ALBERT

Description: Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Description: B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p

Description: Abstract 2547 Introduction. Minimal residual disease (MRD) eradication in patients with chronic lymphocytic leukemia (CLL) treated by standard chemo-immunotherapy regimens correlates with improved outcome. However, there is limited information about the interest of negative MRD after allogeneic stem cell transplantation (allo-SCT). In this study we investigated whether blood phenotypic remission could impact post-transplant outcome in patients with CLL. Methods. We retrospectively included patients who underwent allo-SCT for CLL and with post-transplant MRD monitored by four or six-colour flow cytofluorometry in blood samples (sensitivity≥10−4). Prognostic impact was evaluated on overall survival (OS) and progression-free survival (PFS). Each of these parameters was evaluated according to the best response and to the 12 month-MRD status, using log-rank test. Results. Thirty-three patients from 4 hematology departments were included. Median age at transplant was 54 years (range, 41 to 66 years). The median number of prior chemotherapy regimens was 3 (range, 1 to 6) including autologous stem cell transplantation in 48% of the patients. Status at transplant was available in 27 patients and 11% of them had negative MRD, 26% haematological complete response (CR), 59.% partial response (PR) and 4% had refractory disease. Twenty-two patients (67%) received a reduced intensity conditionning regimen. Conditioning regimen included serotherapy (antithymoglobulin n = 10, alemtuzumab n = 1, rituximab n = 1) in 12 patients. Twenty-two patients (67%) were transplanted with HLA identical sibling donor. The median number of MRD evaluations after transplant was 5 (range, 1 to 23). Response to transplant: After transplant, 16 patients achieved negative MRD, 15 patients achieved haematological CR, 1 PR and 1 did not respond to transplant. Among the 16 patients with phenotypic remission, negativation of MDR was obtained before the cessation of immunosuppressive therapy for 15 of them (94%) and median time to negativation was 7 months (range, 2 to 20 months). In patients achieving phenotypic remission chronic GVHD rate was 75% versus 44% in patients with post-transplant detectable MRD. Post-transplant outcomes: With a median follow-up of 27 months, the 2-y OS and the 2-y PFS were respectively 84% and 53%. Cause of death (n = 8) was progression in 4 cases and transplant related mortality in 4 cases. Impact of phenotypic remission: The achievement of phenotypic remission (whatever the time of evaluation) correlates with better PFS: 2-y PFS was 85% in these patients versus 27% in the other patients (p = 0.012). Considering the 19 patients with MRD evaluation available at 12 months after transplant, 2-y PFS was 100% in 12-month-negative-MRD patients (n = 10) versus 17% in positive-MRD patients at that date (p = 0.003) No relapse was observed in the group of patients who achieved phenotypic remission at 12 months post-transplant (figure). Conclusion. These data suggest that achievement of post transplant negative MRD in patients with CLL is associated with a long-term control of the disease and better PFS. In our series, no relapse occurred in patients with negative MRD at 12 months post transplant. These results could lead to decrease immunotherapy and to administrate donor lymphocytes to patients with post-transplant persistent positive MRD. Disclosures: Leblond: ROCHE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MUNDIPHARMA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Identifying the etiology of neurological symptoms in hematological malignancies is still a challenging issue. Lymphomatous meningitis (LM) is mainly described in aggressive systemic B-cell lymphomas (diffuse large B-cell (DLBCL) and Burkitt lymphomas), and is associated with poor prognosis. Leptomeningeal involvement in small B-cell lymphoproliferation is a rare, poorly described condition, mentioned only in case reports. The diagnosis relies on the combination of non-specific central nervous system (CNS) symptoms (headaches, paraplegia, etc.) or psychiatric symptoms, and the cytological detection of tumor cells in the cerebrospinal fluid (CSF). However, cell analysis has a low sensitivity here due to the small drawn volumes of CSF samples, their poor cellularity and cell viability. Soluble biomarkers can be measured by multiplex techniques on small sample volumes, hence can overcome cytological limitations and seem fairly interesting for further investigations. The aim of the present study was to evaluate the diagnostic value of interleukin (IL)-10 and IL-6 quantification combined with the IL-10:IL-6 ratio in CSF of patients suffering from LM secondary to systemic small B-cell lymphoproliferations. Seventeen patients suffering from LM were included in the study: 4 with chronic lymphocytic leukemia (CLL), 2 with mantle-cell lymphoma (MCL) and 11 with Waldenström's macroglobulinemia (WM). All patients presented CNS symptoms associated with documented LM (revealed either by conventional cytology and/or flow cytometry) at the diagnostic stage and before intrathecal chemotherapy. IL-10 and IL-6 quantifications were performed in CSF using the quantitative Cytometric Bead Array® technique (human IL-10 CBA kit and human IL-6 CBA kit; BD BiosciencesTM) on a FACSCanto II flow cytometer (BD BiosciencesTM) following the manufacturer's recommendations, with a limit of detection of 2.5 pg/ml. All CLL and MCL patients displayed an undetectable level of IL-10 (

