ALBERT

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: To evaluate the prognostic significance of clinicobiologic and pathological features in angioimmunoblastic T-cell lymphoma (AITL), 157 AITL patients were retrieved from the GELA LNH87-LNH93 randomized clinical trials. One hundred forty-seven patients received a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)–like regimen with intensified courses in half of them. Histologically, 41 cases were classified as “rich in large cells” and 116 as “classic” (including 19 rich in epithelioid cells, 14 rich in clear cells, and 4 with hyperplastic germinal centers). Sixty-two cases were scored for CD10 and CXCL13 expression according to the abundance of positive lymphoid cells. Median age was 62 years, with 81% advanced stage, 72% B symptoms, 65% anemia, 50% hypergammaglobulinemia, and 66% elevated LDH. Overall 7-year survival was 30%. In multivariate analysis, only male sex (P = .004), mediastinal lymphadenopathy (P = .041), and anemia (P = .042) adversely affected overall survival. Increase in large cells and high level of CD10 and CXCL13 did not affect survival. Intensive regimen did not improve survival. In conclusion, AITL is a morphologically heterogeneous T-cell lymphoma commonly expressing CXCL13 and CD10 and carrying few prognostic factors. It portends a poor prognosis even when treated intensively. However, AITL is not always lethal with 30% of patients alive at 7 years.

Description: Aims: To evaluate the diagnostic value of bone marrow biopsy (BMB) with immunohistochemistry studies compared to blood flow cytometry (FC) in response evaluation after treatment in CLL. Patients and methods: 75 previously untreated patients with stage B and C-CLL were treated with oral Fludarabine (30mg/m2/d) + Cyclophosphamide (200mg/m2/d) combination, day 1 through 5, repeated every 28 days for 6 courses. Response evaluation was performed 2 months after the completion of therapy, according to NCI criteria. Sixty patients out of 75 achieved partial or complete remission and underwent BMB. Minimal residual disease (MRD) was simultaneously evaluated on paraffin-embedded BMB by anti-CD5, -CD20 and -CD3 immunostaining and in the peripheral blood using a very high sensitive FC method (Maloum et al, Br J Haematol, 2002, 119:970–5). BMB immunostaining was considered positive for MRD in case of concurrent expression of CD20 and CD5 by the same cells assessed on consecutive sections. Twenty nine patients could have been analyzed so far. The median follow-up was of 49 months [range 19–56]. Results: In all these 29 patients, BMB was considered histologically normal according to the NCI criteria. In 23/29 cases, bone marrow immunostaining did not show expressing CD20 cells. Fifteen out of 23 were in phenotypic remission using blood FC (PhR), of whom only 2 relapsed (at 31 and 37 months). The remaining 8/23 patients did not achieve PhR and all relapsed (at 22, 27, 37, 40, 45 and 51 months). In 6/29 patients, immunochemistry on BMB showed persistent B CD5+ cells : 3 did not achieve PhR of whom 2 relapsed (at 32 and 37 months) while 3 were in complete PhR and displayed a persistent clinical complete remission with a follow-up of 39, 47 and 47 months respectively. Interestingly, in the latter 3 cases, FC analysis showed an expansion of polyclonal BCD5+ cells in blood as assessed, which could explain the presence of such normal BCD5+ cells in the bone marrow. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy (ACC) to predict relapse for BMB and FC are given in the following table: Sensitivity (%) Specificity (%) PPV (%) NPV (%) ACC (%) BMB 16.7 76.5 33.3 56.3 51.7 FC 83.3 94.1 90.9 88.9 89.7 Conclusion: These results strongly suggest that BMB should not be indicated in the evaluation of treatment response and is not useful for relapse prediction. In contrast, blood flow cytometry constitutes the best method in routine laboratory to predict clinical outcome and should be included in response treatment criteria in CLL.

