ALBERT

Description: Introduction: Allogeneic stem cell transplantation is a potentially curative treatment for patients with high-risk non-Hodgkin lymphoma (NHL). Fludarabine/busulfan based conditioning regimens are widely used in Europe for this purpose. Busulfan dose intensity discriminates between reduced intensity (FB2, 2 days of busulfan at 4 mg/Kg/d per os or 3.2 mg/kg/d iv) and reduced-toxicity myeloablative (FB3/FB4, 3 or 4 days of busulfan at 4 mg/Kg/d per os or 3.2 mg/kg/d iv) conditioning regimens (Bacigalupo, 2009). While some data have been recently published showing some advantages of higher busulfan dose intensity for myeloid malignancies, there is no such data available in the lymphoid setting. Methods: This was a large retrospective study conducted on behalf of the SFGM-TC including all adults allografted in France between January 2004 and December 2014 for NHL (n=378). Clinical data were obtained through ProMISe (Project Manager Internet Server), an internet-based system shared by all French transplantation centers. We aim to compare various outcomes (overall (OS) and lymphoma free (LFS) survivals, relapse incidence (RI), non-relapse mortality (NRM), acute and chronic GVHD) between those who received FB2 (n=277) or FB3/FB4 (n=101) as conditioning regimens. GVHD free relapse free survival (GRFS) was also studied (defined as alive with no previous grade III-IV aGvHD, no moderate or severe chronic GvHD (cGvHD) and no relapse). Results: Both groups were comparable for the following variables: median follow-up (FB2: 24.9 vs FB3/4: 23 months), gender (male 61% vs 53%), disease type (low-grade lymphoma 25% vs 21%, mantle-cell lymphoma 17% vs 13%, high-grade lymphoma 25% vs 21%, T cell lymphoma 32% vs 45%), disease status at transplant (complete remission/very good partial response 64% vs 62%, partial response 28% vs 31%, active disease 8% vs 7%), donor type (sibling 43% vs 49.5%, matched unrelated 56% vs 47), median number of previous courses of treatment (2 vs 2, p=0.44), stem cell source (peripheral blood 96% vs 95%). FB2 patients were significantly older (median 57.3 vs 53.1 years, p=0.07), have been transplanted more recently (median year of transplant: 2011 vs 2010, p=0.001) and have been more previously autografted (69% vs 50.5%, p=0.001). FB3/4 patients have been allotransplanted earlier during the evolution of their disease (median time between diagnosis and allograft 18.2 vs 33.8 months, p=30 months), there were also no significant differences between both groups in terms of OS, LFS, RI or NRM. In multivariate analysis there was a trend for worse outcome using FB3/FB4 regimens (OS: HR 1.46, 95%CI: 0.96-2.23, p=0.07; LFS: HR: 1.43, 95%CI: 0.99-2.06, p=0.05; RI: HR 1.54; 95%CI: 0.95-2.48, p=0.07). These results were also confirmed using a propensity score-matching strategy including 184 FB2 and 98 FB3/4 patients. Conclusion: This large retrospective study showed that reduced toxicity myeloablative fludarabine/busulfan regimens did not improve outcomes of adults allografted for NHL. FB2 conditioning regimen still should be considered as the standard of care conditioning regimen in this setting. To validate these results, prospective studies are needed, like the French prospective trial currently ongoing for myeloid diseases (NCT01985061). Also, new conditioning regimens and post-allograft strategies should be tested to improve outcomes of patients. Disclosures Peffault De Latour: NOVARTIS: Consultancy, Honoraria, Research Funding; PFIZER: Consultancy, Honoraria, Research Funding; ALEXION: Consultancy, Honoraria, Research Funding.

