were treated by: ATG+CsA+/-splenectomy (n=8); CsA+/-splenectomy (n=9). G-CSF was not administered in any case. Primary response (hematological response) was registered in all 17 pts with the further development of CR in 13 pts; median response duration till the clonal evolution was 78 (10-360) months. Three pts at the time of progression are conducted IST (CsA). Clonal disorders developed at a median of 80 (47-408) months from the time of AA diagnosis. The median age at the moment of clonal evolution was 34 (27-60) years. Abnormalities of chromosome 7 were found in 8 pts (44%). In pts with monosomy 7 the median time response duration till the clonal evolution was twice less than in pts with another abnormalities of karyotype (70 (47-209) months vs 145 (70-408) months). At the moment the clonal expansion was detected all the 17 pts had morphological characteristics of dismyelopoesis, however, bone marrow was hypercellular in 7 cases, hypocellular - in 4; mixed (hyper- or hypoplasia) - in 4 cases. Fibrosis was found in 2 pts, it was difficult to detect karyotype in these cases (not mitosis). The therapy was conducted in accordance to the type of disease and bone marrow cellularity (IST, hypomethylating agents, chemotherapy). Five pts were transplanted from allogeneic donors. The median life duration since clonal evolution was 22 (2-203) months. Five of the 17 pts are alive ( the median - 64(5 -203) months): after allo- HSST (n=3); IST (n=1); "watch and wait" strategy (n=1). Conclusion: Our 20 years study of a big cohort of AA pts has demonstrated low frequence of clonal evolution to myeloid disorders - 4,4%. We suggest that the absence of G-CSF in our IST programs may contribute to this fact. Almost half of AL/MDS cases harboured monosomy 7 and the median time to clonal evolution was twice less to this pts (70 vs 145 months). The only curative approach in case of clonal evolution to AL/MDS in pts with AA is allo-HSST. Disclosures No relevant conflicts of interest to declare.

Background: Approximately 50% of diffuse large B-cell lymphomas (DLBCL) are defined as high-grade by IPI. They are characterized by aggressive course and poor response to standard chemotherapy (CT): 5-years overall survival (OS) rate of less than 30%. R-DA-EPOCH has demonstrated very optimistic results (overall and progression-free survival (PFS) were 90%), but high-risk patients (IPI 3-4) showed only 43% of OS and PFS [1]. The addition of high-dose AraC (12 g/m2) to the upfront therapy of high-risk DLBCL has significantly improved the outcome on Hyper-CVAD/HMA and mNHL-BFM-90 protocols [2, 3]. But high toxicity of these protocols restricts their application. We suggested that addition of courses R-HMA in rotation with R-DA-EPOCH could improve the treatment outcome and decrease toxicity. Aim: To evaluate the efficacy and toxicity of R-DA-EPOCH/R-HMA protocol in patients with untreated high-grade diffuse large B-cell lymphoma. Patients and Methods: 33 untreated DLBCL patients from 4 centers were enrolled in a prospective study between August 2013 - July 2015; stage II-IV; ECOG 0-3; median age 55 years (27-76); age ≥60y/ Results: The median follow-up is 12 months (4-24). There was no mortality associated with toxicity. The main non-hematological toxicities of R-HMA were infections (mucositis, pneumonia, sepsis, enteropathy) grades 1-2 and 3-4 in 90% and 10%, respectively. Hematological toxicity grade 4 for less than 4 days we observed only after courses R-HMA. Complete remission (CR) was achieved in 29 (88%) patients. In 2 patients we observed progression of the disease after first cycle of chemotherapy, in another 2 cases - partial remission after 2-3 cycles and following progression of disease. In patients older than 60 years with doses reduction in R-HMA failures were absent, except one later relapse after 13 month CR. With a median follow 12 months overall and disease-free survival of 33 patients constituted 90,5% and 74% , respectively. In a group of patients older than 60 years results of therapy seemed to be better than in young patients: OS were 100% vs 85,6% (p=0,1), DFS were 80% vs 74,9% (p=0,2), respectively. So the combination of R-DA-EPOCH/R-HMA may be considered as optimal intensive approach in the older patients. Conclusions: TheR-DA-EPOCH/R-HMA protocol demonstrated acceptable toxicity and high efficacy in patients with high-grade DLBCL. This protocol has shown optimistic results in the elderly patients and it could be recommended for further investigation in that group. Ññûëêè: 1. Salit RB, Fowler DH, Wilson WH, et al. Dose-adjusted EPOCH-rituximab combined with fludarabine provides an effective bridge to reduced-intensity allogeneic hematopoietic stem-cell transplantation in patients with lymphoid malignancies.J Clin Oncol. 2012. 10;30(8):830-6. 2. Oki Y, Westin JR, Vega F, et al. Prospective phase II study of rituximab with alternating cycles of hyper-CVAD and high-dose methotrexate with cytarabine for young patients with high-risk diffuse large B-cell lymphoma. Br J Haematol 2013; 163(5):611-20. 3. Magomedova AU, Kravchenko SK, Kremenetskaia AM, et al. Nine-year experience in the treatment of patients with diffuse large B-cell lymphosarcoma. Ter Arkh. 2011;83(7):5-10. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Introduction. Patients (pts) with oncopathology who have hyperexpression of c-MYC demonstrate low response to chemotherapy and poor prognosis. In our study we investigated the level of c-MYC gene expression in pts withmultiple myeloma (MM) and/or monoclonal gammopathy undetermined significance (MGUS) at the moment of MM/MGUS diagnosis. Patients and Methods. 40 patients with newly diagnosed MM/MGUS and 9 healthy bone marrow donors (as a control group) were included in our study. The median age of patients with MM/MGUS was 56 years (29-77), donors - 32 years (24-41). The bone marrow collection for examination was done at the moment of diagnosis. The investigated material was divided into 2 samples: all mononuclears and separated CD138+ cells. Level of c-MYC expression was identified by quantitative real time PCR (qRT-PCR). The whole number of qRT-PCR analysis was 98. Induction therapy (VCD/PAD) was initiated for 36 patients with MM, 4 patients with MGUS remained under observation. Antitumor response was evaluated after induction therapy according to the IMGG criteria. Results. From 98 samples among pts reliable results qRT-PCR and evaluated c-MYC expression were in 36 samples from total mononuclears and 17 from separated CD138+ cells. In all 18 samples from healthy donors: 9 from total mononuclear, and 9 from CD138+ cells results of qRT-PCR were reliable. Mean c-MYC expression level among MM/MGUS pts was 55.014 (2ΔCt) in CD138+ cells and in samples from total mononuclears - 17.585 (2ΔCt). In healthy donors the results were 7.183(2ΔCt) and 1.907 (2ΔCt) respectively. After induction therapy complete response (CR) was demonstrated 6 patients, very good partial response (VGPR) - in 7 patients, partial response (PR) - in 15 and other 5 pts were refractory. Mean c-MYC expression level in samples from separated CD138+ cells in pts with CR was-14.259 (2ΔCt), with VGPR-38.314 (2ΔCt), with PR-50.506 (2ΔCt), and in refractory pts-181.271 (2ΔCt), among patients with MGUS the level was 10.928 (2ΔCt). In samples from mononuclears: 5.138, 30.355, 11.877, 38.911, 7.323 (2ΔCt) respectively. Thus, response after induction therapy was worse among patients with high gene c-MYC expression (Figure 1 a). Such a correlation was not revealed in analysis of data from samples of total mononuclears (Figure 1 b). Conclusion. Mean c-MYC gene expression level in CD138+ cells among MM/MGUS pts was higher than among healthy donors (p Disclosures No relevant conflicts of interest to declare.

Introduction. MYC increases proliferative capacity of malignant B-cells, independently from mechanisms led to increased protein expression. Tumors with solely MYC expression are highly effective curable with doses intensification. Ability of cells to escape apoptosis by several mechanisms like BCL2 or P53 соехpression promotes malignant B-cells growth and survival and represents a big therapeutic problem. Aim. To investigate mechanism of MYC hyperexpression in absence of c-MYC -rearrangement in DLBCL (diffuse large B-cell lymphoma). To analyze efficacy of intensive chemotherapy in pts with DLBCL who underwent modified NHL-BFM-90 (m-NHL-BFM-90) plus rituximab (R) in correspondence with MYC and BCL2 protein expression. Patients and methods. Data of 62 DLBCL pts (35 males and 27 females) who underwent m-NHL-BFM-90+R in National Research Center for Hematology (Moscow) between 2004 and 2013 years were analyzed. Tumor samples were stained with antibody to BCL2 (clone 124, Dako) and MYC (clone Y69, Epitomics). We used a previously reported cut off for MYC expression ≥40% and BCL2 ≥50% (N. Johnson et al., 2012). G-banding data were available in 19 pts. In one case was revealed c-MYC rearrangement into heavy chain locus t(8;14)(q24;q32). In all other cases FISH didn't reveal c-MYC orBCL2 rearrangements with DNA probes Vysis LSI MYC Dual color, Break Apart Rearrangement Probe, Vysis LSI BCL2 Dual color, Break Apart Rearrangement Probe. RQ-PCR was performed in 17 cases to estimate mRNA level of c-MYC expression relatively to ABL. To assess relation between mRNA of c-MYC and MYC protein expression was used correlation analysis (R²). To estimate treatment results were performed Kaplan-Meyer and Cox regression analyses (SAS 9.3). Results: Majority of pts - 48/62 (78%) attended to a high-risk group according IPI (3-5). 9/62 (14,5%) patients had MYC+/BCL2- tumors, 15 (24%) - "double-expressor" (DE) MYC+/BCL2+, 21 (34%) - MYC-/BCL2+, 17 (27,5%) - MYC-/BCL2-. Median age of DE pts was statistically higher than others (61 (25-73) vs 47 (15-73) years old, P Conclusion. We illustrated that MYC protein expression level correlates with c-MYC transcriptional activity in absence of c-MYC rearrangement. MYC hyperexpression alone didn't influence on prognosis, only coexpression of MYC and BCL2 had a crucial role increasing probability of relapse or progression in DLBCL patients. Disclosures No relevant conflicts of interest to declare.

Background: Castleman' s disease (CD) is rare lymphoproliferative disorder with long asymptomatic current and high risk of transformation in malignant lymphoma. For last years, diagnostics of CD improved and uniform classification of an illness was accepted. Depending on morphological features allocate 3 variant of CD: hyaline-vascular (HVV), plasma cell (PCV) and mixed cell. Most cases of the ÑD involve a single or localized group of lymph nodes and can be asymptomatic. In these cases the histologic picture can be characterized as a HVV or mixed cell and PCV morphology. Less often the disease proceeds with multiple lymphoid regions (multicentric CD, MCD) and is usually accompanied by various systemic inflammatory symptoms (fever, weight loss and night sweats). In these cases histologic picture in the involved lymph nodes meet mix cell or PCV of CD. The role of a human herpes virus-8 (HHV-8) in pathogenesis of MCD is studied. It is known that HHV-8 codes in a genome of a cell of the person some regulatory proteins and cytokines, first of all a virus homolog interleukin-6 which stimulates development of human IL-6 and endothelial factor of growth that conducts to a neoangiogenesis in not tumoral lymph nodes. HHV-8-positive MCD allocate separately in due to its extremely aggressive course and a high risk of transformation into HHV-8-positive plasmablastic lymphoma. Aim: Explore the features of a clinical current and of therapeutic approaches at different variants CD. Patients and Methods: In Center for Hematology since 1996 to the present time observed clinical and morphological features of 76 pts with CD. Clinical data were obtained during the retrospective analysis of 17 outpatient clinical records and prospective supervision over 59 pts. The diagnosis was established from results of histological and immunohistochemical examinations of removed lymph nodes in all cases. Frequency analyses were performed using z-tests with SAS 9.1 software. Results: HVV with local lymph nodes involvement was diagnosed in 38 (50%) pts. The average age of pts was 40 years The PCV was also observed in 38 (50%) cases, among them in 17 (22%) pts were found to have local involvement and 21 (28%) had multicentric involvement. Five (24%) pts with MCD were established to be infected with human herpesvirus type 8. Male (37 pts) and female (39 pts) are suffering with an identical frequency. However, HVV is more often diagnosed for female, than at male (71% to 29% respectively), PCV equally often meets both at male, and at female (47% of male, 53% of female) and, at last, at MCD the share of male statistically significantly is more than share of female (86% against 14%, p=0,05) (Figure 1). The basic involvement areas in local HVV and PCV were peripheral (38%), mediastinal (29%), retroperitoneal (18%) abdominal (9%) and small pelvic (6%) lymph nodes (95% CI). Local HVV and PCV proceed benign and in 94.5% of cases are cured by surgical removal of lymph nodes involved in the pathological process, in 5.5% of cases with inoperable cases require followed by radiation therapy. MCD was significantly more common in male, occurs aggressively with severe constitutional symptoms, generalized lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, autoimmune hemolysis, thrombocytopenia, and involvement of extranodal foci in the pathological process. MCD transformation to plasmoblastic lymphoma was observed in 4 of 5 HHV8-positive pts and followed by a poor outcome. The prognosis of untreated MCD was unfavorable. In a number of cases prednisolone monotherapy worsened and the MCD pts receiving timely R-CHOP or R-VD chemotherapy could achieve sustained remission (the 5-year OS was 55%). Conclusion: The prognosis of HVV and local PCV is favorable: the disease is surgically cured in 95% of cases. Chemotherapy according to the program of Non-Hodgkins Lymphomas indicated for the treatment of MCD: sustained remission can be achieved by the use of R-CHOP or R-VD programs. HHV8-positive MCD characterized by high risk disease transformation to incurable plasmablastic lymphoma. Overall, prognosis and therapy choice in HIV-negative pts with CD depend on the histological type of the disease, the extent of a tumor and HHV-8 infection. Figure 1. Gender distribution for three variants of CD; there is male-female imbalance in HVV and MCD groups (z-test, ð Disclosures No relevant conflicts of interest to declare.

Introduction It's generally considered that all alloimmune process such as acute graft-versus host disease (aGVHD) after allo-HSCT are mostly controlled by lymphocytes. The role of neutrophils in systemic alloimmunity after allo-HSCT is still illusive. In 1987 a distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with system lupus erythematosus has been described. There is no LDG's in healthy donors. While the origin and role of LDGs still needs to be fully characterized, we try to describe this population in patients with hematological malignancies after allo-HSCT Patients and methods. Peripheral blood samples were collected in EDTA-tubes before allo-HSCT, on day +30,+60,+90 after allo-HSCT and at day of aGVHD from 47 patients with hematological malignancies (AML=22, ALL n=17, LPD=3, MDS =2; CML=2; 17 with active disease, 30 - in CR) after allo-HSCT (from matched unrelated donor n=34, from matched related donor n=13; MAC = 13, RIC=34). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD66b-PE (Biolegend, USA) antibodies and FSC/SSC were used to determine LDGs cells as FSChigh \SSChigh \CD66b+. 100000 of cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. Results of blood evaluation of 47 patients with hematological malignancies, whose blood was examined after allo-HSCT presented in table 1. Conclusion Despite the fact that we don't get significant differences. LDG's detection in allo-HSCT patients need further investigation. Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Disclosures No relevant conflicts of interest to declare.

