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Introduction Ibrutinib, a once-daily oral Bruton's tyrosine kinase inhibitor, has shown progression-free survival (PFS) or overall-survival (OS) benefit over chemoimmunotherapy (CIT) in multiple phase 3 studies in previously untreated patients with CLL, and significantly longer time to next treatment compared with CIT in previously untreated high-risk patients in a RW setting. We conducted an adjusted comparison of ibrutinib versus RW treatment for previously untreated CLL using patient-level data from the phase 3 RESONATE-2™ (NCT01722487) trial and RW databases from 2 countries. Methods Previously untreated patients with CLL, fulfilling RESONATE-2™ eligibility criteria (age ≥ 65, no del17p) were selected from 2 RW data sources containing electronic medical records for patients with CLL: Centre Hospitalier Lyon-Sud, France; CLLEAR CLL registry from 7 academic centers in the Czech Republic. PFS and OS were compared between patients from the ibrutinib arm of RESONATE-2™ with a median follow-up of 60 months, and those receiving physicians' choice (PC) treatment other than ibrutinib from the RW database, adjusting for differences in baseline characteristics including age, gender, del11q, IGHV status, RAI/BINET disease stage, and Eastern Cooperative Oncology Group (ECOG) score. Hazard ratios (HRs) for ibrutinib versus RW PC treatment were estimated using a multivariable Cox proportional hazards model including available baseline characteristics as covariates, and using an inverse probability weighted (IPW) Cox model with average treatment effect for treatment (ATT) weights derived from propensity scores estimated from a logistic regression including the same covariates. Results The analysis included 136 patients from the RESONATE-2™ study receiving ibrutinib and 920 previously untreated RW patients receiving PC treatment (Lyon-Sud n = 162, CLLEAR n = 758). Baseline characteristics were generally balanced between treatment groups. The most common PC regimens contained CIT and were fludarabine + cyclophosphamide + rituximab (FCR) (n = 227), bendamustine + rituximab (BR) (n = 201), and rituximab + chlorambucil (R + Chlor) (n = 116). Older age, male gender, del11q and advanced disease stage were independent risk factors for PFS and OS. When comparing ibrutinib versus the overall PC cohort, the adjusted HR (95% confidence interval [CI]) was 0.24 (0.16-0.34) for PFS and 0.33 (0.21-0.52) for OS (both p < 0.0001). IPW-based comparative estimates were highly consistent for both PFS (HR = 0.27 [0.18-0.39]) and OS (HR = 0.39 [0.24-0.62]). ATT-weighted survival curves estimating PFS and OS for the RESONATE-2™ population as if treated with PC, showed a median value of 32.1 months and 72.5 months, respectively, while median values for Ibrutinib were not reached. When comparing ibrutinib versus FCR, BR, and R + Chlor, the adjusted HRs (95% CI) were 0.26 (0.17-0.38), 0.27 (0.18-0.41), and 0.27 (0.17-0.42), respectively, for PFS and 0.34 (0.20-0.56), 0.28 (0.16-0.49), and 0.61 (0.32-1.13), respectively, for OS (Figure). Concl usions Adjusted comparisons of the RESONATE-2™ trial and RW patient-level data demonstrates significantly improved PFS and OS for ibrutinib versus physician's choice treatment (predominantly CIT) in previously untreated patients with CLL. PFS and OS benefit for ibrutinib was consistent across a range of common regimens: FCR, BR, and R + Chlor. These results are consistent with data from phase 3 studies and support the use of ibrutinib for first-line CLL treatment. Funding Source: Sponsored by Janssen Pharmaceutica NV, and Pharmacyclics LLC, an AbbVie Company. The RW databases are independently owned. Writing assistance was provided by Emma Fulkes and Liqing Xiao of Parexel and funded by Janssen Pharmaceutica NV. Figure 1 Figure 1. Disclosures Doubek: Janssen-Cilag, AbbVie, AstraZeneca, Amgen, Gilead, Novartis: Honoraria, Research Funding. Smolej: AbbVie, AstraZeneca, Gilead, Janssen-Cilag, and Roche: Consultancy, Honoraria, Other: Travel Grants. Šimkovič: AbbVie, AstraZeneca, Janssen-Cilag, Gilead, Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants. Lysak: Novartis, Janssen-Cilag, AbbVie; AstraZeneca: Honoraria, Research Funding. Bachy: Kite, a Gilead Company: Honoraria; Novartis: Honoraria; Daiishi: Research Funding; Roche: Consultancy; Takeda: Consultancy; Incyte: Consultancy. Ferrant: AbbVie: Honoraria, Other: Travel, Accommodations, Expenses; AstraZeneca: Honoraria; Janssen: Other: Travel, Accommodations, Expenses. Diels: Janssen: Current Employment. Cabrieto: Janssen: Current Employment. Nielsen: Janssen: Current Employment. Salles: Allogene: Consultancy; Regeneron: Consultancy, Honoraria; Velosbio: Consultancy; Takeda: Consultancy; Rapt: Consultancy; Genentech/Roche: Consultancy; Epizyme: Consultancy, Honoraria; Debiopharm: Consultancy; Genmab: Consultancy; Incyte: Consultancy; Ipsen: Consultancy; Janssen: Consultancy; Kite/Gilead: Consultancy; Loxo: Consultancy; Miltneiy: Consultancy; Morphosys: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Beigene: Consultancy; Abbvie: Consultancy, Honoraria; Bayer: Honoraria.

To the editor: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), characterized by the t(15;17) translocation resulting in the PML-RARA fusion transcript. The use of all-trans retinoic acid (ATRA) and/or arsenic has revolutionized the treatment of this disease, which

To the editor: Most hematologic malignancies are preceded by a premalignant condition. In myeloid neoplasms, genetically defined premalignant conditions such as clonal hematopoiesis of indeterminate potential[1][1] reflect the clonal dominance of hematopoietic stem cells harboring oncogenic

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 The median overall survival (OS) for the entire cohort was 126 months with a median follow-up time of 47 months [ 0.2-141]. We found MYC activation (translocation or gain) to be associated with a shorter OS (p=0.03). Regarding MYC and del17p, we identified 3 distinct cytogenetic prognostic groups, with significant differences in OS (p=0.0006) (Fig 2). The patients without MYC activation had the lower risk (n=8, median not reached). Patients with a MYC activation without del17p had an intermediate risk (n=18, 125 months). The highest risk group corresponded to patients with both MYC and TP53 aberrations (n=7, 11 months). We performed drug response profiling on primary B-PLL cells using the ATP-based CellTiter Glo kit (Promega) (n=5). We observed that after 48h of exposure to increased doses, response was heterogeneous, with a majority of samples resistant to fludarabine (n=3), ibrutinib (n=3), idelalisib (n=4), venetoclax (n=3) and OTX015 (n=4). Annexin/PI assays using flow cytometry showed that the induced cell death could be increased by combination of ibrutinib or venetoclax with OTX015 or JQ1, two BET protein inhibitors that target MYC signaling (n=1/2). In summary, B-PLL have complex and highly complex K, a high frequency of MYC activation by translocation or gain, frequent 17p deletion, and frequent mutations in MYC, TP53, BCOR, and MYD88 genes. We identified 3 prognostic subgroups according to MYC and 17p status. Patients with MYC activation + 17p deletion have the shorter OS, and should be considered as a high-risk "double-hit" subgroup. Our results show that cytogenetic analysis is a useful diagnostic tool in B-PLL that improves prognostic stratification. We recommend to perform K and FISH (MYC and TP53) analyses systematically when a B-PLL is suspected. Our in vitro data suggest that drugs targeting the BCR and BCL2 in combination with MYC inhibition may be a therapeutic option in some patients. Disclosures Baseggio: Takeda Oncology: Honoraria.