Description: Introduction Since the first descriptions by Slavin et al, indications of allogeneic haematopoietic stem cell transplantations (AHSCT) with fludarabine and busulfan conditioning regimen (CR) have increased. The use of strongly immunosuppressant products such as antithymocyte globulin (ATG) increase transplant engraftment and limit the risk of GVHD but with a higher risk of infectious complications. Recently, with myeloablative conditioning regimen (MAC), one randomized and one retrospective studies showed a reduction of cGVHD incidence (1,2). In reduced CR (RIC), the results are contradictory. In large series, the use of ATG seems to increase the relapse rate and ultimately influence the overall survival (OS) (3). Therefore, the ATG dose in RIC conditioning remains discussed. In order to clarify this point, we conducted a retrospective study on patients who received a fludarabine-busulfan RIC AHSCT with easer one or tow days of ATG. Methods 168 consecutive patients, followed between January 2000 and December 2011 in 2 French centers were analysed. RIC was based on Fludarabine 150 mg/m² and Busulfan for 2 consecutive days. Selected dose of ATG (Thymoglobuline®) administered were easer 2.5 or 5 mg/kg/day. Information concerning donors, recipients, conditioning regimen, graft harvesting and follow-up were collected using prospectively designed forms from Promise database. Results 108 patients had received 2.5 mg/kg (ATG1) versus 60 patients 5 mg/kg (ATG2). Median follow-up was of 60 months range [18-148]. Median age was 58 years with 99 males and 69 females. Diagnosis included AML/MDS (n=84), ALL (n=7), CML (n=7), lymphoma or CLL (n=52), and myeloma (n=18). Donors were matched sibling (n=74) or unrelated (n= 94). Only 6.5% of transplants were in HLA mismatch. Median time to recovery of polynuclear neutrophils was 17 days (range 3-73.2). Time to platelet reconstitution was 11 days (range 3-39). Median of OS was 39 months. In multivariate analysis, disease status at transplant, aGVHD severity (III-IV) (p=.000044; 3.52 (1.92-6.45) and cGVHD occurrence were the prognostic parameters statically significant. Median of Disease Free Survival (DFS) was 45 months (1-111) with no difference between ATG1 and ATG2 or other parameters. Death attributed to disease progression seemed to be higher in ATG1 group but difference failed to reach statistical significance (25% versus 17%, p=0.21). Incidence of non-relapse mortality (NRM) at 3 months was 5% and the global NRM at 26%. In multivariate analysis, two parameters influence significantly the NRM, the aGVHD incidence (III-IV) (p=.00016; 8.03(2.73-23.65)) and the dose of ATG delivered (31% vs. 16% for ATG2; p=.048; 2.36 (1.01-5.54)). Interesting, NRM was principally significant in late events (〉 3 months) with more death by GVHD or infectious disease in ATG1 group. No difference between ATG1 and ATG2 group was noted for hematopoietic reconstitution, rejection rate (2 in each group of SAL) and incidence of aGvHD. Although cumulative incidence of cGvHD seemed to be higher in ATG1 group, difference failed to reach statistical significance (42% versus 33%, p=0.23). Conclusion Our result show that 2 days of ATG decrease NRM independently of disease, source of donor, graft or GVHD incidence without increasing the risk of relapse or infectious disease. These results are compatible with previous results observed with MAC. According to our results, in RIC, higher dose of ATG might be recommended. 1. Socie G, et al; Blood. 2011 Jun 9;117(23):6375–82. 2. Mohty M, et al; Leukemia. 2010 Sep 30;24(11):1867–74. 3. Soiffer RJ, et al. Blood. 2011 Jun 23;117(25):6963–70. Disclosures: No relevant conflicts of interest to declare.