Introduction. Favorable therapeutic regimen for pregnant women with primary mediastinal B-cell Lymphoma (PMBCL) is determined by tumor mass, somatic status, period of pregnancy and potential risk of teratogenic effects of medications which penetrate through placenta. Aim. To estimate chemotherapy efficacy of PMBCL in case of pregnancy and its toxicity for fetus. Patients and Methods. Since 2004 to 2015 years in National Research Center for Hematology were treated 106 patients with primary mediastinal B-cell lymphoma. In 8 (7,5%) from them disease debuted during 2-3 trimester of pregnancy, one women had two fetuses simultaneously. Before delivery women underwent induction chemotherapy with VACOPB- regimen in 5/8 cases and with R+DAEPOCH in 3/8 cases. Results. After induction treatment had been performed 6/8 women achieved a partial remission, 1/8 had progression, 1/8 - complete remission. High dose chemotherapy was performed in 7/8 women after 1 month following delivery. It was born 9 children (4 boys and 5 girls). Median body weight was 2000 g. (1300 - 3600 g.). Median growth was 47 cm. (40 - 53 cm). In two cases when rituximab had been administered to women before delivery, children were diagnosed with intrauterine pneumonia. One child, born from woman after VACOPB chemotherapy had thrombosis of superior vena cava. All women continue to be in complete remission of PMBCL, children are practically healthy without malformations. The median follow-up of patients was 40 months (15 - 78 months). Conclusions. Pregnancy is not a contradiction for induction chemotherapy of PMBCL in 2-3 trimesters. Rituximab shouldn't be administered before delivery because of a high risk of infectious in fetus. Disclosures No relevant conflicts of interest to declare.

Background: Course of multiple myeloma (MM) is associated with hemostatic disorders that can lead to bleeding or thrombosis. According to previous studies the most risky period of time for thrombotic complication is the first year after diagnosis. Aims: The study was aimed to analyze of hemostasis conditions in newly diagnosed (ND) MM patients (pts) and to compare their initial status with the status after induction therapy (IT) and with the status of healthy volunteers (HV). Patients and Methods: 17 pts with ND MM: 10 males, 7 females at the age of 26-72 (median age - 54) were involved in the study. The distribution of the stages among the participants according to Durie-Salmon system: stage I - 1 pts, stage II - 5 pts, stage III - 8 pts, stage III in 8 pts. Immunochemistry variants: IgG - 11 pts, IgA - 2 pts, MM B-J - 4 pts. Average paraprotein (PP) level was 34g/L (0,8 - 78). HV group consisted of 26 persons without serious diseases, 40 - 70 y.o., (median age 50). Hemostasis analysis was taken place twice: before and after the IT which included 4-8 bortesomib-containing cycles (PAD, VCD, VD). After the IT 6 pts achieved CR, 5 pts - VGPR, 4 pts - PR and 1pt was resistant. Average value of PP decreased to 8 (0,1 - 29,7). Routine tests: activated partial thromboplastin time (APTT, normal rate 25-38 sec), international normalized ratio (INR, normal rate 0.85-1.15), D-dimer concentration (normal rate 0-500 mkg/l). Two global tests: thrombin generation test by using endogenous thrombin potential (ETP, normal rate 760-1450 nM*min) and thrombodinamics (TD), characterized by the initial clot growth velocity (Vi, normal rate 36-56 um/min), stationary velocity (Vst, normal rate 20-30um/min), and density of a fibrin clot measured by light scattering from clot (D, normal rate 16000 - 32000 conventional units). Hypocoagulability wasassessedby data APTT > 38 sec, INR> 1.15, ETP < 760 nM*min, Vi < 36 um/min, Vst < 20 um/min, D < 16000 cu. Hypercoagulability was considered in cases when APTT < 25 sec, INR < 0,85, D-dimer > 500 ng/ml, ETP > 1450 nM*min, Vi > 56 um/min, Vst > 30 um/min, D > 32000 cu. Statistics. Ðaired t-test and Pearson correlation coefficient (r) have been got with SAS 9.1. 95- percent CI has been used. Results: ND MM APTT evaluation demonstrated normal coagulation in 13pts and extended one in 4pts (48 sec [38-57]). INR was normal in 13 and increased in 4 cases (1.3% [1.2-1.5]). Increased concentration of D-dimer was revealed in 7pts (3810 ng/ml [350-7270]). ETP was normal in 12 and increased in 5 cases (1592 nM*min [1406-1779]). Vi was normal in 11 and increased in 6pts (62 um/min [58-65])). Vst was reduced in 1pts (15um/min), normal in 10pts and increased in 6pts (36um/min [28-44]). Density of clot was normal in 8 pts and decreased in 9pts (9769 UE [6379-13160]). (Tab. 1) After IT only 1 pt had elongated APTT (44sec), the results of the others were normal. INR was normal in all cases. D-dimer concentration was normal in 12pts and increased in 5pts, but this data was statistically less (1080ng/ml [500-1003]) than in ND MM. ETP was normal in 13pts, elevated in 4 pts (1604 nM*min [1099-2109]). Vi was normal in 12pts and increased in 4pts (61 um/min [57-65]). Vst was decreased in 2pts (13 and 14um/min), normal in 13pts and increased in 2 (32 and 43um/min). Density of clot became normal in 14pts, but clot remained not dense enough in 3 pts (9512 [13606-15779]). (Tab. 1) We found that inverse linear relationship between PP level and blood clot density was more significant in ND MM (Fig. 1, fig.2). Statistically significant difference was revealed between clot density in ND MM and pts in PR or CR and between them and HV (Fig. 3). Furthermore, a significant difference was identified between D-dimer concentration in ND MM pts and after IT (Fig. 4). Conclusions: MM pts have complex disorders of hemostasis characterized by a tendency to hyper- and hypocoagulation at the same time. In spite of an increased tendency to thrombosis confirmed by D-dimer, ETP, TD, pts are under risk of bleeding related to the formation of defective clot and elongated APTT. According to the obtained results one can assume that PP embedded in the clot and disorder its structure. However, further studies are needed to confirmthat assertion. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

Introduction The majority of clinical recommendations on MDS indicate the use of IST for low-risk hypoplastic MDS patients. ATG followed by cyclosporine A (CSA) is recommended for younger pts with normal karyotype or with +8, CSA - for older patients with autoimmune phenomenon or hypoplastic bone marrow. There are no reports on the use of CSA with splenectomy as a combined IST for MDS patients. Here we report the long term (20 years) results of the single center trial comprising 61 MDS pts treated with CSA as the first or the second line treatment + splenectomy. Patients and methods. Since 1995 till June 2015, 61 patients were included in the combined IST protocol. There were 34 males, 27 females; median age constituted 39 years (16-74), refractory cytopenias with multileaneage dysplasia = 46, RAEB1/2 = 15; IPSS - low-risk= 4, intermediate 1 =40, intermediate-2 =9, high =4; no risk evaluation - 4 patients. All patients had trepanobiopsy evaluation, half of them - bilateral. It worth to note that bilateral trepanobiopsy permitted to redefine cellularity in 15% of patients. Pure hypoplastic forms of MDS were registered in 38 patients (63%), combination of hypoplasia with hyperplasia or normal cellularity - in 10 (16%), hyperplastic forms - in 13 (21%); non-clonal lymphoid nodules in b/m were detected in 29 pts (46%); fibrosis - in 7. Cytogenetics was done in 57 pts, 32 had normal karyotype; 5 had trisomy 8; 8 - high risk cytogenetics (-7/7q-, complex); 8 - different intermediate. Cyclosporine A was applied at a dose 5 mg/kg per day, followed by reevaluation of response at 2-3 months. In case of response CSA was continued, if there was no response or it was inadequate - splenectomy was done followed again by CSA; if progression occurred - CSA was stopped. We consider splenectomy as a component of the programmed IST as it leads to removement of a big lymphoid organ. For younger patients ATG for 20 mg/kg for 5 days could become the 2nd or the 3rd line of treatment. For patients with sibling donors allogeneic HSCT was an option. Analysis was carried out in June 2015. The patients were grouped for analysis according to cellularity (hypo vs hyper, in case of combination hypo with hyper/normal the case was considered as hypocellular), % of b/m blast cells (<> 5%), IPSS index (low/int-1 vs int-2/high), cytogenetics, the presence of lymphoid nodules. Results. CSA was started as the 1st line treatment in 42 patients, as the second line (after ATG or splenectomy or ESA) - in 20. 5 patients were transplanted from sibling donors. 35 patients received CSA as monotherapy (16 of them had a progression or a transformation to AML). Totally splenectomy was performed in 20 patients, in 8 patients it was followed by additional immunosuppression with ATG (9 patients) or MMF (1 patient). Splenectomy was performed by laparoscopy, no deaths or sever complications were noted. Half of the splenectomized patients achieved sustained response after operation and continuation of CSA. On the whole 18 patients had progression to RAEB or transformation to AML (1 from low-risk group, 7 - int1, 6 - int2, 5 - from high risk). Complete remission was achieved in 15 patients (24%), with overall response rate of 56% (n=34) with a median duration of response - 21 months (1-230 mo). The 20-years survival data revealed highly statistical difference between hypoplastic forms of MDS vs hyperplastic (38% vs 0%), bone marrow blast cells less vs more than 5% (43% vs 5%), IPSS categories low/int1 vs int2/high (50% vs 0%), cytogenetics favorable vs intermediate vs high (62% vs 35% vs 0%) (Pic.1). No differences were detected in patients with or without lymphoid nodules in b/m (38% vs 35%). Conclusions Our small but prolonged study has demonstrated a substantial long-term efficacy of the combined IST. CSA followed by splenectomy is a reasonable treatment approach in MDS patients with hypoplastic features in bone marrow, normal or intermediate karyotype, low blast cell count, low/int1 IPSS score. Pic.1 Overall survival of MDS patients treated with combined IST depending on cellularity, blast cells count, cytogenetic features and IPSS score. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Allogenic bone marrow transplantation (allo-HSCT) with high-dose, post-transplantation cyclophosphamide (CY) nowadays has become an alternative for standard immunosuppression. Many groups have shown that high dose CY after allo-HSCT selectively deplete alloreactive T effector cells and downregulate Granzyme B expression in CD8+ T and NK cells after allogeneic HSCT. Despite that fact there is no data about other T cell populations, such as Tregs and their immunosuppression abilities after post-transplant CY. We studied Granzyme B expression in Treg on day +14 after allo-HSCT with post-transplant CY and standard immunosuppression. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +14 after allo-HSCT in patients with hematological malignancies with post-BMT-CY alone (n=8) and patients with standard immunosuppression (post-HSCT CSA, MMF or MTX at standard dose) (n=18). The anti-CD4-PE-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine Treg cells population as CD4+ CD25high CD127low. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms and etc. CD4- lymphocytes (certainly containing CD8+ and NK-cells population that obligatorily express granzyme B) were used as internal positive control to define population of CD4+ CD25high CD127low GranzymeB+ cells and gMFI (geometric mean fluorescence intensity). 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. Mann-Whitney U test was used to test for differences between Granzyme B+ Treg cells after post-transplant-CY alone and in a group of standard immunosuppression. The percent of CD4+ CD25high CD127low GranzymeB+ cells among CD4+ cells at day +14 after post-transplant CY alone was statistically higher (30.1±21,9; p=0.018*) than in patients with standard immunosuppression (3,52±1,56) (see Chart 1). There is no differences in Granzyme B expression (gMFI) in CD4+ CD25high CD127low cells after post-transplant-CY (2131,8±412,7) alone and in a group of standard immunosuppression (1718,17±316,8; p=0.541). It's worth to note that probably this mechanism in combination with depletion of alloreactive T effector cells and downregulation of Granzyme B expression in CD8+ T and NK cells help to prevent aGVHD in this group of patients. Conclusion We suggest that post-transplant CY spares Granzyme B expression in Tregs in a patients with post-transplant-CY and help to prevent aGVHD development in this group of patients. Despite the fact that the analyzed group is small, obtained data is important and needs further investigation. Figure 1. Granzyme B expression in Tregs after post-transplant-CY alone and in a group of standard immunosuppression. Figure 1. Granzyme B expression in Tregs after post-transplant-CY alone and in a group of standard immunosuppression. Disclosures No relevant conflicts of interest to declare.

Aim. To evaluate late nephrotoxicity of intensive modified program NHL-BFM-90 (mNHL-BFM-90) in adult poor-prognosis patients with diffuse large B-cell lymphoma (DLBCL). Materials and methods. The data of laboratory tests of blood and urine of 40 patients with DLBCL which received mNHL-BFM-90 protocol in the Hematology Research Center in the period from 2002 to 2009 years was analyzed. Group consisted of 20 men, 20 women, aged from 31 to 76 years, median age - 56.5 years at the time of the survey, the median period after therapy- 6 years. Individual cumulative dose of ifosfamide - 8-12 g / m 2 , cyclophosphamide - 2-3 g / m 2 , methotrexate - 6-9 g / m 2 . Comparison group included 19 patients (8 men and 11 women), aged from 39 to 78 years, median age - 70 years at the time of the survey, who received therapy to CHOP or R-CHOP. The median period after therapy - 5,5 years. Individual cumulative dose of cyclophosphamide - 3-6 g / m 2 . Laboratory tests of blood and urine were performed before chemotherapy and after 5 years after treatment. Results. From all 40 patients with DLBCL received mNHL-BFM-90, signs of chronic kidney disease were found in 32 (80%) patients, only 2 (5%) of them showed a decrease in creatinine clearance daily. In comparison group signs of chronic kidney disease were found in 12 (63%) patients, 2 patients (10.5%) showed a reduction in the daily creatinine clearance. No significant differences in the manifestations of nephrotoxicity in the long term between comparison groups were found. No significant differences in the manifestations of nephrotoxicity in the long term period were found between two groups of patients (who underwent and not underwent acute renal failure during high-dose chemotherapy). Amount of nephrotoxicity signs was significantly dependent on the presence of hypertensive disease in history (p = 0.02). Conclusion. Nephrotoxicity of high-dose mNHL-BFM 90 exceeds significantly nephrotoxicity program CHOP / R-CHOP. Conducting high-dose chemotherapy in 10 (25%) patients complicated by acute renal failure, 2 (20%) patients required hemodialysis in contrast to the group of standard chemotherapy without such complications (p=0,01). However, in the long term observations there weren9t significant differences in the nephrotoxic manifestations between two groups. Impaired renal function in the long term period observed in 100% of patients undergoing acute renal failure during treatment and only in 71% of patients without acute renal failure (p=0,04). It was comparable repairing of renal function in the long term in patients undergoing high-dose therapy between patients with and without acute renal failure among those who showed signs of chronic kidney disease. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma. Activated B-cell like (ABC) molecular subtype DLBCL is characterized by more aggressive behavior and poor clinical outcomes with standard R-CHOP chemotherapy. Approximately 30% of all patients (pts) with ABC DLBCL have recurrent mutation in the MYD88 gene (a change from leucine to proline at position 256 - L256P). We report the impact of the MYD88 mutation status on the clinical course and outcome of pts with ABC DLBCL. METHODS In present study MYD88 mutation status was investigated in untreated pts with ABC DLBCL. The diagnosis of ABC DLBCL was established according to the WHO 2008 classification. DNA from 41 cryopreserved and 12 formalin-fixed paraffin-embedded tumor samples were tested in our study. Sanger sequencing and real-time allele-specific PCR was used to assess MYD88 L265P mutation status. RESULTS We report data for 53 pts (34 male, 19 female) with a median age of 53 (18-74) years. MYD88 L265P mutation was detected in 26,4% (14/53) of pts. The international prognostic index (IPI) score was 4-5 in 7/14 (50%) of pts with MYD88 mutated DLBCL and 12/39 (30,8%) pts with MYD88 wild type DLBCL, whereas nearly half of all pts presented with Ann Arbor stage IV disease. The majority of pts 13/14 (93%) with MYD88-positive DLBCL had elevated lactate dehydrogenase levels versus 26/39 (66%) pts with MYD88 unmutated DLBCL. Extranodal was more common in pts with MYD88 mutation than in pts with MYD88 unmutated DLBCL (78,5% vs 54%). All pts received high-dose chemotherapy and were followed for 50,5 (11-95) month. Pts with MYD88 mutation had similar complete response rate comparing to pts without MYD88 mutation (78,6% vs 84,6%). The disease relapsed in 45,4% of pts with MYD88 mutation and in 24,2% of pts without MYD88 mutation. Mortality was in 45,4% of pts with MYD88 mutation and in 24,2% of pts without MYD88 mutation. Overall survival (OS) was better in pts with MYD88 unmutated DLBCL than in pts with MYD88 positive DLBCL (5-year OS, 73% vs 31%). CONCLUSION IPI high risk and extranodal involvement is common in ABC DLBCL pts with MYD88 L265P mutation. MYD88 L265P mutation can be a predisposing factor for poor OS probability in pts with ABC DLBCL. Disclosures No relevant conflicts of interest to declare.