Description: New treatment approaches in CLL aim at lowering as much as possible the level of minimal residual disease (MRD) to prolong the duration of response and eventually overall survival. Such a goal should be achieved by combining chemotherapy and monoclonal antibodies. The present phase II study was designed to evaluate the efficacy and the safety of first line therapy with 3 courses of oral fludarabine and cyclophosphamide (FC : F 40 mg/m² and C 250 mg/m² D1 to D3 every 4 weeks) followed by consolidation treatment with alemtuzumab in previously untreated patients (pts) aged 65 to 70 years with Binet stages B and C CLL. Pts achieving complete response (CR) and partial response (PR) after FC were eligible for the second part of the study i.e. consolidation with alemtuzumab administered subcutaneously (SC) at the dose of 10 mg thrice a week for 8 weeks. CMV antigenemia was monitored every week and alemtuzumab was discontinued in pts with more than 3 positive nuclei. MRD was evaluated before and during alemtuzumab treatment using a highly sensitive 6 color flow cytometry technique. Since severe infectious toxicity has been reported by others with similar strategies, FC regimen was limited to 3 courses and the duration of alemtuzumab treatment to 8 weeks with stringent stopping rules for CMV in this trial targeting a population of elderly pts in first line treatment. From June 2004 to June 2006, 42 pts have been enrolled in the study of whom 37 are assessable so far for induction treatment with FC. CR was achieved in 11 pts (30%) and PR in 17 pts (46%) for an overall response rate of 76% (28 pts). FC regimen was discontinued in 5 pts (13.5%) and stable disease was observed in 3 pts (8%). All the responding pts but one (one woman with onset of breast cancer) proceeded to alemtuzumab consolidation after a 2 month rest period. Alemtuzumab therapy has been completed in 16 pts, discontinued in 5 pts because of CMV reactivation as defined above (none of them developed CMV disease) and is still on going in 6 pts. Blood MRD assessment by flow cytometry was centralized in one center (RL) and could be performed in 19 pts with a set of 14 pts having been tested sequentially before alemtuzumab, after 4 weeks and after 8 weeks of treatment. In most cases, there was an impressive decrease of MRD level following alemtuzumab therapy and residual tumor cells were no longer detected. Detailed results of MRD study will be presented at the meeting. In conclusion, 3 courses of FC yielded a high response rate in previously untreated elderly CLL patients and SC alemtuzumab consolidation could thereafter be administered safely in most of them, resulting in a striking reduction of blood MRD.

Description: Introduction: AITL, one of the most common peripheral T-cell Lymphoma portends a poor prognosis. AITL is characterized by neoplastic T cells with a follicular helper immunophenotype, frequent mutations in epigenetic regulators TET2, IDH2, DNMT3A and in RHOA, and a prominent tumor microenvironment that could contribute to lymphomagenesis. Aiming to target this microenvironment and given the promising activity of lenalinomide (Len) in a relapsed setting (PMID: 23731832), we postulated that AITL patients (pts) might benefit from a treatment with Len combined with a classical CHOP regimen. This multicenter, open label, phase 2 trial (NCT01553786) investigates this treatment in previously untreated elderly pts. Patients and methods: Patients older than 59 years were treated with 8 cycles of Len + CHOP 21 (Len 25 mg/day (d), d1 to 14) and received intrathecal methotrexate prophylaxis. Thromboprophylaxis with low molecular weight heparin was mandatory. PET CTs at diagnosis and at the end of treatment were centrally reviewed. The primary objective was to evaluate the complete metabolic response rate according to the Lugano 2014 Classification. Secondary endpoints were safety, progression-free (PFS) and overall survival (OS). Mutations in TET2, IDH2, DNMT3A, RHOA, CD28, PLCG1, STAT3 and STAT5B were analyzed by deep sequencing (1000X) using DNA extracted from formalin-fixed paraffin-embedded tumor samples by PGM technology and were correlated to clinical parameters. Results: Between November 2011 and March 2017, 80 pts were enrolled, and 78 were evaluable. Central pathology review confirmed the diagnosis of AITL in 72 cases (92%). Median age was 69 (59-80), 52 % were female, 68% had a performance status of 0 to 1, 94% an Ann Arbor stage ≥III, 82% IPI≥3. Forty-five patients (58 %) completed the 8 planned cycles (mean number of cycles delivered, 5.9). Of the 624 