Introduction Ibrutinib, a first-in-class, oral, covalent inhibitor of Bruton's tyrosine kinase, is approved in many countries for treatment-naïve (TN) and previously treated CLL, supported by improved response rates, progression-free survival (PFS), and overall survival (OS) vs chlorambucil in RESONATE-2™ (NCT01722487; Burger JA, et al. N Engl J Med. 2015;373:2425-2437) and ofatumumab in RESONATE™ (NCT01578707; Byrd JC, et al. N Engl J Med. 2014;371:213-223). We analyzed survival outcomes for ibrutinib vs RW treatments for TN and relapsed/refractory (R/R) CLL by del11q status, an adverse prognostic factor linked with poor patient outcomes despite conventional chemoimmunotherapy. An adjusted comparison restricted to patients with confirmed del11q status was conducted using patient-level data from RESONATE-2™ and RESONATE™ and RW databases from 2 countries. Methods The Lyon-Sud RW database holds medical records for CLL patients diagnosed between 1980 and 2017 from the Centre Hospitalier Lyon-Sud, France; the Chronic Lymphocytic Leukemia Registry (CLLEAR) RW database holds medical records for CLL patients diagnosed between 1988 and 2017 from 7 academic centers in the Czech Republic. TN CLL database patients were selected using RESONATE-2™ criteria (which excluded those aged < 65 years or del17p positive). For the R/R group, patients ≥ 18 years of age or those with del17p were allowed per RESONATE™ inclusion criteria. PFS and OS outcomes by del11q status were compared between the ibrutinib arms of RESONATE-2™/RESONATE™ and physicians' choice (PC) treatment in the pooled RW databases (excluding ibrutinib). A multivariate Cox proportional hazards model was fitted on pooled RCT/RW data to estimate adjusted hazard ratios (HRs) for effect of ibrutinib vs RW PC treatment using age, sex, and treatment line as covariates. The unit of observation for the RW databases was the treatment line (rather than patient) number; RW patients receiving multiple lines of therapy contributed to multiple observations, and baseline was defined as the line-specific treatment start date. Results For the RW TN cohort, 466 treatment lines were analyzed; 134 (28.8%) patients were del11q positive. In RESONATE-2™, 29/136 (21.3%) TN patients were del11q positive. The table below shows adjusted HRs for PFS and OS for patients with/without del11q. For the del11q-positive subgroup, adjusted HRs for ibrutinib vs RW PC first-line (1L) therapy were 0.02 (95% confidence interval [CI], 0.00-0.17; p = 0.0002) for PFS and not estimable for OS (no deaths on ibrutinib). For the del11q-negative subgroup, adjusted HRs were 0.34 (95% CI, 0.2-0.57; p < 0.0001) for PFS and 0.74 (95% CI, 0.37-1.49; p = 0.3957) for OS. For second-line (2L) PC treatment in the RW R/R cohort, 385 treatment lines were analyzed; 135 (35.1%) were from del11q positive patients. In RESONATE™, 13 (37.1%) 2L patients were del11q positive. For the del11q-positive subgroup, adjusted HRs for ibrutinib vs RW PC of 2L therapy were 0.03 (0.00-0.21; p = 0.0003) for PFS and 0.08 (0.01-0.46; p = 0.0050) for OS. For the del11q-negative subgroup, adjusted HRs were 0.27 (0.14-0.51; p < 0.0001) for PFS and 0.50 (0.24-1.05; p = 0.0670) for OS. For 2L and beyond (2L+) treatment in the RW R/R cohort, 727 treatment lines were analyzed; 291 (40.0%) were from del11q positive patients. In RESONATE™, 63/195 (32.3%) 2L+ patients were del11q positive. For the del11q-positive subgroup, adjusted HRs (95% CIs) for ibrutinib vs RW PC of 2L+ therapy were 0.13 (0.08-0.20; p < 0.0001) for PFS and 0.15 (0.08-0.26; p < 0.0001) for OS. For the del11q-negative subgroup, adjusted HRs were 0.20 (0.14-0.29; p < 0.0001) for PFS and 0.33 (0.21-0.52; p < 0.0001) for OS. Concl usions Adjusted comparisons of the registration trial (RESONATE-2™/RESONATE™) and RW patient-level data suggest that OS and PFS improvements with ibrutinib vs PC therapies are pronounced for patients with CLL harboring del11q compared with those who are del11q negative. The findings inform physicians of the comparative effectiveness of ibrutinib in the RW for patients with this high-risk cytogenetic abnormality. Funding Source: This project was sponsored by Janssen Pharmaceutica NV, and Pharmacyclics LLC, an AbbVie Company. The real-world databases are independently owned. Writing assistance was provided by Emma Fulkes of PAREXEL and funded by Janssen Pharmaceutica NV. Disclosures Salles: Takeda: Honoraria; Acerta: Honoraria; Epizyme: Honoraria; Pfizer: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Gilead: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Servier: Honoraria; Janssen: Honoraria; Merck: Honoraria; Morphosys: Honoraria. Besson:Janssen Pharmaceutica NV: Employment. Doyle:Janssen Pharmaceutica NV: Employment. Garside:Janssen Pharmaceutica NV: Employment. Spacek:Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Doubek:Novartis: Consultancy; AbbVie: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Affimed: Research Funding; Gilead: Consultancy, Honoraria, Research Funding.