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is a rare tumor derived from follicular T-helper and characterized by low response rate and relapsing course to standard therapy. CHOP-like therapy shows 70-80 % of overall response rate, though 2-year progression-free survival is observed only in 30-40 % of patients. Unsatisfactory results of treatment angioimmunoblastic T-cell lymphoma by ÑÍÎÐ-like therapy require searching new approaches to therapy. Aim: To define a rational approach to treatment of angioimmunoblastic T-cell lymphoma. Patients and methods: Ñlinical outcomes of 23 patients with newly diagnosed AITL treated between June 2002 and July 2015 we retrospectively analyzed. The diagnosis was verified according to WHO classification. Results: Among the 23 patients 14 men and 9 women were included with a median age of 63 (range, 29-81). Twenty-two of 23 patients had Ann Arbor stage IV. Bone marrow involvement was detected in 21 of 23 cases by histological investigation. Nine of 23 patients received CHOP-like therapy; other 14 patients were treated prolonged therapy. Eight (89%) of 9 patients after CHOP-like therapy had treatment failure. Time to disease progression ranged from 1 to 7 months (median 3). Fourteen of 23 patients were treated by prolonged therapy consisting of low dose cytotoxic drugs weekly and supporting the therapy by interferon-A and thalidomide. Supporting therapy has administrated after achieving complete remission and was continued in 2 years. Prolonged therapy wasn't finished in 3 patients with severe infections and somatic pathology. Eleven (79%) of 14 patients treated by prolonged therapy achieved complete remission, treatment was finished only in 5 of 11 patients; follow-up varies from 3 to 67 months, a median 29. Univariate analysis of progression-free survival curves showed that prolonged therapy is more effective than CHOP-like therapy (p < 0.01) (Fig.1). Prolonged therapy was performed in 4 of 8 patients with progression disease after CHOP-like therapy; 2 patients achieved a complete remission, continuing 41 and 77 months. Conclusion: Although, this was a small retrospective study, the results show that efficiency of prolonged therapy is better than CHOP-like therapy in patients withangioimmunoblastic T-cell lymphoma. Prolonged therapy allows preventing early treatment failures. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a heterogeneous subgroup of PTCL with no consistent immunophenotypic, genetic and clinical features. PTCL-NOS represents less than 29% of all T-cell lymphomas in adults and often has an aggressive course with extranodal and systemic involvement. This study is focused on the application of clonality testing methods for PTCL-NOS diagnosis and monitoring. Patients and Methods: Biopsies, blood and bone marrow samples were tested for 30 patients with PTCL-NOS: the ratio of male/female was 15/15, median age was 56 years (32-75), and 22 of 30 patients had IV stage of the disease. TCRG and TCRB gene rearrangements were evaluated for T cell clonality analysis. We used PCR-based methods (BIOMED-2 standardized clonality protocol) followed by GeneScan analysis on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Results: Clonal TCR gene rearrangements were found in 29 of 30 patients (97%): TCRG in 27 of 30 (90%) and TCRB in 29 of 30 (97%). Primary biopsies or bone marrow samples showed oligoclonal pattern of rearrangements (more than 3 clonal peaks in TCRG or TCRB GeneScan profiles) in 13 of 30 patients (43%). New clonal rearrangements in affected tissues (skin, tonsil, CSF, lymph nodes and etc.) during disease progression were found in 15 of 24 (63%) patients. Various representations of clonal rearrangements in different tissues indicate the presence of several tumor clones in the majority of cases (19 of 30 patients; 63%). The presence of several clones correlated with the number of tissues examined (rs = 0.6, p Conclusion: Oligoclonality, clonal evolution and the appearance of new clones in the progression of the disease are typical features of PTCL-NOS. Multiple clonal rearrangements complicate diagnostics and assessment of minimal residual disease in this disease. Disclosures No relevant conflicts of interest to declare.

Background: Except multiple myeloma secretion of monoclonal paraprotein may be associated with several malignant conditions (lymphomas), they are produced by abnormally expanded single clone of plasma cells in an amount that can be detected in serum, urine, or rarely in other body fluids. A secretion of monoclonal paraprotein related to the neoplastic clone, was detected in more than 10% of newly diagnosed diffuse large B-cell lymphoma (DLBCL). M-protein in DLBCL might be easily missed, the rarity of associated clinical signs and the rapid fading during treatment. This group of DLBCL showed homogeneous morphologic and immunohistochemical features consistent with the non GCB-type. FISH analysis was negative for c-MYC gene rearrangements. M-protein in DLBCL patients, might be related to a very poor non GCB-type subset who should be given upfront intensified therapies [ASH 2012, abstract 2659]. The role and biological relevance of monoclonal paraprotein in diffuse large B-cell lymphoma is unknown. Aims: to compare the clinical and biological features of DLBCL patients with and without secretion of paraprotein. Patients and methods: We included in the study 86 high-risk DLBCL patients, diagnosed between July 2007 and September 2014. All patients were treated with intensified therapy (mNHL-BFM-90 or R-DA-EPOCH/R-HMA). 14 cases (16%) with monoclonal secretion paraprotein was identified. The Hans algorithm was used in order to classify cases of DLBCL as GCB-type and non GCB-type at the moment of diagnosis. Bone marrow involvement was identified by histological and B-cell clonality study. Results: We detected different types of protein in DLBCL: Mk (in 5 patients), Bence-Jones k (3 pts), Gk (2 pts), Ak (1 pts), Mλ (2 pts), Bence-Jones λ (2 pts), Gλ (2 pts), Aλ (1 pts). Four cases have shown 2 different clones simultaneously. Secretion ranged from trace to 27,1 g/l. 12 of 14 cases with monoclonal secretion were classified as non-GCB-type DLBCL. Characteristic of patients with monoclonal secretion were: stage III-IV; ECOG 2-3; median age 57 years (28-71); age ≥60y/ Conclusions: Our study has shown that monoclonal secretion of paraprotein was associated with bone marrow involvement in patients with diffuse large B-cell lymphoma. Detection of secretion of paraprotein could help to suspect the cases with bone marrow involvement before the results of histology and when histology not informative. Diffuse large B-cell lymphoma with bone marrow involvement is characterized by aggressive course and poor response to standard chemotherapy. Disclosures No relevant conflicts of interest to declare.

Introduction. Primary mediastinal B-cell lymphoma (PMBCL) amounts from 0,9 to 4% of Non-Hodgkin Lymphomas. Predominantly suffer young women. All relapses in PMBCL occur as early events. Taking into account unfavorable prognosis in case of relapse or progression of PMBCL (2-year overall survival (OS) is about 15% [Kurvilla J., 2008]), it's necessary to achieve a complete remission (CR) in first line treatment. Aim. To compare an efficacy of R-DA-EPOCH followed by R-DHAP and autologous peripheral blood stem cell transplantation (autoSCT) with BEAM conditioning regimen with modified (m) NHL-BFM-90 chemotherapy protocol in pts with partial remission (PR) of PMBCL. Patients and Methods. In 107 pts (34 males, 73 females) diagnosis of PMBCL was established according to WHO criteria. All pts had stage II of disease according to Ann Arbor classification. 30 pts received R-DA-EPOCH, 77 pts - m-NHL-BFM-90 ± R. Median age of pts in R-DA-EPOCH group was 28 years old (from 20 to 67 years old); in m-NHL-BFM-90 ± R-group - 77 years old. Increased LDH concentration was revealed in 24 from 30 (80%) pts in R-DA-EPOCH group and in 75/77 (97%) pts from m-NHL-BFM-90 ± R group. Bulky mediastinal disease was in 30/30 (100%) pts in R-DA-EPOCH group and in 59/77 (76%) - m-NHL-BFM-90 ± R group. In case of CR after 6 courses according to computer tomography and PET scanning treatment was completed. In case of PR pts underwent 2 R-DHAP courses with autoSCT (BEAM conditioning regimen). All pts from m-NHL-BFM-90 ± R group independently from PET-results stopped treatment. All cases of treatment failure occurred till one year of observation. Kaplan-Meier method was used to calculate OS and relapse-free survival (RFS). OS was defined as time from diagnosis to death from any reason; RFS was defined as time from diagnosis until relapse or death from any cause. Results: After 6 R-DA-EPOCH therapy CR was achieved in 24/30 (80%) pts, 6/30 (20%) pts had PR and underwent 2 R-DHAP courses followed by auto-SCT (BEAM). 2- RFS and OS was 100%. Median follow up was 16 months. In m-NHL-BFM-90 ± R group 10-year RFS and OS were 85% and 92%, respectively. Median follow up was 53 months. Conclusion: High dose pulsed m-NHL-BFM-90-chemotherapy as well as R-DA-EPOCH allows getting high efficacy of primary mediastinal B-cell lymphoma therapy. Although future analysis will show more distant results of R-DA-EPOCH chemotherapy. Disclosures No relevant conflicts of interest to declare.

Background. Patients with myeloproliferative neoplasms (MPN) have a high risk of portal thrombosis. This problem leads to the disability of the patient without prompt treatment, or with resistance to conducted therapy. The aim of this publication is to present own experience with antithrombin III, which subsequently will form the basis for the development of new treatment approaches for acute and subacute portal thrombosis resistant to therapy with anticoagulants (heparin, low molecular weight heparins (LMWH)) in patients with MPN. Material and methods. At the outpatient department of National Hematology Research Center (Moscow) were observed 5 patients with MPN (3 patients with primary mielofibrosis, 2 essential thrombocythemia). Course of the disease was complicated by acute and subacute thrombosis of the portal system, resistant to treatment by anticoagulants. Antithrombin III was used for treatment of these patients in the outpatient department first time. Antithrombin III was administered regardless of the level of endogenous antithrombin III in combination with LMWH in therapeutic or prophylactic doses. The drug was used at a dose of 1,000 IU by intravenous bolus injections of 3-5 in 3 days (total dose rate of 3000-5000 IU). Results. Treatment was performed on an outpatient basis, the side effects were not observed. In all 5 cases, use of the antithrombin III in combination with LMWH observed positive clinical effect as a quick relief of pain, and early recanalization of the thrombosed vessels (from 3 weeks to 1.5-2 months) (Table 1). In all cases it was normalization of increase of endogenous antithrombin to normal values​​; it was observed positive changes in other parameters: the value of D-dimers, fibrinogene, XII-dependent fibrinolysis ([Figure 1][1]). Conclusion. Antithrombin III therapy is indicated for the management of acute and subacute portal thrombosis in patients with MPN who are resistant to treatment with anticoagulants (heparin and LMWH). The advantage of the therapy is the possibility of therapy on an outpatient basis without the risk of side, including hemorrhagic complications. | Patient | Diagnosis. Clinical features. | Localisation of thrombosis | Preliminary treatment. Duration of the preliminary treatment | Treatment with antithrombin III, 1000 IU. | | -------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------------------------------- | ----------------------------------------------------------------------------------------------------- | ----------------------------------------- | ---------------- | | Number of injections | Total dosage | The efficiency. Duration of treatment | | 1. L.T | PMF, JAK-2 - 31%. Thrombocytosis, leukocytosis. | The portal and superior mesenteric vein (complete occlusion). | Heparin, clopidogrel, aspirin. 2 weeks. | 3 | 3000 IU | Pain relief after the first injection. Complete recanalization of the portal and superior mesenteric veins in 3 weeks | | 2. D.T. | PMF, JAK-2 - 83%. Ðistory of bleeding from the duodenal ulcer. | Occlusive thrombosis of the portal and splenic veins. | The patient received long-term antiplatelet therapy. | 5 | 5000 IU | Partial recanalization of the portal vein, complete recanalization of the splenic vein in 8 days. Complete recanalization of the portal vein in 4 weeks. | | 3.V.T. | ET, Varicose veins of the esophagus and stomach grade 2-3. History of gastric bleeding. Splenectomy with secondary thrombocytosis. | Occlusive thrombosis of the main part and the left branch of the portal vein. | Heparin, LMWH, aspirin, clopidogrel. 1.5 months. | 5 | 5000 IU | Recanalization of the left branch of the portal vein in 10 days, partial recanalization of the portal vein in 1.5 months. | | 4.Ts.I. | PMF, JAK-2 - 25%. History of radiation therapy of the spleen. | Thrombosis of the main part and the left branch of the portal vein, splenic vein. | Heparin, LMWH - 5 months with partial recanalization of the portal vein, the persistent pain. | 5 | 50000 IU | Pain was stopped in 2 weeks, recanalization of the left branch of the portal vein, the appearance of the blood flow in the hepatic portal vein in1 month. | | 5. I.R. | ET, JAK-2 – 34%. Genetic thrombophilia - prothrombin gene mutation G20210A (het). Pregnancy 22-23 weeks | Hepatic vein thrombosis (Budd-Chiari syndrom). Ascites, hepatomegaly, splenomegaly. | Heparin LMWH - 2 months with reduction of ascites, no changes of thrombosis. | 3 | 3000 IU | Absence of pain and ascites, partial recanalization veins of the liver, a significant decrease liver and spleen size, a significant improvement in coagulation in 2 months. | Abstract 5098. Table 1. Dynamics of clinical parameters in therapy with Antithrombin III. ![Figure 1.][2] Figure 1. Disclosures No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes

Background: Patients (pts) with multiple myeloma (MM) have an increased risk of thrombosis. Previous studies have shown that 3.7–4.6% of pts undergoing peripheral blood stem cell (PBSC) mobilization developed thrombotic complications. Aims: The aim of the study was to investigate the blood coagulation status in MM pts during PBSC mobilization. Methods: 20 pts: 12 males, 8 females at the age of 27 – 64 years (median 53) were included in the study. After bortezomib-containig induction therapy 5 of them achieved CR, 11 – VGPR and 6 – PR. PBSC mobilization was performed by using cyclophosphamide (CY, 4g/m2) and granulocyte colony-stimulating factor (G-CSF, 5mcg/kg/day). A central venous catheter (CVC) was placed on the day before CY administration and heparin-sulphate continuous infusion (500 IU/hour) as thromboprophylaxis was started. Subcutaneous injections of G-CSF were administrated when WBS decreased less 1 x 109/l. The procedures of stem cell collection were carried out when the number PB CD 34+ cells exceeded 10 x 103/ml. Hemostasis analysis was performed 5 times, namely before (point 0) and 24 hours after heparin administration (point 1), the next day after CY infusion (point 2), before G-CSF administration (point 3) and on the day of PBSC collection (point 4). Condition of hemostasis was assessed by the results of activated partial thromboplasin time (APTT, normal rate: 25 – 38 sec) and thrombin generation test, namely by using endogenous thrombin potential (ETP, normal rate: 760 – 1450 nM*min) in every point. Hypercoagulation was considered in cases when APTT < 25 sec and ETP > 1450 nM*min. And hypocoagulation was estimated by data APTT > 38 sec and ETP < 760 nM*min. Time series analysis performed using TIMESERIES procedure in SAS 9.3 (SAS Institute Inc. Cary, NC). Significance level of alpha was set at 0.05. Results: Before heparin administration (point 0) APTT evaluation showed normal coagulation in 18 pts (90%) and hypocoagulation in 2 pts (mean 34 sec, 95% CI 32 – 36 sec) (Figure 1). ETP was normal in 14 pts and increased in 6 cases (mean 1441 nM*min, 95% CI 1304 – 1578 nM*min) (Figure 2). Pts in CR demonstrated no significant difference in coagulation status compared to pts in VGPR or PR. On the next day after CVC incorporation and starting thromboprophylaxis (point 1), APTT and ETP have significantly changed (p < 0,05) to hypocoagulability in response to heparin infusion: mean data APTT became 38 sec (95% CI 36 – 41 sec) and mean ETP - 938 nM*min (95% CI 801 – 1074 nM*min). On the next day after CY administration (point 2) APTT was statistically significantly decreased (p < 0.05) reaching 35 sec (95% CI 34 – 36 sec). ETP had also changed but to a lesser degree, the mean was 1079 nM*min (95% CI 929 – 1229 nM*min). Hemostasis at point 3 was characterized by APTT mean 38 sec (95% CI 37 – 40 sec) and ETP mean 1057 nM*min (95% CI 911 – 1203 nM * min). On the day of PBSC collection mean values of measured parameters have not changed as compared to the previous point: APTT was 40 sec (95% CI 37 – 43 sec) and ETP was 1046 nM*min (95% CI 916 – 1176 nM*min) (Table 1). Thrombotic events were diagnosed in 2 cases of the total of 20 pts. There were two females, 54 and 56 years, both in CR MM. The first pt developed CVC-associated thrombosis. She had additional risk factors such as obesity (BMI – 44) and heterozygous MTHFR polymorphism. The second pt demonstrated acute violation of cerebral circulation which arose due to occlusion of the right internal carotid artery. She was a hard smoker and heterozygous FV Leiden polymorphism. Having confirmed thrombotic complications, the dose of heparin had been increased. Besides, folic acid was administrated for the first pt and combination of aspirin and pentoxifilline for the second. PBSC collection was successful for both pts. Summary: The results of our study identify latent hypercoagulation status in 30% pts of confirmed by elevated ETP. Those changes were registered despite of achievement CR or PR MM. Thrombotic complications have developed in 10% from our group. Notably, one should consider individual risk factors for each pt. Figure 1 Figure 1. Figure 2 Figure 2. Table 1. Measurements data of parameters of hemostasis Measuring point Mean APTT (95% CI) Mean ETP (95% CI) 0 33.6 (31.2-35.9)* 1441.1 (1304.3-1577.9)* 1 38.6 (35.7-41.6)* 937.6 (801.3-1074.0)* 2 35.2 (33.9-36.5)* 1079.0 (928.8-1229.2)* 3 38.1 (36.7-39.5) 1057.2 (911.4-1203.1) 4 39.6 (36.5-42.8) 1046.2 (916.0-1176.3) * Means in the same column are significantly different (P Disclosures No relevant conflicts of interest to declare.

Introduction Proposed in 1916 by Shlofferom Katznelson, splenectomy has been widely used since then in the treatment of immune thrombocytopenic purpura. ITP seems to be the most common indication for SE. According to the available literature up to 50-80% of laparoscopic SEs are being performed in patients with ITP, in our Centre - up to 20%. Laparoscopic SE should be considered as the treatment of choice for recurrent thrombocytopenic purpura after the initial response to steroid therapy. Indications for SE include all forms of immune thrombocytopenic purpura, requiring repeated courses of hormone therapy, as well as the cases of developed life-threatening complications, as for example intracranial hemorrhage. Earlier indications for splenectomy included lack of the hormone replacement therapy effect for 6 months. The dimensions of the spleen in patients with ITP are usually within normal or slightly increased limits. Thus these patients may get all the benefits of minimally invasive surgery. Goal: 1. to assess the results of the SE in the treatment of ITP 2. to clarify its place in the treatment of ITP. 3. to investigate the influence of the multiple lines drug therapy on the efficiency of SE in patients with ITP. Materials and Methods 87 SE have been performed in patients with ITP in 2008 – 2013, among them 63 female, 24 male. Median age 28 years old. Duration of the disease before surgery was from one month to 51 years, in 69 patients - over 6 months. 62 patients (71.3%) before SE received only corticosteroids. 25 (28.7%) received besides corticosteroids: 14 - GCS + immunoglobulin 5 - GCS + rituximab 7 - GCS + romiplostim 3 - GCS + cyclosporine 1 - GCS + eltrombopag 1 - GCS + azathioprine Results and Discussion Average duration of the laparoscopic SE was 109 min (from 50 to 250 min), on the average 100 min with the mean blood loss of 300 ml (median 200 ml). In 20 patients intraoperative blood loss was negligible, in 14 – over 500 ml. In patients treated with only corticosteroids blood loss over 500 ml was in 16%, in pretreated- in 20%. Intraoperative complications (5.7%): • pneumothorax-1 • intraoperative bleeding led to conversion-2 • acute respiratory failure-1 • bleeding from the bed of the spleen, stopped by conservative measures-1 Postoperative complications occurred in 13,8% (n = 12): •patients treated with corticosteroids 11.3% •patients receiving multiple lines of therapy - 24%. •thrombotic complications (3.4%) •portal vein thrombosis 1 •tibial vein thrombosis 1 •ischemic stroke 1 •pneumonia 8 (9%) •intestinal bleeding-1 (1.1%) In 13 (14.9%) patients, operated on with thrombocytopenia < 20x109/l, average blood loss was 320 ml (100-1200 ml). The operation was complicated in 2 patients by intraoperative bleeding required conversion. One patient in the postoperative period developed acute adrenal failure, pneumonia, septic shock. In 49 (56.3%) patients, operated on with thrombocytopenia 20-100x109/l, mean blood loss was 240 ml (0-1100 ml). Postoperative complications developed in 7 (14%) of them (1 portal vein thrombosis, acute respiratory failure 1, pneumonia 5). In 26 (29.8%) patients, operated on with the platelet count of more than 100x10, average blood loss was 370 ml (0-2000 ml). One intraoperative complication - injury to the diaphragm. Postoperative complications (1 bleeding, pneumothorax, 1, 1 pneumonia, thrombosis of the saphenous veins of the lower extremities 1) developed in 4 (15%) pts. In 69 patients with the duration of the disease over 6 months the efficiency of SE was 84%. In 17 patients with the duration of the disease less than 6 months - 70.6%. In patients treated with corticosteroids, efficiency of SE was estimated as 85.5% In pretreated - 60%. Nonefficient SEs were observed in 10 pts. In 10 patients the platelets count after SE remained Administration of romiplostim (n = 7) led to an increase of the platelets count up to 60-80 x 109/l in 7 patients (100%) in the first week after surgery Conclusion • SE results in a full or partial response in 79.6% of patients with ITP • Optimal time for SE is 6 - 12 months after the onset of the disease • Risk of complications of the procedure increases with the delay of SE • A history of multiple lines of drug therapy reduces the efficiency of SE in ITP from 79.6% to 60.0%. Disclosures No relevant conflicts of interest to declare.

Introduction The presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube. Objectives: improvement of PNH cells detection technique. Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group. The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed. Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1). Figure 1 Figure 1. In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations. Table 1. Average value of PNH clone on leukocytes in groups of patients both techniques PNH clone Gr, % (mean) Mon, % (mean) CD24-/FLAER- CD157-/FLAER- CD14-/FLAER- CD157-/FLAER- 0%-0,99% 0,178 0,349 0,323 0,378 1%-9,99% 3,98 3,76 9,54 9,98 >10% 71,52 71,52 71,85 71,34 Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Follicular lymphoma (FL) is one of the most frequent types of B-cell lymphomas and accounts for about 22% of all non-Hodgkin lymphomas in Russia. The disease is usually a long-term condition with frequent relapse. At the same time 15-20% of pts have fast-tumor progression. Patients (pts) of this group die within the first 1.5-2 years from the time of diagnosis. High-dose chemotherapy (HDT) followed by ASCT in the presence of nonfavorable characteristics of FL (fast growth of tumor volume, B-symptoms, third citological grade of tumor, large tumor size, absence of anti-tumor response to R-CHOP courses in the first remission) increases the overall survival and progression-free survival of pts. AIM: To estimate HDT with ASCT efficiency among pts with FL in front-line therapy who received treatment in the National Research Center for Hematology during 2000-2013. PATIENTS AND METHODS: The results of ASCT among 12 pts with FL have been analyzed: 8 male and 4 female aged from 31 to 50 with median age 43. Patients in the main group (11/12) were on the IVth stage of tumor growth. In 6/12 cases the tumor size was more than 6 sm. Among 7/12 pts, besides lesions of lymphatic nodes, extra nodal lesions were found: infiltration in psoas (1/12), kidney (1/12), stomach (1/12), lungs (1/12), thyroid (1/12), pancreas (1/12). In 2/12 cases leukaemization was observed. Based on FLIPI criteria all the pts were divided into three groups: in the first risk group - 5/12 pts, in the second - 3/12, in the third - 4/12. B-symptoms were in the majority of cases (9/12). 9/12 pts were diagnosed with I-II cytological grade of FL, among 3/12 - III A/B. Based on the character of tumor growth the dispersion was the following: nodular tumor growth - 5/12, nodular-diffuse tumor growth-6/12, diffuse tumor growth - 1/12. Immuno-chemical investigation of protein serum and urine was performed in 9/12 cases, and among them rising serum β2-microglobulin above the norm was observed in 4/9 pts. A rise in lactate dehydrogenase concentration above the norm was observed among 4/12 pts. Lesion of bone marrow at the beginning of the disease was identified among half of the pts (6/12). Induction courses were performed on the R-CHOP programs, with intensive therapy - on protocol mNHL-BFM-90. RESULTS: Anthracycline courses were prescribed for nearly all of the pts as an inductive therapy: R-CHOP (11/12). mNHL-BFM-90 protocol was chosen as an inductive regime because of the fast growth of tumor size, IIIB grade of FL in 1/12 case. This tumor growth behaved like diffuse large B-cell lymphoma. In three cases medical-diagnostic splenectomy was undertaken before chemotherapy. After the inductive regimes the pts were given HDT based on the following scheme: two courses R-DHAP, course with rituximab and high-doses cyclophosphamide, a course with rituximab and high doses of etoposide, R-BEAM. All 12 pts, who were given ASCT are in full remission on the main disease. The period of observation is from 13 to 164 months. CONCLUSION: This first experience of intensive therapy in FL in Russia demonstrates that ASCT as a front-line therapy leads to complete remission of FL among pts who have nonfavorable prognosis factors. Disclosures No relevant conflicts of interest to declare.

Introduction Hematopoiesis is maintained in close contact with bone marrow (BM) stroma. Malignant hematopoietic cells probably affect the hematopoietic microenvironment, as well as high dose chemotherapy. The goal of this study was to analyze the alterations in the characteristics of human multipotent mesenchymal stromal cells (MSC) and their more differentiated progeny – fibroblastic colony forming units (CFU-F), derived from the BM of acute myeloid and lymphoid leukemia (AML and ALL) patients. Methods 26 newly diagnosed cases (18 AML, 8 ALL) and 35 patients (20 AML, 15 ALL) before and during 1 year after allogeneic hematopoietic stem cell transplantation (alloHSCT) were involved in the study after informed consent. BM was aspirated prior to any treatment in the newly diagnosed group and before the conditioning and at 6 time points during 1st year after the alloHSCT. MSC were cultured in aMEM with 10% FCS. Cumulative MSC production was counted after 3 passages. CFU-F concentration was analyzed in BM samples by standard protocol. The relative expression level (REL) of genes was measured by RT2 Profiler PCR Array (Qiagen) and TaqMan RQ-PCR. MSC and CFU-F from 50 healthy donors of BM for alloHSCT were used as a control after informed consent. Results The CFU-F concentration in the BM of patients at the moment of diagnostics was 1/3 of the donor’s for AML and ≈1/10 of ALL (9±5.2 and 3.9±2.3 versus 31±3.5 per 106 nucleated cells in donor BM, p Conclusions During the leukemia development malignant cells alter the stromal precursor cells leading to the decrease in the REL of many regulatory genes and in the number of more differentiated stromal precursor cells in the BM. Chemotherapy used for induction of the remission in these patients restore functional ability of MSC and CFU-F, while gene expression levels remained altered. Conditioning regiments used for the alloHSCT significantly damage both types of studied stromal precursors, and the effect lasted at least for 1 year. So, both leukemic cells and chemotherapy affect BM hematopoietic microenvironment. Disclosures No relevant conflicts of interest to declare.