Description: The benefit of radiotherapy (RT) following chemotherapy in limited-stage DLBCL remains controversial. Before the Rituximab era, 4 randomized trials have been reported with conflicting results (ECOG 1484 and SWOG 8736, GELA 93-1 and 93-4 studies). More recently, the German Unfolder study prematurely closed the R-CHOP without RT arm in bulky limited-stage DLBCL due to an excess of relapse. In 2005, we conducted a randomized trial in patients with non-bulky (defined by a tumor size 50% but a persistent positive FDG-PET) after C4, 2 additional cycles of R-CHOP followed by RT (even if not initially allocated) were recommended. The primary objective was EFS at one year after the last randomization, and secondary objectives were the impact of interim FDG-PET on EFS and the toxicity of RT. From May 2005 to December 2013, 313 patients were randomized and 301 patients are currently evaluable. Median age was 56 yr (20-75). There were 181 males and 120 females: 106 patients (35%) were older than 60 yr. Most patients had normal LDH (82%), PS=0 (80%), and no B symptoms (96% of cases). Modified IPI score was as follows: IPI =0 (n=170), IPI=1 (n=113), IPI=2 (n=16), IPI=3 (n=2). Main tumor sites were cervical (n=159), Waldeyer’s ring and sinus (n=36), inguinal (n=29), axillary (n=25), mediastinum (n=21). Extra-nodal sites were observed in 121 patients (40%). One hundred and fifty patients were randomized in the R-CHOP arm and 151 in the R-CHOP + RT arm. After 4 cycles, 253 patients (84%) were in CR and 43 in PR (14%). Three patients had stable disease. Thirty-four patients (79%) out of the 43 partial responders received 2 additional cycles of R-CHOP followed by RT (including 12 patients not initially allocated to RT arm). At the end of treatment, CR and PR rate were 94% and 3%, respectively. Seven (4%) out of the 151 patients randomized in the RT arm declined radiation. With a median follow-up of 51 months (2-110), there were 20 relapses: 12 in the R-CHOP arm and 8 in the R-CHOP+RT arm (p=ns). Sixteen patients died. Causes of death were as follows: relapses (n=9), toxic (n=1), secondary malignancies (n=3), unknown (n=3). Median time of relapse was 21 months (2-110 months). EFS and OS are not statistically different between the two arms. In an intent to treat analysis, 5y-EFS is 87% n the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.55), p=0.13, and 5yr-OS is 90% in the R-CHOP arm versus 95% in the R-CHOP + RT arm (HR=0.60), p=0.32. For patients in complete response after the 4 cycles of R-CHOP (84% of the patients), 5yr-EFS is 89% in the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.59), p=0.24. In this prospective study, the results demonstrate that in non-bulky limited-stage DLBCL, R-CHOP alone (4 to 6 cycles) induces very high CR rate with a very good overall survival and a very low relapse rate. With the current follow-up, the addition of radiotherapy is not significantly superior to R-CHOP alone and should be reserved to the minority of patients who do not reach CR after R-CHOP. Disclosures Gyan: Roche: Research Funding.

Description: Background Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for MDS patients (pts). Due to transplant-related mortality, only pts with short life expectancy are referred to HSCT, usually those with IPSS int-2 or high risk. The aim of this prospective study was to compare outcome in candidates for HSCT according to donor availability: no donor, HLA-matched sibling donor, 10/10 HLA-matched unrelated donor and 9/10 HLA-mismatched unrelated donor. Method SFGM-TC and GFM centers that had agreed on general recommendations concerning the management of MDS pts participated to the study (16 centers). Transplant indications were int-2 or high IPSS, int-1 with refractory thrombocytopenia or proliferative CMML. Pts were registered in this study if older than 50 years, when they acquired an indication for HSCT and in the absence of comorbidity contraindicating HSCT. A donor research was initiated at registration including HLA-matched sibling donor, HLA-matched unrelated donor (10/10) or mismatched donor for one allele. Other alternative donors (mismatched unrelated cord blood or 〉 1 mismatched donor) were not accepted. Transplantation was scheduled upfront if bone marrow blasts 〈 10% at inclusion or after