INTRODUCTION: The benefits of ibrutinib (ibr) have been demonstrated by the phase 3 RESONATE and RESONATE-2 trials for patients (pts) with R/R and TN (≥ 65 years) CLL, respectively. In these studies, ibr showed significantly improved progression-free survival (PFS) and overall survival (OS) vs approved comparators (ofatumumab [ofa] in RESONATE and chlorambucil [clb] in RESONATE-2). However, a number of other treatment regimens are widely used in clinical practice for CLL based on individual patient and disease characteristics; it is currently unknown if ibr results in better survival outcomes when compared directly with each of these other treatments. AIM: In the absence of a head-to-head comparison, we investigated the relative efficacy of ibr vs physician's choice (PC) in pts with R/R or TN CLL by comparing pt-level data from RESONATE and RESONATE-2 with data from pts in a real-world setting (the Lyon-Sud database). This database holds electronic medical records for 390 pts with CLL from the academic Lyon-Sud Hospital in France; nearly all patients were diagnosed between 1990 and 2014. This is the first analysis of ibr vs PC in a real-world setting for pts with TN CLL. METHODS: Pt-level data from RESONATE (ibr, n = 195; ofa, n = 196) were compared with data on pts with CLL from Lyon-Sud who received 2nd(n = 107) or later-line treatment (3rd [n = 62], 4th [n = 43], and subsequent [n = 51] lines). Similarly, RESONATE-2 (ibr, n = 136; clb, n = 133) pt-level data were compared with those of pts aged ≥ 65 years who received 1st-line treatment (n = 131). In order to account for non-comparability due to lack of randomization, a multivariate Cox proportional hazards model was used to compare PFS and OS between treatments, including line of therapy, age, sex, disease stage (based on Binet/Rai), and deletion 17p or 11q mutations as covariates. For the definition of PFS, missing data for date of disease progression for pts in Lyon-Sud who initiated subsequent therapy were replaced by the conservative proxy of date of initiation of next treatment. Predicted PFS and OS curves for the Lyon-Sud cohort were derived from the multivariate model. RESULTS: For R/R pts in Lyon-Sud, across all treatment lines the most frequent regimens used were rituximab (R) + chemotherapy (n = 46), bendamustine + R (BR; n = 28), fludarabine + cyclophosphamide + R (FCR) (n = 27), clb + R (n = 19), R monotherapy (n = 21), and R-CHOP-based (n = 19); the remaining percentages comprised various other therapies, each used in < 5% of pts. Median age at treatment initiation for Lyon-Sud and RESONATE was 69 and 67 years, respectively; median number of prior therapies was 2 and 3, and median follow-up was 30.0 and 21.9 months. More lines of prior therapy, older age, male sex, advanced disease stage (Binet C/Rai III/IV), and presence of deletion 17p/11q were independent risk factors for worse PFS and OS outcome. After adjustment for differences in baseline characteristics, PFS and OS hazard ratios (HRs) for ibr vs PC were 0.18 (95% confidence interval [CI], 0.13-0.26) and 0.28 (0.17-0.46), respectively. Adjusted HRs for ibr vs the most frequent treatments ranged from 0.09 (R monotherapy) to 0.38 (FCR) for PFS and 0.21 (R monotherapy) to 0.33 (FCR) for OS; all were statistically significant. For TN pts in Lyon-Sud, the most frequent treatment regimens used were FCR (n = 40), clb + R (n = 21), clb monotherapy (n = 17), R-(mini)CHOP (n = 11), and BR (n = 11). Median age at treatment initiation was 73 years for both Lyon-Sud and RESONATE-2; median follow-up was 36.9 and 28.1 months, respectively. Older age, male sex, and advanced disease stage were independent risk factors for worse PFS and OS outcome. After adjustment for baseline differences, the PFS and OS HRs for ibr vs PC were 0.25 (0.14-0.47) and 0.41 (0.17-0.99). Predicted PFS and OS curves for both R/R and TN patients are presented in Figure 1. CONCLUSIONS: Investigation of survival outcomes suggests that ibr administered to pts in RESONATE and RESONATE-2 was more effective than PC used in a real-world cohort of French pts with R/R or TN CLL, suggesting a 4.8-fold improvement in PFS and a 3.4-fold improvement in OS in R/R pts and 4-fold and 2.4-fold improvements in TN pts, respectively. These results further support the growing evidence that ibr significantly improves both PFS and OS vs commonly used regimens in the R/R and TN settings, and could have important implications for improving treatment of CLL in clinical practice. Disclosures Salles: Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Sarkozy:Sandoz: Research Funding; Janssen Research & Development: Honoraria; Gilead: Honoraria; Takeda: Research Funding. Ghesquieres:Mundipharma: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche France: Research Funding. Diels:Johnson & Johnson: Employment, Equity Ownership. Besson:Janssen: Employment. MacDougall:Janssen Research & Development: Research Funding; IMS Health: Employment. Hermans:Janssen Research & Development: Research Funding; IMS Health: Employment. Healy:Janssen Research & Development: Employment. Garside:Janssen Research & Development: Employment. Iraqi:Janssen Research & Development: Employment.

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of VH3 and VH4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM—a higher incidence of t(11;14) (33% vs 16%; P

Marginal zone B-cell lymphoma (MZL) is a recently individualized lymphoma that encompasses mucosa-associated lymphoid tissue (MALT) lymphoma, splenic lymphoma with or without villous lymphocytes, and nodal lymphoma with or without monocytoid B-cells. If the clinical description and outcome of MALT lymphoma is well known, this is not the case for the other subtypes. We reviewed 124 patients presenting non-MALT MZL treated in our department to describe the morphologic and clinical presentation and the outcome of these lymphomas. Four clinical subtypes were observed: splenic, 59 patients; nodal, 37 patients; disseminated (splenic and nodal), 20 patients; and leukemic (not splenic nor nodal), 8 patients. These lymphomas were usually CD5-, CD10-, CD23-, and CD43-, but the detection of one or, rarely, two of these antigens may be observed. Bone marrow and blood infiltrations were frequent, except in the nodal subtype, but these locations were not associated with a poorer outcome. Splenic and leukemic subtypes were associated with a median time to progression (TTP) longer than 5 years, even in the absence of treatment or of complete response to therapy. Nodal and disseminated subtypes were associated with a median TTP of 1 year. However, in all these subtypes, survival was good with a median survival of 9 years, allowing these lymphomas to be classified as indolent. Because of the retrospective nature of this analysis, no conclusion may be drawn on the therapeutic aspects, but conservative treatments seem recommended for leukemic and splenic subtypes.

Introduction Among recently discovered B cell activators responsible for signaling events leading to B-cell activation and maturation, of particular interest are the Toll-like receptors (TLRs). TLR expression is heterogeneous and variable among B-cells. In addition, abnormal TLR levels/signaling may play an important role in the pathogenesis of lymphoma, particularly in splenic marginal zone lymphoma (SMZL), since TLR pathways are recurrently targeted by genetic changes in this lymphoma. Methods Frozen spleen tissue specimens from patients with SMZL (n=13) and splenic diffuse red pulp lymphoma with villous lymphocytes (SDRPL, n=5) were analyzed for the expression of TLR1 to TLR10 in comparison to control cases (traumatic spleen, n=7) using multi-parametric flow cytometry and quantitative mRNA Taqman assay. To identify the B-cell subset, the samples were also stained with anti-CD19 and anti-CD3. All cases were studied by morphological, immunological, cytogenetic and molecular analysis. Results The TLR profile obtained at protein level by flow cytometry was closely related to that obtained at mRNA level. The SMZL/SDRPL B-cells expressed all TLRs, but with variable levels of protein expression (low for TLR1, TLR2, TLR3, TLR8, TLR9, TLR10, and high for TLR4, TLR5, TLR6 and TLR7). On the other hand, distinct TLRs profiles were observed according lymphoma subtypes. The SDRPL cases showed indeed a significant TLR2 under-expression and TLR4/TLR7 over-expression in comparison to SMZL cases and control B-cells. SMZL cases exhibited a significant TLR4/TLR8 over-expression in comparison to control B-cells. These TLR profiles were not associated with specific cytogenetic features (presence of del7q or trisomy 3) and/or immunological profile (expression of the CD11c/CD27/isotype of immunoglobulin). But, in both entities, TLR4 expression was higher in cases with mutated IGHV than in cases with unmutated IGHV. The overexpression of TLR7 MyD88-dependent signaling molecules has been reported as pathogenic mechanism for autoimmune diseases; however no more autoimmune disease or circulating auto-antibodies were associated with SDRPL cases as compared to SMZL cases. As previously reported, all cases but one SMZL case presented unmutated MyD88 (L265P mutation), and MyD88 protein evaluated here by flow cytometry was similarly expressed in both entities. While TLR7 stimulation is known to induce CD38 expression, all SDRPL cases were CD38 negative; while in SMZL cases, higher TLR7 expression was observed in CD38 positive as compared to CD38-negative cases. This may suggest the existence of an abnormal TLR7 pathway in SDRPL as opposed to SMZL. Conclusion TLR profiling should allow a better understanding of the mechanisms involved in SMZL/SDRPL pathogenesis. Present data suggest an abnormal TLR pathway involving NF-kB and/or MyD88 in the SDRPL entities. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of chromosome 7q have rarely been reported in chronic B-cell lymphoproliferative disorders.[1][1] Chromosomal translocation leading to overexpression of the CDK6 gene, which is located in 7q21, has recently been described in splenic marginal zone lymphoma (SMZL).[2][2],[3][3] CDK6