Introduction: Detection of bone marrow involvement (BM) is a factor of poor prognosis for diffuse large B-cell lymphoma (DLBCL), since DLBCL BM is characterized by aggressive course and low response level to standard chemotherapy (CT), and 5-year overall survival (OS) rate for this group is less than 30% after ÑHOP-like CT. Intensification of therapy in this group of patients can improve the results, except patients with bone marrow involvement at diagnosis. Bone marrow involvement is a poor prognostic factor for patients treated with high-dose chemotherapy. Bone marrow involvement in DLBCL by histology is detected in 10-30% patients. The importance of B-cell clonality examination in bone marrow as prognostic and staging factor has not been described in the literature. Aim: To evaluate the significance of the immunoglobulin heavy chain gene rearrangement analysis performed by PCR for identification of the bone marrow involvement frequency and its value for staging and prognosis in patients with de novo DLBCL treated with high-dose chemotherapy (HDC). Patients and methods: We performed a pilot prospective trial, including 175 consecutive adult patients (median age 45 years, range 18–67) with newly diagnosed DLBCL who were enrolled in HDC protocol (mNHL-BFM-90 program or scheme R-EPOCH/R-HMA) since June 2007 till July 2014. Their clinical characteristics included such factors as IPI and phenotype DLBCL by immunohistochemical study. Out of the 175 patients, 85 had a GCB phenotype and 90 - non-GCB; 36 patients (20%) had low IPI risk, 40 patients (23%) had low-intermediate, 38 patients (22%) had high-intermediate, 61 patients (35%) had high risk. B-cell clonality was evaluated using PCR amplification by IGH (FR1, FR2, FR3) and IGK (Vκ-Jκ, Vκ/intron-Kde) gene rearrangements with multiplex BIOMED-2 primer sets and subsequent fragment analysis using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).In 105 of 175 patients the study by BIOMED-2 multiplex polymerase chain reaction protocol of B-cell clonality of bone marrow was fulfilled. Statistical analysis was done using JMP ver. 10.0 (SAS, Cary, NC). Results: Histological and immunohistochemical studies validated bone marrow involvement in 19 patients (10.8%), including: concordant in - 12 patients (63%), and discordant - in 7 patients (37%). In 14 of these patients the B-cell clonality detection was performed and confirmed immunoglobulin (IG) heavy chain gene rearrangement in all cases. In 26 out of 105 patients, bone marrow involvement (24.7%) was revealed: in 14 patients bone marrow involvement was identified by histological examination and in 12 patients only by clonality of bone marrow. 12 patients with only B-cell clonality in bone marrow were classified by IPI: 4 (33%) patients had high IPI, 6 (50%) patients - high-intermediate IPI and low-intermediate IPI - 2 (17%) patients. So, in 17% patients had to change the risk group according to bone marrow involvement. Thus, 31 patients with bone marrow involvement signs were identified. Univariate analysis of the entire cohort (n = 175) revealed that both IPI and phenotype were not of prognostic significance (p > 0.05). In multivariate analysis, detection of B-cell clonality in bone marrow was an independent predictor of OS and relapse-free survival (RFS) (p < 0.005). (Fig.1-2). OS, RFS in patients with histological detection bone marrow involvement and with only B-cell clonality in the bone marrow did not differ. Conclusion:Detection of B-cell clonality in the bone marrow seems to be an independent predictor of outcome in de novo DLBCL pts, treated with high-dose chemotherapy, while IPI and phenotype DLBCL cannot be considered as risk factors for this group of pts. It seems to be reasonable to include the detection of B-cell clonality in the bone marrow in primary diagnostics of the DLBCL for staging and predicting response in HDC. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Introduction. Most studies have found that hyperleukocytosis (HL >100*109/l) is a poor prognostic factor in acute myeloid leukemia (AML). It is diagnosed in 5-13 % of cases and is more common in AML with monocytes’ differentiation (M4, M5), as well as in cases with poor prognostic molecular markers, such as FLT3-ITD and others. According to the literature the early (during the first week) mortality in AML due to HL may reach 90%. The main cause of death are leukostasis, leading to neurological symptoms, respiratory distress syndrome (RDS) with lung lesions, disseminated intravascular coagulation (DIC) syndrome with the development life-threatening bleedings and massive tumor lysis syndrome during induction therapy. Aim. National Research Center for Hematology (NRCH) has initiated a prospective clinical study aiming to develop a feasible clinical approach preventing severe tumor lysis syndrome during induction in AML patients with initial WBC >100*109/l by using serial plasmapheresis (PA). Patients and Methods. From Jan 2010 till June 2014, 92 patients with de novo AML were treated in the NRCH according to AML-10 protocol (ClinicalTrials.gov Identifier: NCT01587430). Initial white blood cell (WBC) count more than 100x109/l was detected in 18 patients (19,5%): median age - 38 y (17-56yy), M/F - 10/8, WBC - 130x109/l (100-408x109/l), LDH -1765 µ/l (720-6653). 2 patients were in the favorable, 13 – in the intermediate, 2 - in the poor cytogenetic risk group. 83,3% (15/18) patients had hepatosplenomegaly and lymphadenopathy, 38.8 % (7/18) - gingival hyperplasia, 16,6 % (3/18) - neuroleukemia, 16,6% (3/18) - skin leukemids. All patients had infectious complications at diagnosis. Lung infiltrates due to leukostasis were revealed - in 77,8% (14/18), including 5 patients (27,8%) with RDS; neurological symptoms (headache, blurred consciousness) - in 33,3% (6/18), DIC syndrome - in 8/18 (44,4%) with 1 intracranial hemorrhage. All patients received allopurinole 600 mg/day. PA procedures were started during cytoreductive therapy (hydroxyurea 10 mg/kg/day 1-2 days) sometimes with leukapheresis (LA) and were continued within 1 or 2 days of conventional 7+3 (ARA-C 100 mg/m2 bid iv 1-7 d, DNR 60 mg/m2 x 3d) 2 hours after short (30 min) ARA-C infusion. DNR was applied on days 3 to 5 or 5 to 7, thus PA did not interfere with its pharmacokinetic properties. All patients concomitantly have got intensive hydrating (3-4 l) and diuretic therapy with electrolytes and albumin support. Results: Cytoreductive therapy with hydroxyurea was used for a median 2 days (1-7 dd). Half of the patients (9/18) have got 1-2 procedures of LA (median 2). Median 2 (1-4) procedures of PA were performed in 17/18 patients. The volume of removed plasma for 1 PA procedure was 1200 ml (900-1500 ml), the volume of compensation was – plasma 1100 ml (500-1600 ml) +/- 20% albumin 50-150,0 and 0,9% NaCl 500-1000,0. Median WBC count after cytoreductive therapy at the 1st day of 7+3 was 60x109/l (5,6-124x109/l). DNR was administered on days 5-7 in 16 and on days 3-5 - in 2 patients. No severe cytolysis with the development of multiple organ failure was observed. Only 1 patient with massive extramedullary involvement has developed just laboratory signs of acute renal failure without stopping the urinary function. Less than third of the patients (5/18) developed the transient signs of volemic overload within the first days of chemotherapy treated successfully with diuretics and plasmapheresis. CR after the 1st induction was achieved in 50% of (9/18) patients. Additional 7 patients achieved CR after the 2nd induction, resulting in 89% overall CR rate. Only 1 patient (5,5%) died with severe infectious complications during aplasia. Allogeneic bone marrow transplantation was performed in 8 of 16 CR patients. The median OS and DFS was 28 and 23 months, 3-years OS and DFS was 32% and 35% respectively. Conclusions. Our small study brought us to a conclusion that serial PA procedures during cytoreductive phase and the first days of 7+3 may prevent the development of tumor lysis syndrome and its complications. This clinical approach is feasible, easy to perform and decreases early mortality. Disclosures No relevant conflicts of interest to declare.

Introduction Intensive “pediatric oriented” treatment with heavy cytostatic load, incorporation of allogeneic HSCT is now considered a backbone approach in adult ALL therapy. Highly myelosuppressive treatment is more toxic and less reproducible, so RALL has initiated non-intensive but non- interruptive protocol “ALL-2009”and started a prospective multicenter trial for adult Ph-negative ALL based on: 1) the replacement of prednisolone (Pdn) 60 mg/m2 with dexamethazone (Dexa) 10 mg/m2 if blast cells are >25% in bone marrow after 7 days of prephase; 2) de-intensified but non-interruptive treatment with doses modification according to the myelosuppression with total treatment duration of 127 weeks; 3) prolonged implication of L-asparaginase (total proposed dose 590.000 IU/m2); 4) autologous hematopoietic stem cell transplantation (HSCT) after non-myeloablative BEAM conditioning followed by prolonged maintenance – for T-cell ALL patients. Allo-BMT was an option for high risk patients with sibling donors. The study is registered on the ClinicalTrials.gov public site; NCT01193933. Methods and patients From Jan 2009, till June 2014, 30 centers enrolled 250 pts: median age 30 years (15-60 years), 115f/135 m; in 2,4% phenotype was unknown (n=6), B-lineage ALL - 63,2% (n=158), T-lineage ALL - 34% (n=85), biphenotypic - 0,4% (n=1). Cytogenetics was available in 62,4% of patients (n=156) and 41% of them (n=64) had normal karyotype (NK). 25,2% of patients (n=63) were in the standard risk (SR) group (WBC 30 for B-lineage, >100 for T-lineage; EGIL BI, T-I-II-IV; LDH > 2N; late CR; t(4;11)-positive), 39 patients were not qualified by risk group. The analysis was performed in June 2014. The median time of follow-up was 26,1 mo. Results Prednisolone was replaced with dexamethazone after prephase in 68,1% of patients (135 of 198). The portion of non-responders to PRD in SR and HR groups was 54% and 67%, respectively (p=0,06). CR rate in 228 available for analysis patients was 87% (n=198), induction death occurred in 10,5% (n=24), resistance was registered in 2,5% (n=6). The majority of CR patients (91%) achieved it after prephase (7,1%, n=16) and the 1st phase of induction (83,9%, n=164). Late responders constituted 9% (n=18). None of those factors (PRD sensitivity, initial risk group, immunophenotype, late response) influenced overall (OS) or disease-free survival (DFS). OS rate in older ALL patients (>30) was substantially less than in younger ones (52,7% vs 73,6%, p=0,0009). DFS was comparable - 61,2% vs 71,5%, p=0,1. 24 of 75 (32%) CR T-ALL patients underwent autologous BMT after BEAM conditioning at a median time of 6 mo from CR and proceeded to further maintenance. No relapses were registered in this group so far. Allogeneic BMT was performed only in 14 patients (Ò-ALL-7, B-ALL-7) on the protocol. Totally 47 patients (20,6%) relapsed (16 with T-lineage, 31 with B-lineage ALL). At 48 mo OS for the whole group constituted - 65,6%, DFS - 69,3%. OS and DFS differed in B-ALL patients with NK in comparison with abnormal karyotype: 80% vs 57%,(p=0,01) and 81% vs 61%, respectively (p=0,02), but not in T-ALL patients. Conclusions Our data demonstrate that the proposed treatment approach is rather effective and reproducible. OS and DFS did not depend on various common risk factors (initial risk group, WBC, LDH, immunophenotype, late response, PRD resistance). The only independent risk factor for OS was age (>30 y). In B-cell ALL abnormal karyotype became an independent adverse risk factor for OS, DFS, relapse incidence. Fig.1 Overall survival (A) according to age in the whole cohort and disease-free survival (B) in B-cell ALL according to karyotype Table 1 (A) OS in different age groups (B) DFS in B-cell ALL Figure Figure. (A) OS in different age groups Figure Figure. (B) DFS in B-cell ALL Disclosures No relevant conflicts of interest to declare.

Introduction. The vast majority of AML clinical trials incorporate high-dose ARA-C (HDARA-C) as a basic approach. Though it was recently proved by a controlled prospective comparison that different treatment strategies in patients with AML did not show clinically relevant outcome differences (Th.Buchner, JCO, 2012), the RALSG initiated a randomized multicenter AML-10 trial (ClinicalTrials.gov Identifier: NCT01587430) aiming to evaluate the necessity of HDARA-C in consolidation in the context of high total dose of different anthracyclines/anthracenedione (660-720 mg/m2). Materials and methods. The patients aged 16-60 yy with de novo AML (except APL) were randomized before treatment start to different types of consolidation after two induction 7+3 with daunorubicin 60 mg/m2x3 and ARA-C 100 mg/m2 bid iv (1-7d) in the 1st course and 200 mg/m2/d (1-7d) continuous infusion in the 2nd course: (1st arm) two courses of 7+3 with Idarubicin (Ida) 12 mg/m2x3 and with Mitoxantrone (Mito) 10 mg/m2x3, in both ARA-C 100 mg/m2 bid iv (1-7d); (2nd arm) two courses of HDARA-C 1g/m2 bid iv 1-3 days with Ida 8 mg/m2 3-5 days and Mito10 mg/m2 3-5 days. After consolidation all pts proceeded to the maintenance 5+5 courses (ARA-C 100 mg/m2 bid iv 1-5 days with 6-mercaptopurine 50 mg/m2 1-5 days). Allogeneic HSCT was indicated to patients from intermediate and poor cytogenetic risk groups, late CR, WBC > 100*109/l. Results. From Jan 2010 till Jan 2014, 250 AML patients from 20 centers were randomized: (1st arm) 125 pts (m.age 45 y, 17-59 yy; 73f / 52m; LDH=674 IU (128-6653); cytogenetics favorable - 17,3%, intermediate - 66,7%, poor - 16%) and (2nd arm) 125 pts (m.age 43y, 16-60yy; 69f/56m; LDH=704 IU (123-8159); cytogenetics favorable - 20%, intermediate - 58,6%, poor - 21,4%). No molecular testing in cytogenetically normal pts was done. The analysis was performed in May 2014. The follow-up data were available in 199 pts. CR was achieved in 72,9% (n=145), early death was registered in 12,7% (n=25) and refractory disease - in 12,4% (n=24). Death in CR did not differed in a randomized groups (1st) 13,9% and (2nd) 13,7%. 17% of CR pts (n=20) were withdrawn from the protocol due to refusals (3,5%), infectious complications (13,5%). No relevant cardiotoxicity was registered on both arms. 12% (8 pts on the 1st arm, 9 - on the 2nd) were transplanted in 1st CR from HLA-identical donors. 3-years OS and DFS by intent-to-treat analysis were identical on both arms: (1st arm) 43% and 62%, (2nd arm) - 38% and 51%, respectively. For those patients in whom consolidation was fulfilled the comparison of DFS in different cytogenetic groups demonstrated equal efficacy of each consolidation arm: favorable - 85% (1st) and 85% (2nd), intermediate -65% (1st) and 57% (2nd), poor - 20% (1st) and 22% (2nd). In a multivariate analysis only cytogenetic group (HR=3,01, p=0,005) and CR achievement after the 2nd induction course (HR=2,83, p=0,0007) adversely influenced DFS. As the land-mark (5 mo of CR) analysis have shown, the bad prognosis of late CR could be modified by allo-HSCT in 1st CR: DFS of transplanted patients = 86%, non-transplanted=27% (p=0,03). Conclusion. Our interim analysis has demonstrated that conventional 7+3 consolidation is equal in long-term outcome to high dose ARA-C in case of the high total doses of different anthracyclines/ anthracenedione in induction/consolidation. CR after the 2nd induction became independent adverse prognostic factor (even inside cytogenetic risk groups) defining patients who should be transplanted in 1st CR. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B – mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17). The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4+CD25high. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms, FoxP3-positivity on activated nonregulatory CD4+ cells. CD4- lymphocytes (CD8+ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4+CD25highGranzymeB+ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4+CD25highGranzymeB+ cells among CD4+cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4+CD25highGranzymeB+ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future. Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4+CD25highGranzymeB+ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation. Disclosures No relevant conflicts of interest to declare.

