Background: Recent evidence suggests that MSCs might improve survival during sepsis in animal models. However, no study has investigated the effects of MSC therapy on the survival of pts with sepsis and SS, especially severe-neutropenic pts. Aim: The aim of this study was to investigate the efficacy of the administration of MSCs for the treatment of SS in neutropenic pts. Patients and Methods: This prospective, single-center, randomized Russian clinical trial of MSCs in severe neutropenic pts with SS (RUMCESS) (NCT 01849237) was approved by the local ethics committee and was begun in December 2012. Neutropenic pts (WBC < 0.5x109/l) with SS were enrolled on to the study. The pts were randomly assigned (1:1) to receive either conventional therapy (CT) of SS (CT group), or CT plus donor MSCs at a dose of 106/kg intravenously within the first 10 hours after SS onset (CT+MSCs group). Written informed consent was obtained for all pts. All pts were admitted and treated in the ICU of the National Research Center for Hematology (Moscow). The Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sepsis-related Organ Failure Assessment (SOFA) score were employed to determine the severity of illness. Pts were followed up for 28 days after enrolment in the study, and 28-day all-cause mortality was assessed. Pts characteristics and complication rates were compared using Fisher's exact test. The Kaplan-Meier method with the log-rank test, and Cox proportional hazard regression model were used to determine the statistical significance of the relationship between overall survival (OS) and the treatment regimen. Statistical analyses were performed using SAS 9.1. Results: Of the 27 neutropenic pts with SS, 13 received CT and 14 received CT+MSCs. There were no statistically significant differences in the demographic variables between groups . The CT group included 7 males, 6 females, aged 33-81 yrs, median 55 yrs. The CT+MSCs group included 6 males, 8 females, aged 30-75 yrs, median 48 yrs. Hematological disorders were also similar in the two groups: AML (4), NHL (4), HL (1), MM (3), MDS (1) in the CT group, and AML (5), NHL (7), MM (1) in the CT+MSCs group. In all pts, except for one with MDS, neutropenia developed after chemotherapy. In 8/13 pts in the CT group and 9/14 pts in the CT+MSCs group blood cultures were found positive, mostly gram-negative. Baseline APACHE II scores (34.2 [95% CI 28.3-43.1] and 32.2 [95% CI 26.2-37.5] in the CT- and CT+MSC-groups, respectively), and SOFA scores (17.9 [95% CI 13.5-22.2] and 15.1 [95% CI 11.0-19.2] respectively), were similar in the two groups. 28-day survival rates were 15% (2 out of 13 pts) in the CT group and 57% (8 out of 14 pts) in the CT+MSCs group (P=0.04) (Figure 1). The significant increase in 28 days OS of the pts in CT+MCSs group was associated with SOFA score decrease, which was started in three days after onset of SS. Despite higher 28-day survival rates only 3 pts treated with CT+MSCs remained alive after 3 months, and 5 of 8 pts from the CT+MSCs-group who survived 28 days died later because of sepsis-related organ dysfunction. Conclusions: Administration of MSCs in the first hours of SS might improve short-term survival in neutropenic pts, but does not prevent death from sepsis-related organ dysfunction in the long term. Perhaps repeated administration of MSC is required. Figure 1. Comparison of OS rates between the two groups of pts in the ICU. There was a statistically significant increase of the 28-day OS rates (42% vs. 15%; P=0.04) and a statistically significant decrease of the risk of death (HR 0.35; 95% CI 0.13 - 0.91; P=0.04) in the CT+MCSs group vs. the CT group. Figure 1. Comparison of OS rates between the two groups of pts in the ICU. There was a statistically significant increase of the 28-day OS rates (42% vs. 15%; P=0.04) and a statistically significant decrease of the risk of death (HR 0.35; 95% CI 0.13 - 0.91; P=0.04) in the CT+MCSs group vs. the CT group. 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.

4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. 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 in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

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