Abstract 1594 Background: Follicular Lymphoma (FL) is the most frequent low-grade NHL. Clinical course is heterogeneous, some patients (pts) presenting an indolent clinical course with overall survival (OS) over 15 y and others developing a more aggressive disease with shorter survival. The Follicular Lymphoma International Prognostic Index (FLIPI) or FLIPI-2 are commonly used to predict pts outcome, but fail to identify pts with a really poor prognosis. At diagnosis, few FL pts present with detectable leukemic phase (FL-LP) and this characteristic has been seldom described. Aim: The aim of the study was to describe the clinical features and outcome of FL-LP pts. Results: Among 499 pts diagnosed with FL according to the WHO criteria in the Centre Hospitalier Lyon Sud (transformation and grade 3b excluded) and treated between 01/1992 and 01/2012, 37 (7.4%) had characteristic FL-LP detected by cytological blood smears analysis with confirmation by flow cytometry (kappa/lambda clonality). Median age was 58 y and FLIPI score repartition was 4 pts in low, 16 in intermediate, and 17 in high risk groups. Splenomegaly was present in 23 pts, high tumour burden (GELF criteria) in 11, B symptoms in 8, and ECOG PS>1 in 3. Seven pts had anaemia, 17 platelets UNL, and 11 LDH >UNL. The circulating lymphoma cells expressed the CD10 in 29/37 cases and surface Ig expression was detected in 31/35 cases, mainly IgM or IgG isotype. The median count of circulating lymphoma cells was 1.95.109/L, ranging from 0.6 to 129.109/L, 15 pts having count >4.109/L and 6 pts >10.109/L. Cytogenetic data were available for 21 pts, 20 carried the t(14;18), or its variant t(18;22), 16 of them having complex karyotype. Two pts were on watchful waiting for 24 and 82 m and 35 received a chemotherapy regimen at diagnosis including rituximab in 27 cases. Overall response rate was 83% (29/35) with 23 CR/CRu. Median progression-free survival (PFS) was 29 m. PFS and OS estimates were 37% and 86% at 5 y and 31% and 68% at 10 y, respectively. Splenomegaly (P=.035), high tumour burden (P=.017), lymphoma cells count greater than 4.109/L (P=.028), β2-m>UNL (P=.036), and thrombocytopenia (4.109/L (HR 5.92; P=.000916) as independent prognostic factors. After progression, 12 pts received high-dose therapy (HDT) with HSCT as salvage with a long second PFS (68% at 10 y) compared to 8.3 m median PFS in first line. Pts not receiving HDT at salvage had a median second PFS of 27 m. To further evaluate the impact of FL-LP on pts outcome, a 1:3 matched analysis was performed. The FL-LP 37 pts were successfully matched with 111 newly diagnosed FL without FL-LP according to FLIPI score, age, treatment type (abstention vs chemotherapy, with or without rituximab) and treatment period (before or after 2000). In these 111 matched pts, 5- and 10-y OS was 97% and 91%, respectively. Considering all 148 pts, high FLIPI score, presence of FL-LP, and β2-m>UNL were all significantly associated with a worse PFS. In a Cox regression model for PFS (120/148 pts with complete data), high FLIPI score (P=.0034; HR=2) and presence of FL-LP (P=.0085; HR=2.2) remained independently associated with shorter PFS. High FLIPI score and presence of FL-LP were also associated with a shorter OS. Interestingly pts with less than 4.109/L of circulating lymphoma cells had a similar PFS than those without. When circulating lymphoma cells >4.109/L as variable (see abstract figure) instead of FL-LP were tested in the Cox regression model for PFS including β2-m>UNL and FLIPI score as variables, the most significant predictor for a shorter PFS was circulating lymphoma cells >4.109/L (P=.0004; HR=3.56) as compared to FLIPI score (P=.051; HR=1.6) and β2-m (P=.09; HR=1.52). Conclusion: The presence of circulating lymphoma cells in FL is a rare event and is associated with shorter PFS independently of FLIPI score and β2-m level. A validation of these findings on pts from the PRIMA study is on progress. However, this population is not homogenous and pts with circulating lymphoma cells >4.109/L have a poorer outcome. Although ∼1/3 of the pts experience long term PFS, these pts should be monitored carefully during and after first line treatment to consider HSCT as a therapeutic option to achieve a sustained response. Disclosures: No relevant conflicts of interest to declare.