Introduction Allogenic bone marrow transplantation (allo-BMT) with high-dose, post-transplantation cyclophosphamide (CY) to promote graft-host tolerance has become an alternative for standard immunosuppression. This post-BMT CY-based immunosuppression regimen has comparable efficacy with other immunosuppressive regimens including cyclosporine A and mycophenolate mofetil. Previously we reported a significant reduction of acute GVHD rate by using MSC infusion for GVHD prophylaxis (n=19, p=0.009) [Abstract 1256, 52nd ASH Annual Meeting abstract]. So we studied high-dose, post-transplantation cyclophosphamide and mesenchymal stem cells (MSC) infusion for graft-versus-host (GVHD) prophylaxis for patients with advanced disease after FLAMSA (where amsacrin was substituted for idarubicin) and FluBuATG conditioning regimen. Patients and methods 7 patients with a median age 38 years (24-59 years; 5 males, 2 females; 4 donors/recipient pairs was sex matched, 3 was sex mismatched). All patients with advanced disease (relapsed or refractory AML n=4, ALL n=2, MM n=1) with a median blast cells 17.4%, underwent allo-BMT (n=5 from HLA-identical related donor, and n=2 from unrelated donor). Conditioning regimen was composed of fludarabine, busulfan, and horse anti-thymocyte immunoglobuline for 2 patients, in 4 patients FLAMSA was applied, and 1 patient has no conditioning regimen at all (he was in profound prolonged aplasia after previous course of chemotherapy). For GVHD prophylaxis CY at dose 50 mg/kg daily at day +3 (n=2) and at day +3,+4 (n=5) was used. All patients also received MSC for acute GVHD prophylaxis at day of recovery (WBC>1*109/l) at dose 1*106/kg. No other immunosuppression was used. Results Neutrophil engraftment was achieved at a median day +20 (range from 17 to 44 days). Acute GVHD occurred in 3 patients (43%) at a median day +45; all cases were Grade 2-3 (with only skin n=2; and with skin and liver involvement n=1); there was only 1 patient with late refractory GVHD in this study treated with prednisone at a dose 2 mg/kg; all other patients responded to initial treatment with cyclosporine A at 3 mg/kg. Overall mortality rate was 42.6%. 1 patient died in CR at day +60 due veno-occlusive disease (TRM - 14%). Relapses occurred in 2 patients (28.6%) at a 3,8 and 2.4 months. The overall survival is 51.4% with a median of follow-up 3,9 month. Conclusion Despite the fact that we study small group use of cyclophosphamide with MSC for GVHD prophylaxis is a very attractive approach for patients with advanced disease, and needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Background Bone marrow involvement occurs in 10–20% of cases of diffuse large B-cell lymphoma (DLBCL). According to its morphological characteristics bone marrow involvement can be concordant (CBM) or discordant (DBM). DLBCL with bone marrow involvement (DLBCL BM) is characterized by aggressive course and poor response to standard polychemotherapy, with the worst results observed in the group of patients with CBM: 5-year overall survival rate is less than 10%. Application of the mNHL-BFM-90 treatment program in DLBCL BM patients resulted in only partial remission. In many cases there was persistent residual bone marrow infiltration, whereas the tumor fully regressed in other locations. In these cases it seemed reasonable to apply consolidating high-dose sequential chemotherapy (sHDT) and autologous stem cells transplantation (ASCT). Aim To evaluate the efficacy of the mNHL-BFM-90 treatment program with sHDT and ASCT in DLBCL BM patients. Patients and Methods We performed a pilot prospective trial, including 15 consecutive adult patients (median age 44 years, range 23–57, female=4, male=11, CBM=5, DBM=10) with newly diagnosed DLBCL BM who were treated with sHDT and ASCT as a part of the mNHL-BFM-90 program from Feb 2005 till July 2013. All patients underwent 4 courses of mNHL-BFM-90 chemotherapy program, including A blocks (methotrexate 1,5 g/m2 day 1, ifosfamide 800 mg/m2 days 1–5, vincristine 2 mg day 1, doxorubicin 50 mg/m2 day 3, etoposide 100 mg/m2 q 12 hrs days 4–5, cytarabine 150 mg/m2 q 12 hrs days 4–5, dexamethasone 10 mg/m2 days 1–5) and C blocks (methotrexate 1,5 g/m2 day 1, cytarabine 2 g/m2 q 12 hrs days 2–3, vinblastine 10 mg/m2 day 1, etoposide 100 mg/m2 q 12 hrs days 4–5, dexamethasone 10 mg/m2 days 1–5). After 4 induction courses patients continued receiving treatment according to the sHDT scheme, including 2 DHAP courses and 3 high-dose courses with the hematopoietic stem cells (HSC) mobilization and collection: cyclofosfamide 4 g/m2 day 1, methotrexate 8 g/m2 day 15 and vincristine 2 mg day 15, etoposide 500 mg/m2 q 12 hrs days 31–34. In cases of sufficient quantity of collected CD34+ cells (≥ 2x106/kg), the BEAM conditioning and the ASCT were applied. The programed therapy was fully implemented in 6 out of 15 patients. The average amount of harvested HSC was 4x106/kg (range 2–7,2x106/kg). Due to unsufficient HSC collection, ASCT was not carried out in 9 patients. If complete remission (CR) was achieved only after sHDT, one additional Dexa-BEAM course was carried out (4 patients). The other patients completed the therapy after the sHDT (5 patients). The rates of overall survival (OS) and probability of relapse were estimated (±SE) by using the Kaplan-Meier method. Statistical analysis was done using JMP ver. 10.0 (SAS, Cary, NC). Results Two patients achieved CR after 4 courses of chemotherapy program mNHL-BFM-90, while the other 13 patients achieved PR; however, they had residual disease only in the bone marrow. All patients achieved CR after sHDT therapy. There were 2 patients with early relapse (8 months): one of them had CBM and another had DBM. One of these cases of relapse was registered in patient after ASCT. Remission has been maintained in 4 of 5 patients with CBM. There were no cases of death from the chemotherapy toxicity. At the median 44 months (range 19–95) follow-up the overall survival was 82±12%, and probability of relapse was 15%. Conclusions DLBCL with bone marrow involvement has a very poor prognosis with the CHOP-like therapy, and we consider it as the basis for high-dose chemotherapy. Our trial showed that the long persistence of the tumor in the bone marrow requires the prolonged treatment. The primary-intensive mNHL-BFM-90 treatment program with the sHDT and ASCT was used for the first time in the DLBCL BM patients, including patients with CBM, and has shown high efficacy. Disclosures: No relevant conflicts of interest to declare.

Background Follicular lymphoma (FL) is characterized by clinical heterogeneity. There is a need for easily quantifiable prognostic biomarkers. No consensus has been achieved regarding the prognostic significance of the microvessel density, number of macrophages and cytotoxic lymphocytes within the tumor. Objectives To characterize the microvessel density, number of macrophages and cytotoxic lymphocytes within tumor tissue using immunohistochemical (IHC) analysis of sections from paraffin blocks of lymph node biopsies in two groups of patients with different clinical courses of FL. Patients and methods The study included 59 patients with FL: 39 women (67%) and 20 men (33%). The median age was 53 years (range: 27–83 years). Forty nine patients were observed in the National Research Center for Hematology (Moscow), and 10 patients in Cancer Research Center (Moscow), from April 2001 until May 2011. Group 1, “good outcome”, included 28 patients who were followed for 2 or more years after the therapy, had remission, or developed relapse in five or more years from the start of treatment (late relapse). Group 2, “poor outcome”, included 31 patients who died due to tumor progression in the first 1–2 years from the time of diagnosis, had primary tumor resistance, or developed FL relapse in the first year from the start of treatment (early relapse). Patients in both groups received the same initial treatment with rituximab. Five year OS rate in group 1 was significantly higher than in group 2 (83±7% vs 28±13%; ð=0.03). In all 59 cases the samples for IHC analysis were selected by using a table of random numbers, without knowing to which study group they belonged. Each study parameter (microvessel density, number of cytotoxic lymphocytes and macrophages within a tumor) was evaluated and then assigned to prognostic group 1 or 2. The vascularization of tumor tissue was assessed by IHC on sections from paraffin blocks of tumor lymph node biopsies. CD34 and D2-40 (podoplanin) antibodies were used for the visualization of blood and lymphatic vessels, respectively. CD68 antibody was used to detect activated macrophages. Granzyme B antibody was used to visualize cytotoxic lymphocytes. Morphometric analysis was performed using light microscopy and a Leica digital camera (400x). The pictures were processed by the computer program “VideoTesT-Morphology 5.2” in order to estimate the percentage of vessels in the tumor tissue. IHC specimen evaluation was carried out using a table of random numbers. Number of macrophages and cytotoxic lymphocytes in nodular and nodular-diffuse types of tumor growth was evaluated by light microscopy (400x): the cells were counted in 1 mm2 of tumor tissue, and the number of positive cells was determined in the intrafollicular and interfollicular space. Results Blood vessel density and lymph vessel density in group 2 were significantly higher than in group 1 (p=0.03). The cut-off point for blood and lymphatic vessel density in 1 mm2, which differentiates group 1 from group 2, was 0.04. The number of cytotoxic lymphocytes in intrafollicular space, interfollicular space and diffuse area of tumor nodules in group 1 was significantly higher than in group 2 (ð=0.05). The cut-off point for tumor-associated cytotoxic lymphocytes in 1 mm2, which differentiates group 1 from group 2, was 100 (Figure 1). The number of macrophages within tumors with nodular and diffuse growth in group 2 was significantly higher than in group 1 (ð=0.01) (Figure 2). The cut-off point for macrophages in 1 mm2, which differentiates group 1 from group 2, was 250. Statistical analysis were done using JMP ver. 10.0 (SAS, Cary, NC). Conclusion Our results demonstrate an association between higher angiogenic activity in the tumor and poor prognosis. A low number of cytotoxic lymphocytes and a high number of macrophages within the tumor were also associated with a poor prognosis. We suggest that these results will help to predict clinical response in FL using rituximab. Disclosures: No relevant conflicts of interest to declare.

Relapses after allogeneic stem cell transplantation (allo-SCT) are the main cause of treatment failure in acute myeloid leukemia (AML). Chemotherapy with subsequent donor lymphocyte infusions (DLI) is considered to be the optimal approach for complete remission (CR) achievement and induction of “graft versus leukemia” effect. There is a tendency to use low-dose chemotherapy (i.e. Low-dose Ara-C) in this setting because of less toxicity. However the efficacy of low-dose chemotherapy is not satisfactory and leads to 45-67% CR rate. In this study we apply intensive chemotherapy (7+3) followed by DLI in aplasia in with relapsed AML. Aim To investigate the efficacy of DLI perfomed in neutropenia after reinduction chemotherapy 7 +3 (Cytarabine 100 mg/m^2 every 12 hours daily for 7 days, and Idarubicin 12 mg/m^2 daily on days 1, 2, and 3) in AML patients (pts) with overt relapse after allo-SCT. Methods The study comprised 16 AML patients. The median age was 31 years (16 - 57 years), male – 11 female - 5. Twelve patients underwent allo-SCT in first remission, 4 pts - in overt relapse of AML. Allo-SCT was carried out from HLA-matched sibling donor in all pts. Myeloablative conditioning regimen was performed in 10 pts. Reduced intensity conditioning (RIC) - in 6 pts. AML relapse occurred at a median 4,7 months (range from 1 to 51 months) after allo-SCT. Patients received DLI at day 7 (7-14 days) after chemotherapy during myelotoxic agranulocytosis. The number of infusions ranged from 1 to 4 (median 2 DLI) per patient. DLI after chemotherapy were carried out twice in 1 patient due to the second relapse. So we analysed 17 cases of relapse in 16 pts. Total amount of the CD3+cells varied from 1 to 16,7x10^7 CD3+cells/kg (median 6,0x10^7 CD3+ cells/kg). The interval between DLI was 1-4 weeks. All pts received 2 - 6 MUE Interleukin-2 (IL-2) subsequently after DLI. Chimerism was monitored by PCR analysis (VTTR and STR) and by FISH – analysis for centromers of X and Y – chromosomes after DLI each 2-4 weeks up to 6 months, then every 3 months. Results Complete remission with 100% donor chimerism was achieved in 14 (82%) out of the 17 cases. There were no toxic deaths, 3 pts died in leukemia progression. All pts developed severe infections in neutropenic phase (mucositis, pneumonia, sepsis), but they were cured. Eight pts (57%) out of 14 developed a relapse in 5 months after DLI (from 1 to 17 months) and 6 pts (35%) remained in remission. Follow-up period was 12 months (1 - 124 months). Median overall survival constituted 15 months. Acute graft versus host disease (GVHD) after DLI was diagnosed in 8 patients 47% (6 - I-II grade; 2 - III-IV grade). Chronic GVHD was diagnosed in 8 pts (47%): limited -6 pts (35%), extensive - 2 pts (12%). Conclusion Our data show that despite myelotoxicity and infections 7+3 and subsequent DLI+ IL-2 is an effective treatment for AML pts with overt relapse after allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Background Splenectomy (SE) at the Myelofibrosis (MF) is one of the difficult operations on the techniques and perioperative complications. There are a lot of complications such as inflammatory and adhesive processes, a massive bleeding associated with disorders of hemostasis in thrombocytopenia or thrombocytosis, enlargement of varicose veins, concomitant pathology. A SE doesn’t cure myelofibrosis but may be performed in some cases. Success is unpredictable, and the operative mortality of SE is on the order of 10%. Objectives Evaluate the clinical and hematologic SE efficiency in patients with MF, resistant to traditional treatment. Patients and methods We analyzed the medical case history of 52 patients with MF, which were observed in the National Research Center for Hematology from 2004 to 2013. All patients were performed SE. 47 patients diagnosed with primary MF, and 4 – post-polycitaemic MF and 1 patient post-trombocitaemic MF. PMF was diagnosed according to the criteria established by WHO (2008), which include bone marrow fibrosis, and splenomegaly leyko-eritroblastosis in peripheral blood. The average age at diagnosis was 47 years (24 – 78 years), and before the SE – 53 (25 – 79 years). Women and men ratio was 1.3:1. According to the predictive model of the Dynamic International Prognostic Score System - (DIPSS) 38 (73%) patients before the SE attributed to intermediate risk 2, 14 (27%) patients – high risk. BCR-ABL gene not found a single patient, and the gene mutation JAK2V617F at different stages of the disease is detected in 53% of cases. Genetic adverse factors are not taken into account because of the small number of studies. Massive and huge splenomegaly was present in 37 (71%) patients. Spleen weight 0.9 to 2.9 kg is removed in 15 (29%) patients, from 3 to 7 kg is removed in 35 (67%) patients. In two cases, removal of the spleen weight of 10 and 11 kg. Statistical analysis were done using SAS 9.3 and JMP 10.0 (SAS Institute Inc., Cary, NC). Results Disease duration was about 76 months and ranged from 1 to 240 months. Surgical complications occurred in 21 patients, including bleeding 3, thrombosis 1, infection 2. Surgical complications from SE in intra-and early postoperative period was 8% from 1978 to 2003. Surgical complications from SE in intra-and early postoperative period was not fixed from 2004 to 2013. After SE complications were hepatomegaly, resistant thrombocytosis (platelet count ≥ 600 × 109/L), leukocytosis (white blood cell count greater than 10 x 109/l) and blast transformation of the disease. More than 80% of patients after SE had no symptoms of intoxication. 22 patients with MF had transfusion dependent anemia, 11 (50%) had disappeared transfusion requirements after SE in and the effect lasted for about a year. Only 4 of the 15 patients with thrombocytopenia who underwent SE due to refractory thrombocytopenia were observed a significant increase in platelet count above 100h109 / L, for an average of 6 months. The possibility of the resumption of long-term cytoreductive therapy appeared in 30 (58%) patients. Currently alive 19 (37%) patients, mean follow-up was 37 months (4 to 72 months) after the SE and of 105 (12-264) months after diagnosis. 33 (63%) patients died during the observation period after SE (1 – 84 months, mean – 27 months). Among these, 27 (82%) patients died of transformation disease and 6 (18%) died of comorbidity. 19 (37%) patients, with a mean follow-up of 37 months after SE (from 4 to 72 months) continue treatment with hydroxyurea. Median survival after SE is 3 years (Figure 1), with a median overall survival - 11 years (Figure 2). Conclusion Despite the potential complications SE is an effective palliative method for patients with MF in the absence of leukemic progression. SE indicated in patients with drug refractory MF, acute constitutional symptoms, anemia, transfusion-dependent, and portal hypertension. Modern methods of surgery, as well as supporting therapy in the perioperative period can safely perform the SE. Disclosures: No relevant conflicts of interest to declare.

Inhibition of normal hemopoiesis-peripheral blood pancytopenia and complete absence of normal colony forming cells in bone marrow [3, 4], is a regular finding in acute (AML) myeloid leukemias [1]. However, this inhibition is reversible. Polyclonal normal hemopoiesis is reestablished in more than two thirds of cases of AML during complete remission, demonstrating the existence of normal stem cells [15]. A few pathogenic mechanisms have been proposed to account for this process: cell crowding, synthesis by leukemic cells of inhibitor substances, cell contact processes and deficiency in the stromal cell microenvironment [2]. Several humoral inhibitors were found to be produced by leukemic cells such as acidic isoferritins, LAI and TNF-alpha [4, 5, 6]. Very little is known about the role of the microenvironment in the inhibition of normal hemopoiesis.