treatment with AML like anthracycline-aracytine chemotherapy (CT) or azacitidine (AZA) if marrow blasts 〉 10% ideally within 6 months if a donor was identified. Recommended reduced intensity conditioning regimen consisted in fludarabine, busulfan and anti-thymoglobulin with peripheral stem cells (PB) as source of stem cells. Characteristics of pts and disease were compared within 3 groups: no donor, HLA-matched donor (sibling or 10/10), HLA-one mismatched donor (9/10). Overall and disease free survivals (OS, DFS) were compared using Kaplan Meier estimates. Cumulative incidences of complete remission and disease-related mortality were compared using Gray-test. Results From April 2007 to January 2013, 163 pts were included: 34 (21%) pts had no donor; 115 (71%) pts had an HLA-matched donor (34% sibling and 37% unrelated) and 14 (9%) pts had an HLA mismatched donor. Groups were well-balanced for age, gender, time from diagnosis to inclusion, WHO classification, bone marrow blasts at time of inclusion, cytogenetic (IPSS) and IPSS classification. WHO classification at time of inclusion was: AML post MDS in 12, RAEB1 in 29, RAEB2 in 82, CMML1 or 2 in 20, RCMD in 14 and other MDS in 6 pts. Cytogenetics were favorable in 49 (30%), intermediate in 37 (23%) and poor in 74 (45%) pts. IPSS was int-1 for 8%, int-2 for 69% and high for 23% of pts. Median follow-up was 38 months. 117 pts were treated by AZA and 40 by CT. Bone marrow blasts 〈 10% were achieved in 68% and 57% for pts without and with donor, respectively. 69% of pts with HLA-identical donor and 57% of those with HLA-mismatched donor were transplanted. Some pts with donors were not transplantation because of excess of marrow blasts in most pts (〉 10% in 3, 〉 20% in 20 pts), comorbidities contraindicating the transplantation acquired after inclusion (9 pts), early deaths (7 pts) and other causes in 3 pts. Four other pts scheduled for transplantation have not been transplanted yet. Probability of complete remission 12 months after inclusion was: 39% (95% CI: 22-55), 49% (95%CI: 40-58) and 46% (95%CI: 17-71) for pts without a donor, with an HLA-compatible donor and with HLA-mismatched donor. Disease-related mortality at 48 months was not significantly different in the 3 groups: 50 % (95%CI: 29-68), 38% (95%CI: 27-49), 51% (95%CI: 17-77). DFS was also not different in 3 groups: 18% (95%CI: 7-44), 25% (95%CI: 16-37) and 9% (95%CI: 1-57). In contrast, OS at 48 months was better in pts with HLA-compatible donor than in pts without donor or those with HLA-mismatched donor: 35% (26-49), 17% (6-43) and 8% (1-55), respectively, p=0.011 (Figure 1). The poor survival observed in pts transplanted with an HLA-mismatched donor may be due to their small number and requires further analysis in a larger cohort. Outcome was not different in pts receiving PB from HLA-matched sibling or from HLA 10/10 matched unrelated donor. Conclusion In a prospective study, we observed that int-2 or high risk MDS pts with a HLA-matched donor have an improved life expectancy as compared to pts without donor. This study confirmed that they should be referred to the transplantation before acquisition of comorbidity contraindicating HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Background Unrelated cord blood transplantation (UCBT) after reduced intensity conditioning regimen (RIC) has extended the use of cord blood in elderly patients and those with co-morbidities without an HLA identical donor, although relapse post transplant remains a concern in high risk AML patients. HLA incompatibilities between donor and recipient might enhance Natural Killer (NK) cell alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT). We previously observed that the quality of NK cell reconstitution was impaired after haploidentical HSCT, impacting on graft versus leukemia (GvL) effect, but was preserved after UCBT in a small cohort of patients. Methods To evaluate RIC-UCBT in patients with acute myeloid leukemia (AML), a prospective phase II multicentric trial was conducted in France, whose primary objective was to show a reduction in non-relapse mortality (NRM) from 40% (based on registry data) to 20%. Seventy-nine patients were enrolled for a de novo or secondary AML in complete remission (CR). The conditioning regimen consisted of