Description: Introduction New insights in the pathogenesis of the splenic marginal zone lymphoma (SMZL) has been recently enlightened by next generation sequencing technology discovering recurrent mutations in several genes implicated in major signalling pathways. In 2 recent reports (JEM 2012), mutations in NOTCH2, a gene encoding a protein required for marginal zone B-cell development, were identified in nearly 25% of the patients, with controversial impact on clinical outcome. The aim of our analysis was to analyse the incidence and the prognostic impact of somatic NOTCH2 mutations in a large cohort of patients with SMZL homogeneously treated. Methods A series of 105 consecutive SMZL patients requiring treatment and referred in 2 expert centers was analysed. Genomic DNA was extracted from frozen tumor samples of involved spleen or blood or bone marrow obtained at diagnosis. Tumor cell clonality was established by amplification of the rearranged IGH genes. Targeted sequencing of the NOTCH2 C-terminal coding exons 26, 27 and 34 was performed using Sanger sequencing and primers described by Kiel MJ et al (J.Exp.Med.209:9,2012). All mutations were verified in at least two independent PCR amplification and sequencing reactions. Log-rank tests were used to compare survival times -overall survival (OS) and progression-free survival (PFS)- between patients with mutated NOTCH2 and patient with wild-type NOTCH2. We controlled the effects of prognostic factors on outcome using multivariate Cox model analysis. Results The median follow-up was 7 years. At diagnosis, median age was 63 years. Sex ratio was M/F 1:1.35. Nearly all patients presented with a disseminated disease and a good performance status. Forty-four percent of the patients had a high LDH level. A monoclonal component was detected in 31% of the patients. An immunological event such as haemolytic anemia, thrombocytopenia, neutropenia, coagulation disorder, and cryoglobulinemia, was observed in 27% of the patients. None of the patients was infected with hepatitis C virus. Patients were scored according to the age-adjusted International Prognostic Index (n=95), and to the IIL score system (n=76). Splenectomy was realized in 97% of the patients. Three patients were treated with R-Bendamusine without splenectomy. Adjuvant treatment after splenectomy was proposed for 24% of the patients, and included CHOP+/-R (n=16), HLX-VP16 (n=1), chlorambucil (n=4), and rituximab alone (n=1). Five and 10-year OS was estimated at 82% and 69%, respectively. Five and 10-year PFS was estimated at 61% and 46%, respectively. Age〉60 was significantly associated to a shorter OS (p=0.0135). We identified NOTCH2 mutations in 10 (10%) of the patients. The detected mutations occurred only within the exon 34 (TAD and PEST domains) and represented mostly truncating or insertional events leading to frameshift mutations (8 of the 10 cases) (also 1 nonsense and 1 missense mutations). Comparison of the clinical characteristics at diagnosis between patients with or without NOTCH2 mutations did not show any significant differences. Patients with NOTCH2 mutations were characterized by a significantly worse OS (at 5 years, 32% vs 87%, p

Description: Key Points B-PLL is tightly linked to MYC aberrations (translocation or gain) and 17p (TP53) deletion. Cases of B-PLL with MYC aberration and 17p (TP53) deletion have the worst prognosis.