Abstract 2034 Background: Mantle cell lymphoma (MCL) is aggressive B-cell neoplasm diagnosed predominantly among elderly men. (R)CHOP-like schemes are effective in remission induction, but the progression-free survival is disappointingly short (median 16–20 months) with median overall survival of 3–4 years. Upfront use of high-dose cytarabine (12 g/m2), autoSCT and rituximab at all stages of therapy is the most effective treatment but possible only with patients younger 65 years. Decrease in AraC doses to 4 g/m2 per cycle significantly reduce progression free survival. Prominent efficacy of gemcitabine-oxaliplatin combinations and irinotecan in relapsed and refractory MCL patients allowed including these drugs in first-line treatment in cases when the scheme R-HD-Met-AraC (Romanguera J. 2005) is impossible. Aim: Toxicity and efficacy assessment of schemes R-DA-EPOCH/R-GIDIOX and R-DA-EPOCH/ R-HD-Met-AraC in primary MCL patients eligible for autoSCT. Patients and Methods: Since May 2008 35 untreated MCL patients (median 55 years (29–63), males/females 68,5%/31,5%, MIPIb: 34% low, 26% intermediate, 40% high risk) were enrolled. After first R-EPOCH course (Wilson W. 2003) patients were stratified according to toxicity they had received either R-DA-EPOCH/R-HD-Met-AraC or R-DA-EPOCH/R-GIDIOX. In the absence of hematological toxicity grade 4 for more than 3 days, severe infection complications and signs of renal failure patients underwent treatment under the scheme R-HD-Met-AraC (rituximab 375 mg/m2 day 0, methotrexate 1000 mg/m2 24 hours CI day 1, cytarabine 3000 mg/m2 q 12 hrs days 2–3). If there was one of these complications patients underwent treatment under the scheme R-GIDIOX (rituximab 375 mg/m2 day 0, gemcitabine 800 mg/m2 days 1 and 4, oxaliplatin 120 mg/m2 day 2, irinotecan 100 mg/m2 day 3, dexamethasone 10 mg/m2 IV days 1–5, ifosfamide 1000 mg/m2 days 1–5). Further these courses are rotated: either R-DA-EPOCH/R-HD-Met-AraC or R-DA-EPOCH/R-GIDIOX. Depending on the terms of response, patients received 6–8 courses (3–4 cycles) of chemotherapy and autoSCT (BEAM-R) with in vivo purging by rituximab before harvest and reinfusion. Patients with residual tumor after autoSCT were consolidated with local radiotherapy. Rituximab maintenance was performed every three months for 3 years. The protocol was approved by the local ethics committee. Patients were analyzed in an intent-to-treat basis. Overall survival (OS) and event-free survival (EFS) rates were estimated (± standard error) by using the Kaplan-Meier method. Efficacy of the therapy was assessed by Cheson's response criteria (2008). Toxicity assessment was performed 93 R-DA-EPOCH, 60 R-HD-Met-AraC and 46 R-GIDIOX courses. Results: A median follow-up is 23 months (range 3–54). Toward August 2012 26 patients underwent autoSCT: 14 from R-HD-Met-AraC arm and 12 from R-GIDIOX arm. 1 induction death after first HD-Met-AraC course (acute renal failure and septic shock). Maintenance therapy with rituximab was completed in three patients. All patients achieved CR in R-HD-Met-AraC arm. In R-GIDIOX arm OR was 100%: 11 CR and 1 PR (without progression for 26 months after autoSCT). Main non-hematological toxicity of R-GIDIOX was hepatic, with elevated aminotransferases grades 1–2 and 3–4 in 59,5% and 7,1% of courses respectively, without clinical signs. The sources of stem cells were PB in 23 patients and BM in 3 cases of harvest failure after R-GIDIOX. Hematological toxicity of R-GIDIOX course: leukopenia grade 4 was in 71,4% (medium duration was 5,4 days, range 1–13), thrombocytopenia grade 4 was in 42,9%. The estimated 4-years OS rates for the R-GIDIOX group and the R-HD-Met-AraC group were 100% and 68% ± 17%. The estimated 4-years EFS rates for the R-GIDIOX group and the R-HD-Met-AraC group were 90% ± 10% and 69% ± 14%. Conclusions: Our main goal was to incorporate intensive induction, autoSCT and rituximab maintenance in first line therapy MCL patients. However, HD-Met-AraC scheme is highly toxic and its use is possible only in 2/3 of patients younger 65 years. R-GIDIOX scheme is less toxic than R-HD-Met-AraC and equally effective in response induction and mobilizing, so it could be recommended for those in whom high-dose AraC and methotrexate can potentially cause severe adverse consequences. Such an integrated approach might lead to a shift of paradigm of MCL from an incurable to a curable lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4554 Background Severe graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic-stem cell transplantation (allo-HSCT). Steroids are the first-line treatment for established GvHD with a response rate of 30–50%. The efficacy of multipotent mesenchymal stromal cells (MMSCs) in the treatment of steroid-resistant GvHD was reported to be 71–94%. Although it has been shown that the immunomodulatory effect of MMSCs mainly occurs through the secretion of soluble mediators, the exact mechanism of their action remains controversial. Aim The aim of the study was to investigate the influence of immunomodulating factors expressed by donor's MMSCs in vitro on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. IL-6, IL-10, colony stimulating factor 1 (CSF1), indoleamine 2, 3 dioxygenase (IDO1), prostaglandin E synthase (PTEGS) and complement factor H (CFH) were measured as factors playing substantial role in GvHD development. Methods Five patients with steroid resistant acute GvHD were treated with bone marrow derived MMSCs from hematopoietic stem cells donors. MMSCs were infused intravenously at the dose 1×106 per kg of body weight. The efficiency of GvHD therapy by means of MMSCs was scored as: complete response – 3, partial response - 2, clinical improvement – 1, no response – 0. MMSCs were cultured in aMEM with 4% human platelet lysate for 2–5 passages. Total RNA was extracted from MMSCs at 2–3 passages by standard protocol. Relative level of gene expression was estimated by the real-time PCR with previous reverse transcription in MMSCs from 31 donors and determined by normalizing the expression of each target gene to b-actin and GAPDH, calculated using ΔΔCt method for each MMSCs sample. Results Characteristics of patients, donors and graft MMSCs are shown in the table 1. Correlation between relative level of gene expression and efficacy of MMSCs infusion was investigated. The existence of inverse negative relationship between IL-6, CSF1 expression level and treatment score was revealed. The increased level of IL-6 in donor's MMSCs in patients with low and no response (treatment score 1 – 0) could be related to main functions of this factor in inflammation. Increased level of CSF1 in donors' MMSCs could further enhance macrophage activation resulting in GvHD progression instead of inhibition. The augmentation of PTGES expression could be associated with improvement of response to MMSCs therapy, but the correlation was not found. There were no relationship between the expression levels of IL-10, IDO1 and CFH and efficiency of MMSCs infusion. In a patient with no response the relative expression level of all studied factors was altered in comparison with average level of their expression in MMSCs from studied donors: IL-6 and CSF1 increased about 2 fold, while the expression level of IL-10, CHF and PTGES decreased 1.7, 12 and 11 fold correspondingly. So not all MMSCs fit to GvHD therapy that could be associated with their characteristics. Conclusions The data demonstrate the influence of IL-6, CSF1 and PTGES expression on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. As most clinical trials involve non-relative allogeneic MMSCs, analysis of expression level of IL6, CSF1 and PTGES in available MMSCs before infusion could predict the efficiency of acute GvHD therapy and permit the choice of the most proper samples. For successful clinical use of MMSCs further investigation of their properties on cellular and molecular levels are needed. Disclosures: No relevant conflicts of interest to declare. Disclosures: No relevant conflicts of interest to declare.

Abstract 4887 Introduction. Erythropoiesis regulatory proteins – erythropoietin (EPO), hypoxia-inducible factor (HIF-1α), proteins involved in iron metabolism - ferritin, hepcidin (HP), in collaboration with cytokines (IL-2, IL-6 and TNF-α) take part in hematopoietic regulation, in stress response, in apoptosis and are generally involved in the pathogenetic pathways in acute leukemias (AL). We suggested that the existing disbalance in the level of these proteins in (AL) at the time of diagnosis might contribute to the clinical course of the disease (anemia, fever, hypoxia, organomegaly, tumor load) and may be modified by the chemotherapy. Objectives. To reveal changes in the level of regulatory proteins and major interleukins in serum we measured their concentrations in AL patients at different time points: at diagnosis, during the course of chemotherapy (day∼+3-7), at time of WBC nadir after chemotherapy (day∼+14-15), and time of complete remission. We also detected the level of ferritin, HP, HIF-1α and interleukins in homogenates of blast cells in order to reveal the discrepancies in serum and cellular concentrations of these proteins. Material and the methods. We have studied 87 newly diagnosed AL patients. All patients were tested for serum ferritin (SF) by immunoradiometric method; HIF-1α, HP by immunoenzyme method in “the sandwich” version in serum; EPO, IL-2, IL-6 and TNF-α by IFA method. Ferritin, HP, HIF-1α and interleukins were also determined in homogenates blast cells. Results. The data of our measurements are reflected in the table. The values of basic regulatory proteins in serum of AL patients were high already at diagnosis. SF was increased in all patients and its high level was detected through the whole study. In spite of Hb decrease and hypoxia in AL patients the values of HP and HIF-1α are increased due to active proliferation and antiinflammatory processes. It has to be emphasized that high values of HIF-1α in serum have been revealed at time of agranulocytosis (23,7±4,1 pg/ml). High serum values of most important pro-inflammatory cytokine IL-6 (78,4±19,4 pg/ml) and very low values of anti-inflammatory IL-2 (96,2±14,8 pg/ml) were found at diagnosis. Despite low serum concentration, we discovered a high level of TNF-α (179,84±28,3 pg/ml) in homogenates of blast cells. IL-6 level of 1392,7±275,2 pg/m detected in blast cells, 17-fold times exceeded its serum concentration. Value of IL-2 in blast cells remained low at diagnosis as well as at +3-7 days of intensive chemotherapy (115,5±36,5 and 146,4±30,1 pg/ml, respectively). High values of HP and HIF-1α in blast cells were fixed during chemotherapy. HIF-1α concentration has been elevated 15-fold at +3-7 days (85,8 ±24,5 pg/ml). We focus on the fact that there have been high meanings of all parameters studied (SF, EPO, HIF-1α, HP) in complete remission. So our findings indicate the severe discordance in regulatory proteins expression levels in serum and blast cells comparing to donors serum and leukocytes levels. Conclusions. 1. Our study has shown that iron metabolic proteins are integrated in the regulative mechanisms of tumor pathogenesis. 2. The observed changes in the main interleukins levels revealed the severe disbalance in interleukin network in particular regarding the expression in blast cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 4895 Introduction: Minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) can be analyzed by flow cytometry, fluorescent in situ hybridization (FISH) if there are chromosomal abnormalities, polymerase chain reaction (PCR) for chimerical transcripts and immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRG) genes rearrangements. IgH and TCRG rearrangements are the for monitoring of MRD, because it is possible to find specific clonal tumor marker in 80–90% patients. According to the data of different research groups phenomenon of oligoclonality (more than one clonal rearrangement) is identified in 16–80% cases depending on how much molecular markers are used (Scrideli CA, 2001; Gameiro P, 2002; Thorsten Raff, 2006; Csinady E, 2009; Katsibardi K, 2011). Methods and materials: We studied bone marrow samples from 59 patients with new diagnosed B-lineage ALL (43 patients), T-lineage ALL (15 patients) and one patient with bilinear B-T-ALL. Ph-positive ALL was diagnosed in 6 patients, t(4;11) – in one patient. Patients were treated by standard ALL-protocols based on 8 weeks induction remission, 3–5 consolidation courses and maintenance treatment during 2 years. Patients with Ph-positive B-ALL were treated by the same chemotherapy with imanitinib and one patient with mature B-ALL was treated by NHL-BFM-90 chemotherapy. Detection of IgH and TCRG genes rearrangements were carried out by polymerase chain reaction using standard family-specific primers. Synthesis of patient specific primers was based on defined nucleotide sequence of clonal PCR-product. Monitoring MRD was realized by nested PCR using patient specific primers. Results: IgH and/or TCRG genes rearrangements were detected in 50 of 59 patients (84,7%). IgH complete rearrangements were found in 31of 43 patients (72,1%) with B-lineage ALL, IgH incomplete rearrangements were detected in 12 of 43 patient (27,9%) with B-lineage ALL and in one of 15 patient with T-lineage ALL. TCRG rearrangements were found in 13 of 15 patients (86,7%) with T-lineage ALL and in 24 of 43 patients (55,8%) with B-lineage ALL. Seventeen (34%) of 50 patients had one clonal marker. So, phenomenon of oligoclonality was revealed in 33 patients (66%). Two clonal rearrangements were detected in 23 cases, 3 rearrangements were detected in 7 cases and 3 patients had 4 rearrangements. In 3 of 27 patients, in whom all markers were followed, monitoring of MRD showed different pattern of tumor subclones elimination. Monitoring of MRD in patients who had also chromosomal abnormalities such as t(9;22), t(4;11) was carried out by FISH and PCR in Real-time. In one case the patient had 3 IgH rearrangements and chromosomal abnormality t(4;11) at the diagnosis. Later at various stages of chemotherapy we observed a different clearance of IgH rearrangements, however, when cytogenetic remission was achieved and MLL-AF4 expression was absent there were two of three IgH markers. This fact confirms the presence of several tumor subclones, their different clearance during the treatment and suggests persistence of tumor in this patient. Conclusion: In ALL patients with several subclones MRD monitoring should carried out with all markers, as we proved different subclone clearance suggesting different cells sensitivity to cytostatic drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract 1473 The incidence of the secondary neoplasms has increased because of the rising numbers of long-term survivors of tumours. Secondary leukemias (sL) and secondary MDS (sMDS) are among the most common types of secondary tumours. Until recently prognosis in cases of sL and sMDS was considered less favorable than in leukemias de novo. Age at presentation and identified clonal cytogenetic abnormalities are among the most important independent prognostic factors in adult patients with leukemias. It is obvious today that the presence of t(15;17)(q22;q12), t(8;21)(q22;q22), inv(16)(p13;q22)/t(16,16)(p13;q22) predicts a relatively favorable outcome, and in contrast the presence of inv(3)(q21q26)/t(3,3)(q21;q26), del(5q), −5, −7 or a complex karyotype (CK) with 3 or more abnormalities generally suggests a very poor prognosis. The monosomal karyotype (MK) defined as two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in combination with at least one structural chromosomal abnormality is also considered as an adverse prognostic factor according to Breems D.A. et al., 2008; Medeiros B.C. et al., 2010 4-year overall survival in AML patients with MK is very low – 3–4%. Therefore, the purpose of our analysis was to determine the frequency of “unfavorable” and “highly unfavorable” (according to Breems D.A. et al.) clonal cytogenetic abnormalities, identified in our laboratory in bone marrow samples of 143 patients with sL/sMDS and to compare it with the frequency of MK in leukemias and MDS de novo according to a published multicenter study (Haase D. et al., 2007; Medeiros B.C. et al., 2010; Grimwade D. et al., 2010). All examined patients with sL/sMDS had solid tumors or lymphomas in anamnesis, for which they received chemotherapy and/or radiotherapy. sMDS was identified in 81 patients (54 patients – ≤5% blasts in bone marrow; in 27 patients – >5%). sAML was identified in 56 patients, sALL – in 1 patient, sCML – in 5 patients. Abnormal karyotypes were observed in 42 (52%) sMDS patients, in 37 (66%) sAML patients, in all 5 sCML patients, in the only sALL patient. The most frequent abnormality in sMDS was isolated monosomy 7: it was observed in 24.4% of the tested abnormal karyotypes. CK and MK are considerably more frequent in sMDS than in de novo MDS. CK occurred in 12 (30.9%) sMDS patients with abnormal karyotypes. Monosomies or deletions of the long arm of chromosome 7 were detected in 8 of 12 identified CK. Balanced translocations in sMDS were detected in only 9 (21%) of 42 karyotypes; no rearrangements involving 3q26, rather frequently occurred in de novo MDS, were registered. Very rare for de novo MDS t(1;7)(q10;q10) was found in 5 of these 9 cases. In general, chromosome 7 abnormalities (translocations, monosomies and/or deletions) were observed in 58.5% of sMDS cases with abnormal karyotypes. In de novo MDS chromosome 7 abnormalities were detected only in 21% of cases. On the contrary, del(5q) occurred more frequently in de novo MDS than in sMDS (30% versus 12.2%). Monosomic karyotypes occurred in 23.8% of sMDS patients with abnormal karyotypes. “Favorable” anomalies were presented in 5 of 37 sAML cases (13,5%) abnormal karyotypes. t(15;17), as a single anomaly, was detected in 3 patients; t(8;21) was detected in 2 cases. “Unfavorable” abnormalities, such as inv(3)(q21;q26)/t(3;3)(q21;q26) in complex karyotypes were observed in 4 cases. Chromosome 5 deletions in complex karyotypes were found in 5 cases, and only in 1 case - as a single anomaly. Other deletions, del(11)(q23), del(12)(p11), del(13)(q12), were found only as isolated anomalies. Complex karyotypes in sAML were observed in 40% (15 of 37) of cases with abnormal karyotypes, whereas in de novo AML CK occurred only in 18% of patients with abnormal karyotypes. Monosomic karyotypes occurred more often in patients with sAML - 27% compared to 13% of cases in de novo AML. In conclusion, prognostically “unfavorable” and “highly unfavorable” cytogenetic abnormalities account for 60% and 25% of all cases with karyotype abnormalities in sAML/sMDS. Thus, our study shows that “unfavorable” and “highly unfavorable” cytogenetic abnormalities in leukemic clone occur more often in sAML/sMDS than in de novo AML/MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 4420 In the period from 18.01.06 to 25.05.11 we have performed mutation analysis in CML pts with the obvious manifestation of resistance against imatinib. Chronic phase CML patients have been treated by imatinib and monitored by means of RQ-PCR using IS units. At first signs of the evident BCR-ABL level increase (more than 5–10% IS), the mutation analysis has been performed by means of direct PCR fragment sequencing. We have used original primers that enable to search for mutations in the area spanning from a3 to a11 ABL exons of BCR-ABL. Sequencing has been performed for 274 CML pts that were suspected to acquire imatinib resistance. Among them there were 47,45% (130/274) of females (median age 51 years, from 15 to 73), 52,55% (144/274) of males (median age 50 years, from 15 to 74). In the group of mutation negative pts (80/274 – 29,2%) the BCR-ABL level was more than 9% (IS). Interestingly, in this group there were a little bit more females than in the total cohort of pts (58,7% against 47,45%, p=0,034). The median age of the mutation negative females (51 years) was quite the same as in the total cohort, whereas the median age of mutation negative males significantly differed from the one in the total cohort (58 vs. 51 year, p=0,041). 70,8% (194/274) pts tested occurred to be mutation positive (42,8% females (83/194), 57,2% males (111/194)). 214 point mutations of 38 different types have been detected, among them T315I (26/194 – 12%), G250E (26/194 – 12%), T317L (16/194 – 7,4%), F359V (15/194 – 7,0%), H396R (15/194 – 7,0%), M244V (15/194 – 7,0%), E255K (12/194 – 5,6%), Y253H (12/194 – 5,6%), E255V (9/194 – 4,2%), L248V (8/194 – 3,7%), E355G (6/194 – 2,8%), M315T (6/194 – 2,8%), Q252H (5/194 – 2,3%), L387F (4/194 – 1,8%), S348L (4/194 – 1,8%), F311I (3/194 – 1,4%), F359C (3/194 – 1,4%), E255D (2/194 – 0,9%), E275K (2/194 – 0,9%), E279A (2/194 – 0,9%), K247R (2/194 – 0,9%), E292V (1/194 – 0,46%), E334G (1/194 – 0,46%), E450K (1/194 – 0,46%), E459A (1/194 – 0,46%), E459K (1/194 – 0,46%), F359I (1/194 – 0,46%), F486S (1/194 – 0,46%), L383F (1/194 – 0,46%), P441L (1/194 – 0,46%), Q252M (1/194 – 0,46%), Q491L (1/194 – 0,46%), T305I (1/194 – 0,46%), T345I (1/194 – 0,46%), V299A (1/194 – 0,46%), Y312C (1/194 – 0,46%), T520S (1/194 – 0,46%). As rare events we have observed some other types of mutations. In one CML patient with primary imatinib resistance there was a G425Stop (1/194 – 0,46%) mutation which caused a significant truncation of the C-end of the BCR-ABL protein. There were also 2 highly imatinib resistant pts with deletions of exon a7 ABL (2/194 – 0,9%). One more unusual mutation was an insertion of 72 bp between the a8 and a9 exons of ABL. The origin of this insertion was a middle part of the 8th intron of the ABL gene. This sequence is arranged as a typical human exon because it is flanked by donor and acceptor splicing signals. We found this mutation in 3 imatinib resistant CML pts (3/194 – 1,4%). This mutation gives rise to the abnormal stop-codon within the exon a9 due to a CD’s frameshift. In all pts with point L248V mutation (8/194 – 3,7%) we have also observed additional abnormal variant of BCR-ABL gene as a splicing isoform lacking 81 bp of exon a4. The point mutation L248V (742C>G) exchange the motif CAAGCT for CAAGGT and the latter is a strong splice acceptor site. It seems that this cryptic splice acceptor site in the middle of 4a exon carrying 742C>G point mutation may compete with natural splice acceptor site from 4th intron. It gives rise to two BCR-ABL transcripts: one carrying a point mutation and the other one with 81 bp’s deletion of the 3’end of exon 4a, without frameshift. These findings suggest that in the case of mutational analysis the considerable attention should be paid not only to BCR-ABL point mutations but also to the possible emergence of the other changes of the BCR-ABL mRNA structure which may occur due to arbitrary activity of splicing machinery. Our findings also suggest that among imatinib resistant CML pts some of them already acquired mutations that prevented adequate response for the 2nd generation of TKI. In our study 12% of BCR-ABL mutation positive CML pts resistant to imatinib (n=26/194) had T315I which also render nilotinib and dasatinib resistance. 23,8% (n=51/194) were positive for mutations that decreases nilotinib response (E255K/V, F359C/V, Y253H) and 7,5% (n=16/194) were positive for T317L that hinders dasatinib response. Disclosures: No relevant conflicts of interest to declare.