cyclophosphamide (50mg/kg) + fludarabine (200mg/m2) + total body irradiation (2Gy), CsA +MMF as GVHD prophylaxis and GCSF from day +1. Patients were enrolled in 23 centers from October 2007 to September 2009. Engraftment rate was 87 % at day+60. At 2 years, overall survival, incidence of relapse and LFS were respectively 44%, 46% and 35%. Peripheral blood samples were collected following UCBT in order to realize an extensive phenotypic and functional study of NK cells. Studies were started at 1 month (M1) post UCBT with available samples for 62 out of the 69 included patients, and were compared to 20 healthy donors and 15 cord blood (CB). Results Total CD3+ T-cells were 117 /mm3 at M1 (range 0-934), and 465 /mm3 at M3 (range 0-2917). CD19+ B-cells were 36/mm3, (range 0-524) and 342/mm3 (range 0-2990) at M1 and M3 respectively. NK cell recovery was prompt, representing 47% of the total lymphocyte population at M1 (186 CD3-CD56+ NK cells/mm3), 30% at M3 (239/mm3; range 2-767) and decreasing to normal rate at M6 (20% of lymphocytes). At M1 post-UCBT, NK cells exhibited high rate of CD56bright, NKG2A, and KIR2DL4 associated with a decreased expression of CD8 and CD161, compared to CB and healthy donors. These immature characteristics were transient and return to normal value from M3 or M6 post-UCBT. Interestingly, we also observed a significant increased expression of the activation markers CD69, and HLA-DR during the whole period of the study, compared to CB and healthy donors, which probably reflects a persistent proliferation state of the NK cells. On the other hand, NK cells post-UCBT were indistinguishable from CB and healthy donors control samples for other receptor tested such as NKp30, NKp46, NKp80, and NKG2D. Notably, Expression of KIR2DL1 was decreased at M1 and M3 but reached similar values to controls at M6, whereas, KIR3DL1 was increased during the whole study. To determine the significance of these phenotypic features, we assessed polyfunctional ability of NK cells following UCBT by a combined analysis of the degranulation (CD107a), and the production of IFN-γ and TNF-α. This study reveals that NK at M1 post-graft exhibited a transient higher ability to produce IFN-γ than healthy donors (p

Description: The incidence of AT and Fg deficiency and of thrombotic and bleeding events after Asp has been well evaluated in children with ALL, but little is known in adults. In this study, the incidence of these events and the use of coagulation supportive treatments was evaluated in 214 adult patients with ALL (n= 191) or LBL (n=23) included in the GOELAL02 trial (Blood 2004, in press). The induction therapy included steroids (40 mg/m2/d d1-21), vincristine (2 mg d1, 8, 15, 22), idarubicine (5 mg/m2 d1, 8,15, 22) and Asp (7500 UI/m2 d10, 13, 16, 19, 22, 25) delivered through a central venous access. Fresh frozen plasma (FFP), Fg (Clottagen®) and AT (Aclotine®) concentrates were recommended to maintain their levels 〉 1 g/l and 60%, respectively. If no transfusion, Asp infusion was delayed for 48 hours. Platelets were transfused when 〈 20 x 109/l. Heparin prophylaxis was left to institutional guidelines. Symptomatic thromboses and significant bleedings were systematically recorded. Active DIC was present in 10.3% of patients on d1. Fg and AT levels were measured 4098 times (20/patient, von Clauss assay) and 1718 times (8/patient, chromogenic assay) respectively, and evolved between d1 and d35 as shown below (mean ± sd). AT nadir was 〈 60% in 71% of patients with values 〈 40% in 26% of cases. Fg levels were 〈 1g/l in 73% of patients with values 〈 0.5g/l in 9%.Twenty-one thromboses occurred two to 35d after the first Asp infusion (median = 14d) in 20 patients (9.3%), with cerebral vein thrombosis (5), pulmonary embolism (3), upper (5) or lower (8) limb deep vein thrombosis. At the time of event, the median AT level was 48% (mean 60.7) with 12 of 21 thromboses (57%) occurring with AT 〈 60%. Fourty-two bleedings occurred one to 45d after the first Asp infusion (median = 8d) in 31 patients, with CNS hemorrhage (1), epistaxis (24), central venous access bleeding (8), GI bleeding (1), and large ecchymoses (8). At the time of event, median Fg level was 1.3 g/l. Asp infusions were reduced or delayed in 64% of all patients due to low Fg and/or AT levels. FFP, AT