Abstract 582 Chromosomal translocations (t) are usually analyzed as one group, and are associated with poor prognosis in chronic lymphocytic leukemia (CLL). Translocations involving immunoglobulin (IG) genes are recurrent, but uncommon (< 5%) in CLL. The two most frequent IG-partners are BCL2 (18q21) and BCL3 (19q13). On the behalf of the Groupe Francophone de Cytogenetique Hematologique (GFCH), we report an extensive analysis of 75 t(14;18)-CLLs, and a comparison to our previously published series of 29 t(14;19)-CLLs (Chapiro et al, Leukemia 2008). The 75 t(14;18)-CLLs or variant BCL2-t have been collected between 1985 and 2009. The morphological and immunological reviews were performed by KM, CS, and HM-B. All karyotypes were reviewed by the GFCH. Fluorescence in situ hybridization analyses were performed to detect IG and BCL2 rearrangements, trisomy 12, and deletions of 11q22 (ATM), 17p13 (TP53), 6q21, 13q14 (D13S319). IGHV mutation analyses were performed by referring laboratories. Statistical analyses were carried out using the Fisher's exact test, and continuous data using the Mann-Whitney test. Overall survival (OS) and treatment free survival (TFS) calculated from diagnosis were estimated using the Kaplan-Meier method, and the statistic significance was determined using log-rank test. Among BCL2-CLL, the sex ratio was 57M/18F, the median age at diagnosis was 66 years; of 68 patients with available data, 63 (93%) presented with Binet stage A; median lymphocytosis was 14.6×109/l. There were 47/75 (63%) “classical” CLL and 28/75 (37%) “atypical” CLL, with more than 10% of lymphoplasmacytoid cells and/or large cells. All tested cases (58/58) were CD10-, 69/73 (94%) were CD5+, and 44/63 (70%) were CD38-; 57/68 (84%) had a Matutes score > 4, 7/68 (10%) a score = 3, 4/68 (6%) a score < 3. We observed 62 t(14;18) and 13 variant translocations [11 t(18;22), 2 t(2;18)]. The karyotype was complex (> 3 abnormalities) in 15/74 (20%) cases, and the BCL2-t was isolated in 25/74 (34%) cases. There were 33/75 (44%) tri12, 32/68 (47%) del13q14, 1/72 (1%) delTP53, 0/72 (0%) delATM, 0/59 (0%) del6q21. Of 42 analyzed cases, 33 (78%) were mutated. Finally, the median time from diagnosis to first therapy was 24 months (m). Comparisons with the BCL3-CLL showed no difference in sex ratio, age, and Binet stages. The lymphocytosis was lower in BCL2-CLL (14.6 vs 24.4 x109/l, p 4 (84% vs 5/20 (25%), p Comparison to common CLL showed that BCL2-CLLs had more tri12 (p Compared to BCL3-CLLs, BCL2-CLLs have a much less aggressive behavior, indicating that distinguishing the individual translocations and the cytogenetic partners would allow a better patients' stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract 801 The genetic bases of Waldenström Macroglobulinemia (WM) are poorly understood. Because of the difficulty in obtaining tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported in WM. We have studied a cohort of 171 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM) comparing the efficacy of fludarabine to that of chlorambucil, by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 8 probes CEP4, CEP12, 13q14, 11q22 (ATM), 17p13 (TP53), IGH, BCL2 Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. The sex ratio was 2.1M/1F, the average age at inclusion was 66.9 years [40-89]. The mean percentage of lymphoplasmacytic cells was 53% [8-97]. Out of 140/171 successful CC, 65 (46.4%) showed clonal abnormalities. Out of 65 abnormal CC, 19 (29.2%) were complex, with at least 3 chromosomal changes, and 22 (33.8%) showed translocations (balanced and unbalanced). Using CC and FISH, we observed 29/132 (22%) 6q deletion, 18/141 (12.8%) 13q14 deletion, 9/80 (11.2%) trisomy 18, 11/135 (8.1%) TP53 deletion, 10/133 (7.5%) trisomy 4, 10/132 (7.6%) ATM deletion, 4/118(3.4%) IGH rearrangement, and 4/136 (2,9%)trisomy 12. Chromosomal abnormalities were compared to adverse characteristics described by Morel et al (ISSWM, Blood 2009,113(8),4163-70): advanced age (> 65 years), hemoglobin < 11.5g/dl, platelet count < 100×109/l, b2-microglobulin > 3 mg/l, and serum monoclonal protein concentration > 7g/dl. Patients with 6q deletion had significantly more frequently albumin < 3.5g/dl (15/29 (51.7%) vs 24/103 (23.3%), p=0.005), b2microglobuline > 3 mg/l (20/29 (68.9%) vs 48/103 (46.6%), p=0.04). Similarly, patients with trisomy 4 had significantly more frequently b2microglobuline > 3 mg/l (9/10 (90%) vs 59/123 (47.9%), p=0.02). Of note, all patients with trisomy 4 had M-protein concentration > 2 g/dl (10/10 (100%) vs 73/123 (59.3%), p=0.02). Finally, there were significantly more patients with age > 65 years, when ATM deletion was observed (9/10 (90%) vs 65/122 (53.2%), p=0.04). Cytogenetic abnormalities did not influence the response rate but in multivariate analysis, TP53 deletion was associated with a shorter time to progression (15months (m) versus 35m, p=0.0007) and 6q deletion with a longer time to progression (55m versus 24m, p=0.04) in responding patients. Cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. The 6q deletion is frequent compared to chronic lymphocytic leukaemia (CLL) or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. Involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma, is rare in WM. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 22% cases with deletions of 6q21, a lower percentage compared with the literature [38-54%].Conversely to previous publications, 6q deletion was associated with a longer response duration to treatment and did not influence the overall survival. These discrepancies could be explained by the heterogeneity of the previous published series, mixing untreated patients and treated patients in various ways. In our trial, all patients were analyzed before randomization. As in CLL patients, TP53 deletion seems to be associated with a shorter response duration. However, regarding the small numbers of patients with cytogenetic abnormalities, these results must be confirmed in larger series. 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.

The t(14;18)(q32;q21) involving the MALT1 and IGH genes is a recurrent abnormality in MALT lymphomas. So far, molecular genetic characterization of the t(14;18)/IGHMALT1 has only been performed in 2 cases and revealed a fusion of the entire coding region of MALT1 to the IGH locus. We herein report the molecular genetic analyses of 2 new cases of MALT lymphoma harboring the t(14;18)/IGH-MALT1 using fluorescence in situ hybridization (FISH) and we determined the molecular characteristics at the IGH-MALT1 junctions using long-distance PCR (LD-PCR). The first case, a 71-year-old female, presented with an extranodal MALT lymphoma of the conjunctiva, stage IEA. The second case, a 53-year-old-male patient, was diagnosed as having a MALT lymphoma originating from the lung. FISH with PAC clones 117B5 and 59N7 revealed a translocation involving MALT1. Further FISH assays with probes hybridizing to MALT1 and IGH showed the t(14;18)(q32;q21)/IGH-MALT1. By FISH with specific probes for the P53, P16, RB1, and ATM genes no deletions of these genes were found. The IGH-MALT1 fusion was confirmed by LD-PCR on patients’ DNA with nested primers for the MALT1 and IGH genes. Cloning and sequencing of the purified PCR products revealed a fusion of sequences upstream of the coding region of MALT1 to the JH segment of the IGH locus in both cases. The breaks on chromosome 18 were located in the 5′ non-coding region of MALT1, only 13 nucleotides apart from each other. The breaks at IGH were located in the JH4 joining segment in both cases and showed features of a V(D)J-mediated recombination. Deletion and “de novo” nucleotides additions at the point of joining were observed in case 1. Furthermore, a detailed analysis of the “de novo” nucleotides additions in this case revealed the presence of DH segments of the DH gene D3-10 in the JH/MALT1 junction. Our findings indicate that the pathomechanism underlying the t(14;18)/IGH-MALT1 in MALT lymphomas is probably based on an illegitimate V(D)J recombination at IGH, similar to other IGH-associated translocations, such as the t(14;18)/IGH-BCL2 in follicular lymphomas and the t(11;14)/CCND1-IGH in mantle cell lymphomas and that the events leading to the t(14;18)/IGH-MALT1 might take place during an initial DH-to-JH or a later VH-to-DJH joining.

MCL is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 and with a genetic hallmark, the t(11;14) (q13;q32) translocation leading to the overexpression of cyclin D1, considered as the initial oncogenic event. However this entity may show a spectrum of morphological features broader than initially described. Clinically most of the patients have a poor prognosis with a rapid acquisition of chemoresistance. It has been recently described that some patients may follow an indolent clinical evolution, with the possibility of confusion in diagnosis because of overlapping morphological features with other small B cell lymphomas such as marginal zone lymphoma (MZL) and small lymphocytic lymphoma (SLL). The aim of our study was to analyze patients treated for a B cell lymphoma bearing the t(11;14)(q13;q32) who had a good outcome with a long survival. We retrospectively selected 21 patients (pts) with a t(11;14) B cell lymphoma and who followed an indolent outcome defined by an overall survival of more than 5 years after no more than 1 therapeutic regimen. Feature review included morphologic aspect, immunophenotype, cytogenetics, immunoglobulin (Ig) variable heavy chain (VH) gene usage and mutation patterns. Expression of CD5, CD10, CD23, and CD43, were evaluated on CD19+ cells by FCM. Cytogenetic analysis consisted in conventional cytogenetics on fresh tissue samples completed by a FISH analysis against 11q, 12p, 17p, and 18p realized on fresh frozen material. We further analysed the gene expression profiling of available samples using quantitative real-time PCR using microfluidic cards designed with selected genes of 3 independent signatures (Rosenwald A, et al. Cancer Cell 2003 - Ruiz-Ballesteros E, et al. Blood 2005 - Thieblemont C, et al. Blood 2004). Clinical characteristics showed that median age was 61 (range 45–81). All pts had a good PS and presented with a disseminated disease: bone marrow and blood involvement in 19 (90%) and 11/17 (65%) pts, respectively; peripheral and profound nodes involvement in 15 (71%); and splenomegaly in 13 (62%). LDH and b2-microglobulin levels were elevated in 4 pts. None of the pts had a monoclonal component. IPI score was low in 18 (86%) pts. Splenectomy alone or with chemotherapy (chlorambucil or fludarabine in 4; CHOP in 1) was proposed in 7 pts; monochemotherapy (chlorambucil or fludarabine) in 4 pts, CHOP +/− rituximab (R) in 2 pts, CHOP +/− R followed by autotransplant in 8. One pt did not receive any treatment. With a median follow-up at 6.44 years, median overall survival was 9.18 years. Morphologic review distinguished 2 groups of cases: true MCL cases (MCL, n = 8) and BorderLine Cases (BLC, n=13). Among the 8 MCL and 13 BCL, 7 and 12 were CD5+, 1 and 4 CD5/CD23+, 4 and 5 CD43+, respectively. All cases exhibited a t(11;14), associated with a complex caryotype in 5/8 (62%) MCL and in 9/13 (69%) BLC. Chromosome 7q deletion (n=1) and trisomy chr3/3q (n=3) were present only in BLC cases. IgVH gene was unmutated in 10 cases (3 MCL, 7 BLC) and mutated in 4 (2 MCL, 2 BLC). The most common VH families were VH1 (n=4), VH 3 (n=8) and VH4 (n=3). From the gene expression profiling studies we selected 70 genes specific of the diagnosis of MCL, MZL and SLL. A set of 5 MCL, 5 SLL, and 5 MZL control samples allowed us to validate 25 out of these 70 genes. These 25 genes were further used to analyse MCL and BLC samples. All these samples exhibited a more heterogeneous profile than control samples. Three MCL were more similar to MCL samples, whereas two were closer to MZL samples. All the BLC exhibited a profile close to that of MZL. We further analysed these samples with the 18 gene-signature predicting MCL survival. This signature could not discriminate survival of these MCL and BCL pts. In conclusion, pts with B cell lymphoma bearing the translocation t(11;14)(q13;q32) and with an indolent outcome can be distinguished in at least 2 groups with different characteristics in terms of morphology, immunophenotype, caryotype, and molecular profiles. Using a 25 gene-signature, we found profiles similar to either MCL or MZL for MCL samples, and similar to MZL for BCL samples. The signature discriminating the survival of MCL could not discriminate these indolent MCL. These findings will be validated on a larger series.

The genetic bases of Waldenstrom Macroglobulinemia (WM) are poorly understood. We have studied a cohort of 139 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM), by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). The sex ratio was 2.2M/1F, the average age at inclusion was 66 years [40–85]. The mean percentage of lymphoplasmacytic cells was 53% [8–97]. CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 7 probes CEP4, CEP12, 13q14, 11q22 ( ATM), 17p13 (TP53), IGH Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. Out of 110/137 successful karyotypes, 37% showed clonal abnormalities (9 additional cases had numerical changes of sexual chromosomes). Among abnormal karyotypes, there were 14 (34%) 6q deletions, 5 (12%) trisomy 4/partial trisomy 4, 4 (10%) trisomy 18, 3 (7%) trisomy 12. Using FISH, deletions of 6q21 were observed in 23/86 cases (27%), 13q14 in 12/89 cases (13%), TP53 9/85 cases (11%), ATM 6/81 cases (7%). Trisomy 4 was present in 9/82 cases (11%), and trisomy 12 in 4/86 cases (5%). No rearrangement of IGH was observed in the first 27 analyzed cases. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 27% with deletions of 6q21 (FISH), a low percentage compared to the literature [39–54%]. This could be explained either by the difference in the probe used or by the absence of selection of lymphoplasmacytic cells before cytogenetic analyses. Interestingly, we confirmed our recent observation that trisomy 4 was frequent in WM (12% if partial trisomy 4 was included). Furthermore we observed in this large series a frequent 13q14 deletion (13%). In conclusion, cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. 6q deletion is frequent compared to CLL or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. We didn’t observe cytogenetic involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. Searches for correlations with clinical and other biological parameters are ongoing.

Within the group of indolent B cells lymphomas derived from cells of the marginal zone, the pathogenesis of Splenic Marginal Zone Lymphoma (SMZL) remains incompletely characterized, in contrast to MALT lymphoma. However numerous findings recently contributed to a better description of this lymphoma, including its possible association with hepatitis C and the recurrence of 7q deletion. Furthermore, the peculiar pattern of Ig genes mutations and the biased usage of some segments (IGHV1-2) is suggestive of a T-independent Ag driven proliferation, at least at initial steps. The clinical heterogeneity is also marked with a lack of reproducible prognostic factors able to characterize the cases with a more aggressive course or histological transformation. MicroRNA (miRNA) are small non-coding RNA that have a post transcriptional regulation role by inhibiting translation of mRNA in protein and their abnormal expression was already found to contribute to the oncogenesis of various leukemias and lymphomas. The aim of the study was then to identify a miRNA profile of SMZL and to assess if specific miRNAs were associated with biological or clinical characteristics. MiRNA expression profile of SMZL was obtained by quantitative RT-PCR technology (Taqman microRNA Assays Human Panel, Applied Biosystems) which enabled to analyze 365 miRNAs. Six frozen spleen specimens of SMZL (including 2 cases with histological transformation) and 5 frozen specimens of non tumoral spleen (traumatic) were used to compare miRNA levels of expression (2−□□Ct method). Out of these 365 miRNAs, 95 were found to be reproducibly detectable in those samples and were further analyzed. Three miRNA were uniformly overexpressed in SMZL samples as compared to non tumor samples: miR-155 (mean fold change = 3.1), miR-451 (mean = 2.37), miR-486 (mean = 2.7). Conversly, 4 miRNAs were uniformly underexpressed: miR-127 (mean = 0.32), miR-139 (mean = 0.32), miR-335 (mean = 0.26), miR-411 (mean = 0.25). Interestingly, 1 miRNA, miR-21 was specifically found overexpressed only in the 2 transformed cases of SMZL (mean = 3.49) in comparison with the 4 non transformed cases (mean = 0.94). Those results were confirmed in a larger cohort of SMZL patients using a simplex real time PCR (Taqman microRNA Assays Human, Applied Biosystems) on frozen or paraffin embedded samples with a specific overexpression of miR-21 in 10 transformed cases (mean = 14.4), absent in the 6 non transformed cases (mean = 0.9) (Mann Whitney test: p =.0022). MiR-155 plays a key role in B cell differentiation and was already found overexpressed in other lymphomas subtypes. Little is know about the other miR overexpressed except for MiR- 21. MiR-21 is commonly overexpressed in solid tumors and the level of its expression is correlated in tumor growth and metastatic dissemination. It was also found overexpressed in chronic lymphocytic leukaemia and DLBCL (diffuse large B cell lymphoma). In opposite to DLBCL, our results seem to link miR-21’s overexpression with aggressive forms of SMZL. MiR-21 regulates multiple genes associated with apoptosis, migration and invasiveness. Altogether, these data points towards a specific miRNA expression pattern in SMZL and suggests that miR-21 could regulate important oncogenic pathways in transformed SMZL lymphomas.

Burkitt lymphomas/leukemias (BL) are highly aggressive mature B-cell malignancies characterized by a constant MYC rearrangement. MYC deregulation is an oncogenic event known to be necessary but not sufficient in BL pathogenesis. Additional (epi)genetic alterations should cooperate, such as inactivation of the p14arf-MDM2-TP53 pathway which is frequently detected. We recently demonstrated the independent negative prognostic impact of specific chromosomal alterations, del(13q) and +7q, in childhood BL. We also showed by Multi-FISH that 13q abnormalities are more heterogeneous than expected and that13q gains are underestimated by conventional cytogenetics (CC). DNA from 30 BL (15 adults/15 children) was analyzed with 50K Xba single nucleotide polymorphisms (SNP) arrays (Affymetrix), in order to characterize additional allelic imbalances and losses of heterozygosity. 47 gains and 30 losses were found in 22 BL; 57 chromosomal imbalances were cryptic (not detected by CC). A recurring 13q amplification was detected in 6 BL. The minimal amplified region (MAR) of 4.35 Mb includes 3 candidate genes: C13orf25, GPC5 and GPC6. One C13orf25 transcripts contains a miRNA 17–92 cluster. The amplicon was unmasked with BAC probes in 2/8 other screened BL with 13q abnormalities. This amplification was associated with a terminal deletion (minimal size: 5Mb) in half cases. A recurring 11q abnormality was also assessed by FISH with a MAR of 3.2 Mb containing candidate genes such as CBL, BLR1. Acquired partial uniparental disomies (pUPD) are characterized by loss of heterozygosity without chromosomal deletion. 73 pUPD (>5 Mb) were found in 15 BL; 14 were telomeric and probably resulted from one mitotic recombination. The pathogenic role of pUPD remains unclear. Some of them may be polymorphisms also detected in healthy population. Other may reflect genomic instability but in our series, no higher incidence of pUPD was detected in BL with a complex karyotype. Thirdly, non random pUPD may render the neoplastic cell homozygous for a preexisting mutation leading to the activation of an oncogene or inactivation of a tumor suppressor gene (TSG). Four telomeric pUPD on 1p, 9p, 17p and 17q were found in at least 2 BL. We confirmed homozygous mutations of 2 TSG (TP53 and CDKN2A) located on 17p and 9p pUPD, respectively. These pUPD may be another way of p14arf-MDM2-TP53 inactivation. Moreover, these results suggest an involvement of the miR 17–92 cluster known to interact with the MYC oncogene in BL pathogenesis. The expression pattern of the different mature miR 17–92 in BL with and without 13q amplification will be presented at the meeting.

Translocation t(14;19)(q32;q13) juxtaposing BCL3 in 19q13 with IGH in 14q32, is a rare recurrent event found in patients with B-cell malignancies. So far, a few cases of well-documented B-cell neoplasm have been reported. We analyzed 34 patients with t(14;19) and 1 patient with a variant t(2;19)(p11;q13) collected by the Groupe Francophone de Cytogenetique Hematologique on the basis of cytogenetic abnormality. Clinico-biological data, morphological review, immunophenotyping with Matutes’ score, conventional karyotype and FISH analysis with BCL3, IGH, CEP12, 13q14, ATM, TP53, 6q21 probes, and IgVH mutational status were recorded. The sex ratio was 22M/13F, the median age at diagnosis was 61 [39–89], the lymphocyte count was > 4×109/l in 96% of patients, and spleen enlargement was found in 47%. 31% (11/35) were morphologically classified CLL, 37% (13/35) atypical CLL, 20% (7/35) MZL; 79% had features of disease progression; 3 of the 4 latter were Diffuse Large B Cell Lymphoma, possibly transformed from MZL. 87% were CD5+, 72% had a Matutes’ score < 3. The IgVH genes were unmutated in 9/11 cases. The time to treatment was < 1 year in 68% of patients. The BCL3 locus involvement was confirmed by FISH analysis in all cases. 46% of cases showed complex karyotype. The chromosomal abnormalities associated with t(14;19) were +12 (57%), 6q- (27%), +3 (15%), 11q- (15%), 13q- (13%), 17p- (12%), +18 (12%), 7q- (12%). Comparison with published cytogenetic CLL data shows that deletion 6q was frequent and deletion 13q uncommon. Trisomies 3 and 18, and 7q deletion are less common than in published MZL. The independent analysis of our series of CLL/atypical CLL and MZL gave the same tendency. The chromosomal abnormalities associated with t(14;19) are not specific, but their frequencies are between those of typical CLL and MZL suggesting an intermediary status between the 2 malignancies. The t(14;19) identifies a subgroup of B-disorders CD5+ and Matutes’ score < 3, which could be of poor prognosis, based on progressive disease and unmutated status.

Chromosomal translocations involving the MYC oncogene (8q24) are known to occur in Burkitt lymphomas/leukemias (BL) but also in 5–30% of diffuse large B-cell lymphomas which are usually highly aggressive (8q24 DLBCL). Two recent conventional cytogenetics (CC) studies showed that secondary chromosomal abnormalities have a negative prognostic impact, especially13q abnormalities (frequently described as a deletion and usually associated with a complex karyotype, i.e. > 3 chromosomal alterations), mainly in childhood mature B-cell lymphomas, and in a less extent 7q, 3q, 22q and chromosome 17. M-FISH was applied to 120 (74% adults and 26% children) high grade B-cell non-Hodgkin lymphomas (86% BL, 14% 8q24 DLBCL) carrying MYC rearrangement and complex karyotype and/or 13q abnormality in order to find recurrent and/or cryptic chromosomal alterations. Abnormal metaphases were available in 96 (80%) cases. We described ‘new’ (not seen in CC) chromosomal rearrangements in 50 (52%) cases, refined those seen by CC in 28 (29%) cases and confirmed CC abnormalities in 18 (19%) cases. M-FISH allowed to characterize 55 structural 13q abnormalities in 42 patients (21 ‘new’ alterations): 24 der(13q) leading to partial del(13q) and partial gain of different partner chromosomes [mainly 1q or 7q], 15 del(13q) [of 2 minimal regions : q14 & q31q34], 13 gains (only 2 seen by CC) and 3 balanced translocations. Combined results of CC and M-FISH showed that the most frequent abnormalities among patients with complex karyotype involved 1q, 7q, Xq, 3q, 18q, 6q, 17p and chromosome 22 (excluding 8q24 translocations). 79 1q abnormalities were detected in 54 patients (26 % ‘new’): mostly gains (minimal amplified region: q22q31) due to unbalanced translocations with chromosomes 13, 22, 7 (59%) or duplications (25%). 55 partial 7q gains were observed in 42 patients (22% ‘new’), mostly +7 or unbalanced translocations with 13q, 6q or 1q. The other most common abnormalities were: t(14;18) in 8q24 DLBCL, del(6q), der(3q) with various partners leading to partial loss of 3q, monosomy 17 or del(17p) and numerical changes of chromosomes 22 (monosomy) and X. In conclusion, this study confirms the contribution of M-FISH in refining CC results in highly aggressive 8q24 B-cell lymphomas: ‘new’ rearrangements were identified especially in 1q (leading to partial 1q gains), 18q, 6q (leading to partial 6q deletions), 13q ; partial 13q gains were underestimated by CC and both 13q deletions and gains were more heterogeneous than expected. We are characterizing these prognostic additional chromosomal abnormalities with SNP-CHIPS 50K array (Affymetrix) to look for candidate genes and/or cellular pathways involved in Burkitt lymphomagenesis in cooperation with oncogenic effect of MYC. °on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCG-Ho (Belgian Cytogenetic Group of Hematology and Oncology).