Abstract 4996 It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio ( We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample. 19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1 CD34+/Bcl-2+ CD34+/Bax+ CD34+/CD95+ CD34+/ACE+ BM PB BM PB BM PB BM PB AML pts n=10 0 day 38±11,6* 41±14 24,4±7,9 29,2±7,6* 16,4±8,5 23,2±7,8 21,7±9,5 20,8±8,7* 36 day 13,5±3,4** 23,7±5** 46,2±11,5 50,3±11 19,9±5,5 36,4±10 34±6,6 35±9,2** ALL pts n=9 0 day 36±11 33,7±12 46,2±9,4 37,4±3,7* 3,4±1,1* 7,1±2,5* 41±10,9 33,2±9,7* 36 day 18,4±5,8 26±8,9 38±11,8 40,5±10 26,2±9,1** 40,9±9,2** 34±10 62,8±10** Donors n=8 11,7±1,6 26,1±5,9 22,8±4 67,8±6,7 13,4±3,2 47,7±11,6 28±5,3 68,2±10,2 * − p CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar. CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures: No relevant conflicts of interest to declare.

Abstract 1879 Splenectomy in patients with MDS is a treatment option that is beeing applied very rare [Steensma D., et al, Leuk Res.,2003; Bourgeosis E., et al, Leukemia, 2001]. There are anecdotal reports with very few patients demonstrating its efficacy. In most cases splenectomy was indicated for MDS patients with immune related thrombocytopenia. Here we would like to report the results of 33 splenectomies in patients with MDS who have been treated in our Center during 1994–2010. Within this period of follow-up totally 155 patients were diagnosed with different forms of MDS, 35% of them presenting with hypoplasia. The MDS treatment algorithm in our Center incorporates splenectomy as one of the options for pts with hypoplastic forms of MDS with bone marrow blast count less than 10%, refractory to initial cyclosporin A treatment or refractory to transfusions. Among patients who were splenectomised there were 20 females, 13 males with a median age of 40 years (range 18–74). Median time from diagnosis to splenectomy was 12 months (range 4–107). By WHO-classification there were 2 patients with RA, 22 – with RCMD, 2 – with MDS and del (5q), 6 - with RAEB, 1 - with AML after MDS. Cytogenetic analysis was available in 32 cases, and karyotype was normal in 15 patients (47%).The most common abnormalities were: del (5q) - 3, del (20q) - 2, trisomy 8 - 2, tetrasomy 8 - 1, monosomy 7 - 2, complex karyotype - 4. Bone marrow biopsy revealed hypoplasia in 25 patients (75%), myelofibrosis – in 7 (21%). The median WBC count was 2,6*109/L (range 0,6-8,7), hemoglobin 6,9 g/dL (47-119) and platelets 26*109/L (6-170). 27 pts (82%) were RBC transfusion dependent, 22 (67%) - platelets transfusion dependent. 13 pts had received immunosuppression therapy (ATG, cyclosporine A) before splenectomy, 2 - cytotoxic chemotherapy, 3 - decitabine. The majority of splenectomies were done by laparoscopic method - 26 (79%), in one case the convertion was done. In all cases we performed liver biopsy. Postoperative complications (hemorrhage) occurred in 1 patient but there were no deaths due to operation. One death occurred in 7 days after splenectomy due to fulminant progression to AML. Median spleen weight was 180 gms (range 70–930). Median intraoperative blood loss was 250 ml (range 50–9350). Histology was available in 30 patients. Extramedullary hematopoesis was revealed in 3 cases (10%), blast infiltration - in 2 (7%), massive lymphoid infiltration was detected in 5 cases and in one patient in was proved to be clonal (marginal zone lymphoma, MZL). Hemosiderin depositions in the macrophages were seen in half of the cases -16 (53%). One case was characterized by granulomatosis in spleen and liver with negative immunohistohemical staining to Mycobacteria tuberculosis. Splenectomy lead to sustained improvement of cytopenias in 16 cases (48%): decreased transfusion dependence in 14 (42%) and transfusion independence in 2 (6%). After splenectomy 5 patients were followed by “wait and see” approach, 17 continued with immunosuppressive therapy (ATG,CyA), 3 patients were treated with cytotoxic chemotherapy, 1 – with decytabine, 2 received EPO, 1- danazol, 2 - iron chelation therapy, 2 – only transfusions therapy. We did not noticed the infections rate augmentation after splenectomy. Transformation to AML was registered 6 (18%) at median 6 months (0,3 -9). 13 splenectomized patients (39%) died at a median 12 months (range 0,3-84) and the main death reasons were: AML progression, aplasia deterioration followed by infections and hemorrhage. 20 patients are alive with a median follow-up after splenectomy 33 months (2-108). Analysis of our 15-years study data give us a confidence to conclude that splenectomy still may be an adequate option for distinct forms of MDS (hypoplastic forms with bone marrow blast count less than 10%, refractory to initial immunosupressive treatment or refractory to transfusions), producing cytopenia improvement in half of the patients with decreasing transfusion dependance also in half of the patients, sometimes bringing a clear diagnosis (MZL). The mechanism of action is not very clear but we can speculate that splenectomy removes the “cell-destroying” organ, deminishes immune pathways of cytopenias due to large lymphoid compartment deletion, provides the resustainment of sensitivity to immunosupressive agents. Disclosures: No relevant conflicts of interest to declare.

Abstract 4348 Acute leukemia during pregnancy is a rare but not an unique event: 1 case per 75–100.000 pregnant women and the majority of cases are registered in the 2nd/3rd trimester. The main law has to be carried out in this life threatening situation: to save two lives - mother's and child's. Though in the 1st trimester medical abortion is highly recommended, in the 2nd/3rd trimester chemotherapy should be applied without dose corrections. It is considered to be of no major danger to the fetus and gives good chances to the mother. Here we would like to report our 20 years experience with 32 pregnant women (median age 25y (19-35)) with acute leukemias: 13 AML, 5 APL and 14 ALL. 26 (81%) of them were diagnosed with AL in the 2nd/3rd trimester. We used the following approach: 1. up to 12 weeks of pregnancy medical abortions were performed (6 pts - 1 AML, 1- APL, 4-ALL); 2. at 35–40 weeks of pregnancy “cesar sections” were done and then chemotherapy was started (7 pts – 4 AML, 2 APL, 1 – ALL). All 7 children are alive and well; 3. at 13–34 weeks of pregnancy standard chemotherapy was applied according to AL subtype treatment protocol (19 pts – 8 AML; 2 APL, 9 ALL). In AML 7+3 protocol (ARA-C – 100 mg/m2 bid 1–7 days, Daunorubicin 45 mg/m2 (in 3 pts) or 60 mg/m2 1–3 days), in APL – 7+3+ATRA or AIDA (2 pts), in ALL - 8 weeks induction and prolonged post-CR therapy - were used. All together in 13 AML pts there were 9 CR (69%), 2 ED (15,5%), 2 DR (15,5%); in 5 APL pts – 4 CR (80%), 1 ED (20%); in 14 ALL pts – 9 CR (64,3%), 3 DR (21,4%), 2 ED (14,3%). 85% of pts had infectious complications during induction, 1 ATRA-syndrome, 1 emergency “cezar section” was performed at 30th week of pregnancy due to premature placenta unlayment (PPU) with hemorrhage due to L-asparaginase. There was one late spontaneous abortion (+21 weeks), no deaths and no defects were registed among newborns “treated” in uteri (n=18), all of them were followed in micropediatriac departments, neutropenias was registed in half of them and pneumonias in 3 (17%). All children (n=25) are alive and well. The oldest is 20 years old now, the youngest – 10 months. The probability of 5-years overall survival in AML pts was 34,6%, in ALL – 26,7%. In APL only 1 pt is alive in 1st CR due to 1 death in consolidation and two relapses. Within the period of the study we have developed some practical recommendations: 1. Daunorubicin in 7+3 courses should be used at 60 mg/m2 as there were no CR in pts receiving 45 mg/m2. It's a crucial point as CR must be achieved after the 1st course, especially in 30–32 weeks of pregnancy because delivery must be carried out in stable status. 2. Idarubicin can be used safely in resistant to daunorubicin AML pts and in AIDA protocol for APL. AIDA is less toxic than 7+3+ATRA for APL induction and well effective. 5-days mitoxantrone consolidation should be postponed to 3–4 months after delivery. 3. L-asparaginase should not be applied in ALL treatment during pregnancy (only after delivery) due to coagulation disturbances and possibility of premature placenta unlayment (PPU). 4. Delivery during AL treatment must be planned at 34–36 weeks of pregnancy. 60% of our patients had “cesar sections”. 5. Every day gynecologists care is absolutely needed (uteri hypertonus, fetus hypotrophy, PPU, etc). 6. Chemotherapy restart should be planned 3–4 weeks after delivery because immediate (within 5–7 days) continuation of cytostatic treatment caused severe combined infections complications due to postdelivery immunodeficiency and desadaptation. In case of resistant leukemia restart treatment in 2 weeks and not with high-dose protocols. 7. In newborn children all complications can be cured within 1–5 weeks in special micropediatic departments, children grew up healthy and intelligent. 8. The results in ALL pts with pregnancy seem to be worse than in general ALL population. 9. After CR in APL careful monitoring of MRD may provide better outcome and avoid aggressive consolidation courses. The main conclusion that comes up from this data is the obvious necessity to treat a pregnant woman with acute leukemia diagnosed in the 2nd/3rd trimester with adequate chemotherapy, that results in saving the child's life and - in many cases – the mother's. The overall survival in pregnant women with acute leukemia is quite similar to the outcome in all patients, though we wished it to be better. Disclosures: No relevant conflicts of interest to declare.