planned treatment cycles, 458 (72 %) were completed. Treatment was stopped in 8 pts because of progressive disease, and in 15 because of adverse events. Toxicity was within the range expected of R-CHOP therapy with 70% grade 4 neutropenia and 31 % thrombocytopenia. Deep vein thrombosis occurred in 8 pts. Four secondary primary malignancies were reported. Five patients died from toxicity (4 from infection). Len dose reductions or interruptions were applied in 37 (5%) and 59 (9%) cycles, respectively, related to toxic effects. The median dose of Len per patient was 2275 mg (IQR 95-2825)-i.e. 81% of the planned dose of 2800 mg. Doxorubicin and cyclophosphamide were administrated at 98% and 97% of the planned dose. Complete metabolic response was observed in 34 patients (43.6%) (90%CI = [34.0%; 53.5%]), partial metabolic response in 3 (3.8%), no metabolic response in 2 (2.6%) and progressive metabolic disease in 16 (20.5%), the other being not evaluated because of progression (N=20) or death (N=3). With a median follow-up duration of 31.5 months (95%CI = [23.0; 43.7]) at the time of the cut-off, 2-year PFS is 42.3% (95%CI = [30.9%; 53.2%]) and 2-year OS is 60.1% (95%CI = [47.4%; 70.7%]). IPI was strongly associated with the survival (figure 1A). Mutational status was successfully determined in 64 pts with confirmed AITL diagnosis. TET2 mutations were detected in 49 cases (77%), with 28 (43%) pts bearing ≥2 TET2 mutations. RHOAG17V mutations in 34 pts (53%), DNMT3A mutations in 20 (31%) pts, including 6 with the DNMT3AR882X variant and IDH2 mutations in 14 (22%). CD28, PLCCG1 and STAT mutations were detected in less than 10% of pts (figure 1B). TET2 mutations correlated to age〉65 years (p=0.006) and IPI 3-5 (p=0.007). Interestingly, DNMT3A mutations were associated with a decreased response rate (p=0.003), a shorter PFS (p=0.04) and a trend toward a shorter OS (0.08). It is noteworthy that none of the 6 pts with the DNMT3AR882X mutant had response, suggesting that the resistance to anthracycline reported in DNMT3AR882X mutated acute myeloid leukemia (PMID: 27841873) could also occur in DNMT3AR882X mutated AITL. No correlation between other detected mutations and outcome was observed (table 1C). Conclusion A combination of 25 mg of Len for 14 days with CHOP cycles gives acceptable toxicity in AITL elderly pts. However, response rate and outcome appear similar to previous studies. We also confirmed in a prospective study the frequency of mutations in epigenetic regulators and RHOA in AITL and clarified their prognostic impact. Figure Figure. Disclosures Bachy: Gilead Sciences: Honoraria; Sandoz: Consultancy; Janssen: Honoraria; Roche: Research Funding; Takeda: Research Funding; Amgen: Honoraria; Celgene: Consultancy. Cartron:Celgene: Consultancy, Honoraria; Sanofi: Honoraria; Gilead Sciences: Honoraria; Janssen: Honoraria; Roche: Consultancy, Honoraria. Casasnovas:Merck: Honoraria; Takeda: Honoraria; Roche: Honoraria; Gilead Sciences: Research Funding; Roche: Research Funding; Janssen: Consultancy; Gilead Sciences: Consultancy; MSD: Consultancy; merck: Consultancy; takeda: Consultancy; Roche: Consultancy; Janssen: Honoraria; Celgene: Honoraria; Gilead Sciences: Honoraria; MSD: Honoraria. Tilly:Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees. Gaulard:Celgene: Research Funding; Roche: Honoraria; Takeda: Consultancy, Honoraria, Research Funding. Haioun:Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Sciences: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Description: Background: Recent advances in next-generation sequencing (NGS) techniques enable the quantitation of circulating tumor DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin (IG) receptor gene sequence. The aims of this study were 1) To evaluate the clonal heterogeneity of follicular lymphoma (FL) and its variations between tumor and plasma at diagnosis in patients (pts) included in PRIMA trial, and 2) To assess the prognostic value of the level of ctDNA at diagnosis on PFS (median follow up of 6 years). Methods: Using the NGS-based immunosequencing method (Adaptive Biotechnologies, South San Francisco, CA), the lymphoma clonotype was first established in the tumor biopsy using locus-specific primer sets for IGH-V, IGH-D and IGK rearrangements. Clonotypes regarded as originating from the lymphoma clone were present at a frequency greater than 5%. The lymphoma-derived sequences identified in the biopsy sample were then used as targets to assess the presence of ctDNA in plasma samples. The quantity and frequency of the ctDNA in plasma is calculated relative to