and Fg were infused in 31%, 41% and 52% of patients at mean doses of 5.3 ml/kg/infusion, 31UI/kg/infusion and 7.9 g respectively. AT level increased from 58%±16 (n=79) to 86%±23.2 (n=57) after the first AT infusion but was unchanged after FFP. Fg level increased from 0.9g/l ± 0.3 (n=85) to 1.4g/l ±0.5 (n=69) after Fg infusion but was unmodified after FFP. In conclusion, Fg and AT levels are frequently decreased in adults treated by Asp, with often a less than optimal chemotherapy. Bleeding events were not associated with severe Fg deficiency, but thrombotic events could be favored in some patients by acquired AT deficiency. The benefit of AT concentrates to prevent thrombosis and to reduce delay in Asp infusions could therefore be prospectively assessed in adults treated by Asp. d1 (n) d10-Asp 1 (n) d13-Asp2 (n) d19-Asp 4 (n) d25-Asp 6 (n) AT % - 121±16 (79) 83±16 (125) 65±20 (111) 65±22 (77) Fg g/l 3.2±1.7 (142) 1.9±1.0 (159) 1.4±0.7 (195) 1.1±0.4 (169) 1.4±0.6 (118)

Description: Abstract 3447 Second allogeneic stem cell transplants for hematological malignancies are associated with a high incidence of non-relapse mortality (NRM) due to the cumulative incidence of toxicity. On the other hand, the RIC approach represents an attempt to harness the immune graft-versus-leukemia (GVL) effect while attempting to control or overcome toxicity. The aim of this analysis was to assess the outcomes (overall survival: OS, leukemia-free survival: LFS, NRM, and relapse incidence: RI) in a cohort of 103 AML patients (57 males) who have received a second RIC allo-SCT (RIC2) as a salvage therapy after relapse following a first RIC allo-SCT (RIC1), and who were reported to the EBMT registry with adequate data. In this series, the median age at time of RIC2 was 54 (range, 20–69) years. The median time from RIC1 to relapse was 184 (range, 28–2377) days. The median time from RIC1 to RIC2 was 307 (range, 44–3165) days, and the median time from relapse to RIC2 was 84 (range, 7–582) days. For the RIC2 transplant, 58 patients (56%) received an allogeneic graft from an HLA-identical sibling donor, while 45 patients (44%) received an HLA-matched or mismatched graft. The stem cell source was PBSCs in the majority of cases (89%). At time of RIC2 transplant, 82 patients (80%) were in relapsed or refractory disease. Only one patient was in first complete remission (CR), while 20 patients (19%) were in CR2 or CR3. Various RIC regimens as per EBMT definition were used for preparation prior to the second allo-SCT. After RIC2, 89 patients (86%) engrafted and the median time to ANC〉500/μL was 15 (range, 1–40) days. The incidence of grade 3–4 acute GVHD after RIC2 was 14%. With a median follow-up of 11 (range, 2–99) months after RIC2, 22 patients were still alive. At 2 years, the rates of OS, LFS, NRM and RI were 19+/−4%, 13+/−4%, 27+/−4%, and 60+/−4% respectively. In multivariable analysis, no factors were associated with a higher risk of NRM after RIC2. However, multivariate analysis showed that the interval from first transplant (RIC1) to relapse (〉6 months) was the strongest predictive factor significantly associated with improved LFS and a lower RI after RIC2 (P=0.004, HR=1.91, 95%CI, 1.23–2.95; and P=0.0008, HR=0.41, 95%CI, 0.25–0.69, respectively). When comparing patients progressing before or beyond 6 months after the first RIC allo-SCT, LFS and RI rates were 6+/−3% vs. 21+/−6% (P=0.002) and 71+/−7% vs. 48+/−8% (P=0.001), respectively. In all, these results suggest that the initial use of a RIC regimen prior to allo-SCT can reduce overall transplant-related toxicity, thus making a second RIC transplant feasible, especially if compared to historical results achieved in the setting of standard myeloablative conditioning second allo-SCT. Results achieved after a second salvage RIC allo-SCT compare favorably with those achieved when testing new drugs in phase I trials performed in this setting of relapsed AML (e.g. HDAC inhibitors etc.). Of note, AML patients who relapse beyond 6 months after a first RIC allo-SCT are potential candidates for a second RIC allo-SCT with a relatively acceptable rate of NRM. Disclosures: Mohty: Genzyme: Consultancy, Honoraria, None, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; Janssen Cilag: Consultancy, Honoraria; Celgene: Honoraria.