Description: Abstract 2860 Introduction. Histological lymphoma diagnosis of adult patients with bulky mediastinal mass is either HL or PMBL, two distinct diseases with a specific outcome. PET interpretation is often difficult for mediastinal lymphoma because of the volume of the tumor and the presence of blood pool. No specific criteria for interpretation of interim-PET have yet been defined in this clinical entity. The purpose of this study was to investigate the prognostic value of qualitative and semiquantitative evaluations of interim-PET in mediastinal lymphoma. Methods. We retrospectively included 72 patients with either HL (n=48) or PMBL (n=24), previously untreated, aged under 60 at diagnosis and who underwent at least one interim-PET evaluation. Patients with sub-diaphragmatic or medullar localisations of lymphoma were excluded. All PET scans were reviewed. Qualitative evaluation included global visual evaluation (GVE) (positive or negative) and 5 points-scale (5PS). Semiquantitative evaluation consisted in maximum standardized uptake value (SUVmax) and SUVmax reduction between the baseline PET (PET0) and the evaluation performed after two (PET2) or four (PET4) cycles of chemotherapy. Prognostic impact was evaluated on the event-free survival (EFS), defined as disease progression/first relapse (n=18) or death (n=2). ROC (Receiver Operating Characteristic) curve was used to assess the value of SUVmax reduction in discriminating future deaths or relapses, based on area under the curve (AUC). The “best“ cut-off that provides both the lowest false positive and the lowest false negative rates was computed. Results. Median age was 29 (24 - 35), 60% male. Tumoral mass was more than 7.5 cm in 70% of the patients, and M/T ratio〉= 0.35 in 79%. Median SUVmax at baseline, PET2 and PET4 were 12.8 (4.1;33.2), 1.9 (1.7;3.1), and 2.4 (1.7;3.1), respectively. With a median follow-up at 24 months, 2-year event-free survival (EFS) was 67%, without significant difference between HL and PMBL (p=0.98). Except for ECOG performance status in PMBL, neither clinical nor biological feature was predictive for EFS. Using GVE, a negative PET2 (n=36/59) and PET4 (n=24/34), were achieved in 61% and 71%, respectively. GVE and 5PS have a significant negative predictive value (NPV) at PET2 (HR=3.2, 95%CI: 1.2–8.2; p=0.012, and HR=1.9, 95%CI: 1.1–3.3, p=0.01) and at PET4 (HR=13.9, 95%CI: 3.5–55; p=0.0001, and 2.6, 95%CI: 1.5–4.7, p=0.001 respectively), with a 2-year estimated EFS of 69% for PET2-negative patients versus 51% for PET2-positive patients (p=0.012), and of 86% for PET4-negative patients versus 20% for PET4-positive patients (p=0.001). An optimal cut-off of 81% SUVmax reduction from PET0 to PET2 or PET4 yielded a 2-year estimated EFS of 70% in patients with reduction of more than 81%, versus 47% in those with reduction of 81% or less (p=0.004). In the HL subgroup, GVE and SUVmax reduction higher than 81% at PET2 (p=0.0001, p=0.015, respectively) and PET4 (p=0.004, p=0.015, respectively) showed significant prognostic values for EFS. In the PMBL subgroup, neither qualitative nor semiquantitative evaluation at PET2 was predictive for EFS. At PET4, GVE and 5PS were strongly predictive for EFS (p=0.005, p=0.0001). Patients with SUVmax reduction higher than 81% between PET0 and PET4 reached a 67% 2-year EFS, while patients with lower SUVmax reduction had only a 33% 2-year EFS (p=0.13). Conclusion. The cut-off value of SUVmax reduction estimated for predicting EFS with best accuracy in mediastinal lymphoma was 81%. Although the SUV semiquantification helps to reduce the number of false positives, visual global analysis had a significant negative predictive value in interim-PET in mediastinal lymphoma, with a possible very early prediction, as early as PET2 in HL, and a better prediction at PET4 than PET2 in PMBL. Disclosures: No relevant conflicts of interest to declare.

Description: Deletions of the long arm of chromosome 14 are rare (

Description: Introduction Post-transplant lymphoproliferative disorders (PTLD) are a rare but severe complication occurring after organ transplantation as a result of immunosuppressive therapy (IST). Until recently, there was no consensus regarding best treatment practice. In 2006, a phase 2 trial using a monotherapy with rituximab showed an overall response rate of 44%, with a complete response (CR) rate of 28%. In 2012, the PTLD-1 international multicenter trial evaluating rituximab followed by CHOP chemotherapy proved an efficacy of 90% and an overall survival of 6.6 years in PTLD treatment. K-virogref is a French national expert network created in 2011 to structure and improve the management of viro-induced cancers, occurring in adult after transplantation, including PTLD. Within this network, an epidemiological observational study has been set up in September 2013. The aim of our study was to analyze the management of PTLD in France before the creation of the national database of K-virogref. Patients and methods We included solid organ or allogeneic stem cell recipients, aged 18 years and above, with a histologically proven PTLD, diagnosed between January 2010 and September 2013 in 19 French hematology departments. We collected from clinical charts data regarding transplant characteristics, PTLD presentation, first-line treatment and outcomes. Results The study population consisted of 94 patients, 67% were men and median age was 53 years (ranging 18 - 81 years). The majority of patients received kidney transplantation (54%), followed in almost equal proportion by heart (13%), liver (13%) and allogeneic stem cell transplantation (10%). Most of the time (80% of patients), PTLD occurred more than one year after organ transplantation with a median delay of 7.8 years. Histologically, mainly monomorphic PTLD were observed with 66% of diffuse large B cell lymphoma and PTLD was EBV associated in 54%. At the time of diagnosis, 80% were classified as stage IV according to Ann Arbor. The median follow-up time was 28.3 months (ranging 0.2 - 78 months). As far as treatment is concerned, modification of IST was performed in 68% of patients but response was rarely evaluable because a specific treatment was often started at the same time. Surprisingly, first line treatment consisted in rituximab alone or rituximab followed by CHOP chemotherapy in only 32% of the patients. Among these patients, the overall response rate was 81% with 55% of CR. Most patients (56%) received chemotherapy (n=11) or immunochemotherapy (n=46) without previous rituximab therapy. For these patients the overall response rate was 71% with a CR rate of 53%. At 1 and 3 years of follow-up, the relapse-free rates were 56% (95% confidence interval (CI) 46% - 68%) and 47% (95% CI 37% - 60%), respectively. Overall, 49% of patients died during follow-up of which 37% due to infectious complications and 32% due to PTLD progression. Conclusion This is one of the largest cohort study of PTLD in the literature, providing an overview of epidemiological features, treatments and outcomes of this rare disease. The lag between the management of PTLD and literature data emphasizes the usefulness of a national expert network such as K-virogref in order to generalize a less toxic and more powerful attitude. Disclosures Leblond: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria. Choquet:Celgene: Consultancy; Janssen: Consultancy.