the total number of reads in the sample and defined as lymphoma clonotype molecules per million diploid genomes. For 34 FL pts from the PRIMA trial, both tumor biopsy and plasma samples at diagnosis were available. Results: One tumor clonotype or more could be detected in 29 pts (85%) in the tumor diagnostic sample (DNA isolated from fresh frozen tissue). Indeed, with the IGH-V assay, between 1 to 3 different clonotypes were identified in 23 pts. With the IGH-D assay, 1 clonotype was detected in 2 pts. With the IGK assay, 1 to 2 different clonotypes were detected in 17 pts. The tumor clonotypes were detected in diagnostic plasma samples in 25 out of the 29 pts (83%). Moreover, in 14 pts we detected clones in the plasma samples that are related (with point mutations in the V(D)J sequence) to the highest frequency index clone identified in the tumor sample. In half of the cases, we observed a different repartition of the subclonal populations between tumor and plasma samples at diagnosis: for these cases the highest frequency clone in the tumor was not the highest frequency clone in the plasma. Among the 24 pts with an IGH clonotype (IGH-V or IGH-D assay), ctDNA could be detected in 19 cases. We could not find a correlation between peripheral blood dissemination and the presence of a stereotyped sequence or an N-glycosylation motif within the CDR3 region of IGH clonotypes. Interestingly, in one case, two different related clones were detected at high frequency in tumor sample and the putative parental clone, not detected in the tumor sample, was detected in plasma sample as the highest frequency clone. The level of ctDNA ranged from 0 to 345,000/million diploid genomes in the 29 plasma samples (median 39,720). The presence of ctDNA was correlated with bone marrow involvement (p=.005), but not with FLIPI score, LDH level, anemia, bulky disease, presence of circulating lymphoma cells (detected by morphology or flow examination), Ann Arbor stage or β2microglobulin level. The absolute level of ctDNA in diagnostic samples did not correlate with any of the clinical characteristics. We assessed the prognostic value of the level of ctDNA at diagnosis. Pts (14/29) with higher levels of ctDNA at diagnosis (〉40,000/million diploid genomes) had shorter PFS than patients with lower levels of ctDNA (median 15.7 m vs NR, p=.027, Fig 1). In an exploratory multivariate Cox regression model including FLIPI score, bulky disease and β2-microglobuline level, high ctDNA level was the only factor significantly associated with a worse PFS (HR 4.2, p=.039). In the observation arm, pts with high ctDNA levels had a median PFS of only 11.6 months vs NR (p=.01). Whereas in the maintenance arm, the prognostic value of elevated ctDNA levels was erased (p=.6). Conclusion: Using the NGS-based immunosequencing method, we were able to show a variation in clonal heterogeneity between tumor biopsy and plasma samples obtained at diagnosis. The poor prognostic value of elevated ctDNA levels in plasma at diagnosis appeared higher than other clinical parameters in a multivariate cox regression model. These findings need to be validated in larger cohorts. Figure 1. PFS according to the level of circulating tumor DNA Figure 1. PFS according to the level of circulating tumor DNA Disclosures Carlton: Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:adaptive biotech: Employment, Other: stockholders. Salles:Roche: Honoraria, Research Funding.

Description: Abstract 3103 Introduction: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) provides the best chance of long-term survival for patients with intermediate or high-risk acute myeloid leukemia (AML). The major limitation of this procedure is the risk of treatment related mortality (TRM). Use of reduced intensity conditioning (RIC) regimen has become standard practice among older candidates with comorbidities. Although RIC regimen have been used for over a decade in older patients, the benefit of this approach in younger patients with AML compared with the risk of toxicity of standard regimen (MAC) is still discussed. We compared the outcomes for patients with AML over 35 years using RIC or MAC HSCT. Patients, methods, and transplantation characteristics: From January 2000 to December 2010, 132 consecutive patients older than 35 years with AML (18 secondary AML) received HSCT in our center, either from siblings (n=87) or HLA 10/10 allele-matched donors (n=45). MAC (n=72) and RIC (n=60) regimens were defined as previously described (Bacigalupo, 2009). Seventy-three patients were in first complete remission (CR1); 30% of