Description: Background: Unrelated cord blood transplantation (UCBT) after reduced intensity conditioning regimen (RIC) has extended the use of UCB in elderly and unfit patients without an HLA identical donor. KIR ligand incompatibility between donor and recipient might favor Natural Killer (NK) cell alloreactivity after UCBT in AML patients (Wilhemze et al, 2009), although contradictory results were reported (Brunstein et al, 2009). We previously reported the results of the biological NK cell reconstitution after RIC-UCBT in a French prospective phase II multicentric trial (Rio et al, 2015). We showed that NK cells generated from RIC-UCBT exhibited features of transient immaturity and stable activation, correlating with a high ability to produce IFN-γ and a quick restoration of the ability both to produce TNF-α and degranulate (Souchet et al, ASH 2013). The aim of the present study is to analyze the impact of KIR ligand incompatibilities and NK cell reconstitution on OS, DFS and TRM after RIC-UCBT in a prospective trial. Materials and methods: Seventy-six patients with a de novo or secondary AML in complete remission were enrolled in 23 centers from Oct. 2007 to Sept. 2009. Peripheral blood samples were collected during the first year following UCBT in order to realize an extensive prospective phenotypic and functional study of NK cells. DNA samples were also collected in recipient and cords blood to perform KIROTYPE and HLA-C allelic typing. NK biological data were available at M1 for 54 patients. The inhibitory Killer-Immunoglobin Receptors (KIR) KIR2DL1, and KIR2DL2/3 bind KIR ligand C2 and C1 respectively, resulting in inhibition of NK-cell mediated lysis. Recipients and UCB were classified into C1 or C2 family depending on their HLA-C typing (C1-C1, C1-C2 or C2-C2). Results: Among the 54 patients, 35 events occurred (relapse or TRM). Median EFS and OS were 13.2 and 18.3 months, respectively. Recipients C2-C2 had a significant worse EFS and OS than C1-C1 or C1-C2 (median EFS C2-C2=3.8 month vs 15.1 month for C1-x; p=0.002); median OS C2-C2 3.8 months vs 29.9 months for C1-x; HR=6.12, IC95% [2.069; 18.113], p=0.001). High intracellular staining of CD107a, reflecting the capacity of NK degranulation with HLA negative K562 target, correlated with better OS. CD107a expression was divided in 2 groups at median (=51%). Median OS of CD107 (0-50%) was 12.8 months vs 20.9 months for CD107a (51-66); p=0.029. Relapse risk was highly increased in recipients C2-C2 (HR=5.04 (IC 95% [1.23; 20.56], p=0.02). Low expression of CD16 (HR=0.97, IC95% [0.937; 0.999], p=0.043), high expression of HLA-DR (HR=1.08, IC95% [1.031; 1.123], p=8e-04) on NK cells, and recipients C2-C2 (HR=9.44, IC95% [1.311; 67.882], p=0.026) significantly increased the risk of TRM. The inhibitory KIR2DL1 receptor binds to C2 ligands. Of interest, KIR2DL1 was significantly decreased on C2-C2 recipients NK cells at M1, as compared to C1-x recipients NK cells. On the contrary, KIR2DL2/3 and KIR3DL1 restored promptly, suggesting a sequential expression of KIRs. As interaction between inhibitory KIRs and their ligands are essential for NK cells to become functional ("licensing" process), we can hypothesize that the weaker expression of KIR2DL1 on C2-C2 NK cells alters the licensing process, rendering the NK cells hypo-responsiveness. Conclusion: Recipient C2-C2 is correlated with a worse outcome (EFS, OS, relapse, TRM) after RIC-UCBT in a prospective trial for AML patients. Weak capacity of degranulation and low expression of CD16 are associated with worse OS and increased TRM, respectively. These features can reflect an alteration of the NK licensing process and might have impact on clinical outcome after UCBT. Disclosures No relevant conflicts of interest to declare.