Background and Objectives Splenic and nodal marginal zone B cell lymphoma (SMZL and NMZL) have been recently identified as distinct clinicopathological entities in the WHO classification. These lymphomas entities may have a common origin in the marginal B-cell compartment of the lymphoid organs. However the precise cell of origin of marginal zone B cells, its status in the B cell differentiation pathway and the mechanisms involved in lymphomagenesis remain unclear. The most widely held view is that marginal zone B cells are mostly memory B cells. But the origin of these cells, especially the transit through germinal center pathway, remains contradictory. Somatically mutated variable-region of immunoglobulin genes and bcl-6 gene represent at this time faithful markers for exposure to the germinal center. In addition, aberrant somatic hypermutations have been suggested to contribute to the development of B-cell lymphomas, occurring in the 5′ sequence of several proto-oncogenes. Interestingly those mutation do not occur in normal germinal center B cells. Design and Methods: IgVH, BCL-6, PIM1, Rho/TTF and PAX 5 genes, highly mutated in DLBCL and other indolent lymphoma such as B-CLL, were analysed for the presence of somatic mutations from 50 marginal zone lymphoma tissue and blood samples (21 NMZL and 29 SMZL including 10 cases with numerous villous lymphoma cells in peripheral blood). According to the morphological and immunophenotypical analysis, the fraction of malignant cells in the specimen was 70% or more in all cases. Mutational analysis was restricted to the regions previously shown to contain more than 95% of mutations in DLBCL. PCR products were directly sequenced on both sides and perfomed in duplicate in two independent reactions. Results: Out of 18 NMZL cases analysed for IgVH mutational status (3 cases not analysed for IgVH) 15 cases were mutated and 21 out of 28 in SMZL cases. Mutation of BCL-6 was detected in only 1 NMZL patients (1/21) and 1 SMZL patients (1/29). For RhoH/TTF, PIM1, PAX5 the mutation average was also low with only 1 case mutated per group and per gene, with a different case mutated in each for each gene. Conclusion In summary, we demonstrate the low frequency of aberrant somatic mutations in SMZL and NMZL, suggesting that this process is probably not a major contributor to lymphomageneis. However the frequent absence of mutation in BCL6 suggest a particular differentiation pathway, as suggested before in normal marginal zone B cells, possibly without transit through the germinal center. Interestingly the relatively high frequency of VH mutated cases compared with the frequent absence of mutation of BCL6, considered as a specific germinal center tag, could suggest somatic hypermutation outside the germinal center. In addition the absence of hypermutation could be linked with the absence of recurrent translocation in SMZL and NMZL, the translocation process haveing been associated with somatic hypermutation dysfunction.

Non-germinal centre small B-cell lymphomas represent a heterogeneous group of non-hodgkin lymphomas which most frequent histologic subtypes are small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). These three lymphoma entities have very different clinical outcomes but may be difficult to distinguish either histologically or clinically. We previously identified transcriptomic signatures specific of these 3 lymphoma subtypes. We further analyzed these lymphomas using Surface-Enhanced Laser Desorption/Ionisation Time of Flight (SELDI-TOF). A total of 58 tumors, including 20 SLL (all lymph nodes), 20 MZL (1 lymph node and 19 spleens) and 18 MCL (19 lymph nodes and 1 spleen) were analyzed. In addition, we included 7 controls obtained from traumatic normal spleens. The spectra were generated on weak cation exchange (CM10), strong anion exchange (Q10) and reversed-phase (H50) ProteinChip arrays. Protein patterns of all samples were comparatively analysed using two distinct strategies. We first used a binary recursive partitioning method with the Biomarker Pattern software (Ciphergen®), and second a hierarchical clustering method to visualized patterns of protein peaks completed with a supervised method (discriminating score) to point out individual peaks distinguishing the three histological subtypes (SLL, MZL and MCL). Spectra analyses revealed a very homogeneous protein patterns among all lymphoma samples. However specific SLL, MZL and MCL signatures based on 34 protein peaks with differential expression could be identified and allowed to classify 95% of the samples in their respective entity. SLL signature included 9 peaks, MZL signature 16 peaks and MCL signature 9 peaks. The binary recursive partitioning analysis was concordant but identified only the five most discriminant peaks. Further identification of the discriminating peaks is currently realized using SELDI-assisted purification. We are focusing on peaks at 9942 Da for SLL and at 11324 Da for MCL. Functional genomic studies can distinguish non-germinal small B-cell lymphomas at the transcriptomic level (our previous study) and at the proteomic level. This will provide new markers for diagnosis and potentially new therapeutic targets.

Glutathione-S-transferase π (GSTπ), whose gene is located in 11q13, is belonging to a complex multigene family of enzymes that may detoxify electrophilic xenobiotics. Recent gene expression profiling analysis showed that GSTπ transcript is belonging to the mantle cell lymphoma (MCL) signature and constitutes one of the most expressed mRNA in MCL[*][1]. Furthermore it has been reported by immunohistochemistry that overexpression of cyclin D1 in MCL was associated with a high level of GSTπ protein expression. We then looked for the specificity of a high level of GSTπ protein expression in MCL, and particularly when comparing to marginal zone lymphoma (MZL) with t(11;14). Material and Methods: Immunochemistry analysis of GSTπ and cyclinD1 expression was performed 111 non Hodgkin’s lymphoma including MCL, MZL, small lymphocytic lymphoma (SLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL) cases with or without t(11;14). Results: A strong GSTπ expression in more than 50% of the cells was observed in 98% of the MCL. None of the MZL cases with t(11;14) expressed GSTπ, except one case that expressed a weak signal in less than 20% of the cells. None of the MZL without t(11;14) expressed GSTpi. Only two (13%) FL graded 3 according to OMS classification expressed GSTπ in less than 50% of the cells. A high GSTπ expression (>50% cells stained) was detected in 29% of the DLBCL and 43% of the SLL. | | n | GST π expression >50% | GST π expression 5–50% | π GST expression