patients had poor risk cytogenetics (MRC classification). Karnofsky performance status was scored at time of HSCT. Engraftment, acute and chronic graft-versus-host disease (GvHD), transplantation-related mortality (TRM), relapse rate as well as overall survival (OS) at 4 years were compared according to the intensity of the conditioning regimen. First a classical multivariable Cox analysis was conducted. In a second step, baseline confounding factors were adjusted for using inverse probability-of-treatment weighting (IPTW). Results of the comparison: Patient characteristics according to the intensity of the conditioning regimen were similar for AML type (de novo versus secondary), gender, karnofsky performance status, CR#, donor type and number of CD34+ infused. Particularly, cytogenetic risks were comparable in both groups. Patients were younger in the MAC group (median age 44 years [range 35 to 56 years] vs 54[37 to 66] for RIC, p

Description: Autoimmune cytopenias represent a well-known complication of chronic lymphocytic leukemia (CLL). The most frequent one is autoimmune haemolytic anemia (AIHA), occurring in about 5% of the patients during the course of the disease. The prognostic significance of autoimmune cytopenias remains uncertain and controversial. In previous studies, a positive direct antiglobulin test (DAT) was found in up to 35% of CLL cases, however only a minority of the patients would develop AIHA at any time of the disease. The aim of this monocentric study was to explore the clinical and biological characteristics and the outcome of CLL patients showing a positive direct antiglobulin test at any time during the course of the disease. We reviewed all CLL patients seen at our institution who had at least one positive DAT between January 2007 and May 2013. Fifty-four patients were found, representing about 10% of our CLL cohort. The sex ratio was 1,8 (35M/19F). Median age at diagnosis was 66,2 years (range 44,7 to 87,3). According to the Binet classification, 41 (76 %) patients were in stage A, 7 in stage B, and 6 in stage C. Study of usual prognostic parameters evidenced that these patients represented a very high risk group: 82% of cases harbored unmutated IGHV, and CD38 was expressed in 60% cases. Cytogenetic alterations were studied by FISH analysis: 11q deletion, trisomy 12 and 17p deletion were found in 12,5%, 22,9% and 14,3% cases respectively. In this cohort, with a median follow up of 85 months, 41 (76 %) patients required treatment. The first line of treatment was initiated for CLL progression in 33 cases, for symptomatic AIHA along with CLL progression in 5, and for symptomatic AIHA (without CLL progression) in 3. The median time to first treatment was very short (16 months), and the median overall survival of this cohort was 84 months. In 22 cases, the DAT was positive from the time of diagnosis, while in 34 patients it became positive later during the course of CLL. Only 19 patients (35%) developed symptomatic AIHA. DAT specificity was IgG, IgG+ C or C for respectively 36, 10 and 8 patients. Interestingly, there was no impact of the time of first positive DAT during the course of the disease (at diagnosis or later). Moreover, occurrence of a symptomatic AIHA had no impact on treatment free survival or overall survival when compared with cases with positive DAT only. Among these cases, 41 were in stage A at diagnosis and up to 78% harbored unmutated IGHV (as compared with 30 % in our institutional stage cohort). In order to evaluate the impact of a positive DAT on a homogenous population, we focused on the 31 IGHV unmutated stage A cases and compared them with our cohort of IGHV unmutated stage A with consistently negative DAT. We found a significant prevalence of VH1-69 and VH 3-21 use (43 %) and a high percentage belonging to a stereotyped subset (34%). The majority of the cases (19/28 tested) were responders to in vitro B-cell receptor stimulation by anti IgM. In this population, time to first treatment (26 months) was slightly shorter but median overall survival (55 months) was significantly reduced highlighting the poor response to treatment. In conclusion, occurrence of a positive DAT at any time of the course of CLL is associated with poor outcome and has the same adverse prognostic impact as the onset of a symptomatic AIHA. Moreover, study of B-Cell receptor (BCR stimulation by anti-IgM, CDR3 stereotypy) show that these patients may represent a pathophysiological distinct subgroup in which antigen stimulation is likely to play a major role. Disclosures: No relevant conflicts of interest to declare.

Description: Background CD20 is a cell surface antigen commonly expressed on B-cell lineage, but is absent on hematopoïetic stem cells and plasma cells. Therefore, it has been postulated that its targeting should not significantly affect immunoglobulin serum concentrations. However, due to rapid and prolonged B-cell depletion, rituximab-based therapy has been shown to impair humoral response. In addition, protective serologic response to influenza vaccination in lymphoma patients in the 6 months following rituximab-containing regimen has been found to be particularly low1. Because the vast majority of adults individuals have been previously vaccinated against tetanus, we decided to analyse the impact of prolonged rituximab-based therapy on the immune protection rate against tetanus toxin in patients, and conducted in this aim a sub-analysis in follicular lymphoma (FL) patients treated into the PRIMA study. Methods The PRIMA study is a multicentre, phase III, randomized study in patients with advanced FL evaluating the benefit of maintenance therapy with rituximab after induction of response with chemotherapy plus rituximab in comparison with no maintenance therapy, whose results have been previously published2. In a subset of patients, serum samples were prospectively collected and antitetanus toxin antibodies were measured at baseline, end of immunochemotherapy induction and end of maintenance period. Protective titer was defined as 〉 0.1 UI/mL according to standards. Statistical analysis aimed to determine if serum antitetanus antibodies titers significantly changed over time according to different phases of treatment, and if rituximab maintenance arm had a significant impact as compared to observation arm. Results Overall, 104 patients were evaluable for this subanalysis. Fifty-five percent were male. Median age was 57 (range, 27-80), 88.5% had Ann-Arbor stage III-IV disease. All patients had received R-CHOP induction. Among those patients, 56 were randomised into rituximab arm, 48 into observation arm. At baseline, median serum antitetanus antibodies titers in the overall population was 0.8 UI/mL (range, 0–8) and 89.4% of patients had protective titers. At the end of induction, among the 70 evaluable subjects, 90% had still a protective titer with a median of 0.7 UI/mL (range, 0-4), and at the end of maintenance period, among the 62 evaluable patients, 93% had still protective titer with a median of 0.8 (range, 0-3). When comparing the two arms, at the end of maintenance period, median serum antitetanus antibodies titer was 0.8 UI/mL in the rituximab arm, not significantly different from that in the observation arm (0.6 UI/mL) (p=0.17). At the end of the maintenance period, 100% of subjects had still protective titer in the rituximab arm versus 84.6% in the observation arm. Conclusion In this subanalysis of 104 FL treated patients issued from the PRIMA study, rituximab-based induction and maintenance therapy did not significantly affect the immunisation rate against tetanus toxin in previously immunized patients. These results are in contrast with the poor serologic response rate obtained following rituximab-based therapy, emphasizing that common serum vaccinal titers should be controlled, and vaccination updated, when possible, before treatment initiation and the first rituximab administration. References 1-Yri O, Torfoss D, Hungnes O, et al. Rituximab blocks protective serologic response to influenza A (H1N1) 2009 vaccination in lymphoma patients during or within 6 months after treatment. Blood 2011, 118 (6769 – 6771). 2-Salles G, Seymour J, Offner F, et al. Rituximab maintenance for 2 years in patient with high tumour burden follicular lymphoma responding to rituximab plus chemotherapy (PRIMA): a phase 3, randomised controlled trial. Lancet 2011, 377 (42-51). Disclosures Karlin: Sandoz: Consultancy; celgene: Consultancy, Honoraria; Janssen: Honoraria. Delmer:Roche: Honoraria. Brice:Seattle Genetics, Inc.: Research Funding; Takeda Pharmaceuticals International Co.: Honoraria, Research Funding; Roche: Honoraria.

Description: Background The assessment of platelet function is crucial during the course of essential thrombocythemia (ET), as this myeloproliferative disorder is associated with concomitant thrombotic and bleeding complications. In the context of high platelet count, the assessment of platelet function is challenging. Current recommendations suggest the adjustment of platelet count for functional assays. This adjustment does not take into account the entire pool of circulating platelets in patients. This may explain the lack of predictive value of such assessment towards bleeding/thrombotic risk. Our objective was: 1- to set up a new strategy to evaluate platelet function, taking into account the circulating pool of platelets; 2- to evaluate the effect of JAK-2 mutation and of cytostatic treatment on platelet function. Methods Fifty two patients presenting with essential thrombocythemia (ET) were enrolled. ET diagnosis was established according to WHO criteria. Seventeen males and 35 females were included (sex ratio = 0.48, mean age 52 ± 16 years old). Fifteen healthy subjects were enrolled according to the hospital policy. Platelets were analyzed in whole blood flow cytometry (Beckmann-Coulter). GPIIbIIIa, GPIb and P-selectin were quantified on resting and TRAP-activated platelets (Platelet GP Receptors, Diagnostica Stago). Calculation for quantification strictly followed the manufacturer’s instructions and results were expressed as a number of GPs/P-selectin per platelet (mode A). In order to take into account the total amount of platelets, we calculated the total number of GPs/ P-selectin x platelet count (mode B). As vWF binding to GPIb is critical in the prevention of bleeding, we measured the capacity of platelets to bind vWF in response to ristocetin using an original and sensitive assay in flow cytometry. JAK2 V617F mutation analysis, cytostatic treatment and clinical events (bleeding and thrombosis) were recorded. Data were expressed as median [interquartiles] and Mann-Whitney was used for statistical comparisons. p