Description: Background: AZA is the standard of care for patients with either HRMDS or AML with 20-30% blasts (Oligoblastic AML). The response rate is ~50% with a median response duration of 12-15 months. The mechanisms underlying primary and secondary resistance are poorly understood and it remains difficult to accurately predict which patients will respond and in responders, to predict secondary resistance. The main factors demonstrated or suggested to interfere with response to AZA and/or survival include marrow blast percentage, performance status, IPSS cytogenetic risk, presence of peripheral blasts, transfusion dependency, IPSS-R risk, mutations of TET2, P53, IDH1/2, and DNMT3A, elevated expression of BCL2L10, Fas, or PI-PLCbeta1. Deregulation of microRNAs is a hallmark of MDS, yet its consequences on AZA efficacy have not been specifically addressed in HRMDS. Methods: We measured the expression of 754 miRNAs in SKM1 MDS cells resistant or sensitive to AZA. miRNAs of interest were ectopically expressed or specifically inhibited in HEK293T cells which were assayed for AZA sensitivity (MTT). Seven miRNAs were quantified in bone marrow mononuclear cells deriving from 75 patients with HRMDS (n= 50) or oligoblastic AML treated with AZA [median age 74, 24 females, median cycle number 6, (1-39)]. Results: Seven miRNAs (miR-125a-5p, miR-99b-5p, miR-126, miR-126*, hsa-let-7c, miR-34b-3p, and miR-10b*) that include 6 tumor-suppressor miRNAs, were found differentially expressed between AZA-sensitive versus -resistant cells; all being transcriptionally repressed in resistant cells. In silico, 5 of these miRNAs were found to target the 3' UTR of DNA methyltransferase 1 (DNMT1) while Zhao et al. [Arthritis & rheumatism, 2011; 63(5)] have shown that miR-126 directly inhibits DNMT1 translation. DNMT1 is one of the main AZA molecular target, the higher the level of DNMT1 expression, the lower the AZA sensitivity [Li et al., Cancer biology & therapy, 2010; 9(4)]. Microarray and western blotting showed that levels of DNMT1 protein, but not mRNA, were significantly higher in AZA-resistant cells. In HEK293T cells, specific endogenous miR-126/126* inhibition significantly augmented DNMT1 protein expression and triggered AZA-resistance, as measured through MTT proliferation assay. In the same cells, ectopic expression of miR-126/126* decreased DNMT1 protein expression in a concentration-dependent manner. In the 75 patients treated with AZA, a decreased expression level of all but miR-10* was associated with decreased response rate (IWG 2006 criteria) and poor outcome; miR-126/126* having the strongest prognostic impact. When compared with miR-126*High MDS, miR-126*Low MDS had significantly lower overall response rate (37% versus 60%, p=0.04), higher relapse rate (100% versus 71%, p=0.034), shorter progression-free (PFS) (8±8% versus 49±12% at 3 years, p=0.004, log rank test), and overall survival (OS) (16±6% versus 46±9%, p=0.004). Baseline patient characteristics of the 2 groups were similar. Multivariate analyses were performed using the Cox proportional hazards model. Table 1 shows that age, miR-126* expression, and IPSS-R risk independently predicted PFS and OS. miRNAs expression was analyzed over time in 15 patients. The mean expression level of 5/7 miRNAs increased over time in the 10 responders without statistically significant difference (DNS) between the first and latest values. In contrast, the expression of 7/7 miRNAs decreased over time in the 5 patients with treatment failure, the difference being statistically significant for 2 miRNAs (p²0.043, Wilcoxon test). Secondary resistance occurred in 7/10 responders and was found associated with a decreased expression of 6/7 miRNAs (p²0.044 for 4 miRNAs) versus 2/7 (DNS) in the 3 long-term responders. Conclusion: Our results suggest that in HRMDS and oligoblastic AML, i. primary or secondary resistance to AZA is associated with the decreased expression of anti-DNMT1 tumor-suppressor microRNAs that trigger AZA resistance in vitro; ii. measuring miRNAs expression before and under treatment might help to predict primary or secondary AZA resistance and thereby to rapidly offer alternative treatment; iii. increasing AZA exposure might help to circumvent AZA resistance in case of either low or decreased anti-DNMT1 miRNA concentration while using anti-DNMT1 microRNAs as a therapeutic tool might increase or restore AZA sensitivity. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Wattel:AMGEN: Consultancy, Research Funding; PIERRE FABRE MEDICAMENTS: Research Funding; CELGENE: Research Funding, Speakers Bureau; NOVARTIS: Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding.