ALBERT

Description: Introduction Mantle-cell lymphoma (MCL) is of poor prognosis, with a median survival of about 5 years. Besides the t(11;14) translocation, several secondary genetic abnormalities have been shown to correlate with prognosis. However, most studies have analysed patients with heterogeneous treatment, mostly with anthracyclin-based regimens. In 2004, the European MCL Network started the randomized MCL Younger trial comparing R-CHOP followed by high-dose radiochemotherapy and autologous stem cell transplantation (ASCT) versus alternating R-CHOP/R-DHAP followed by a high-dose cytarabine conditioning regimen and ASCT in previously untreated MCL stage II-IV patients up to the age of 65y. The R-CHOP/R-DHAP arm showed improved time to treatment failure (TTF) and, potentially, overall survival (OS) (Hermine et al., ASH 2010, ASH 2012). Our aim was to revisit the prognostic value of some gene copy number alterations (GCNA) in this randomized trial and to determine whether high-dose cytarabine could counteract some of those factors. Methods The inclusion criteria for this biological study were: confirmed histological diagnosis of MCL, the availability of diagnostic tumor DNA and complete clinical data. When no frozen biopsy was available, peripheral samples with more than 50% tumor cells were considered eligible for GCNA analysis. CDKN1B, CDK2, and MDM2 were analyzed using quantitative multiplex PCR of short fluorescent fragments (QMPSF) (Jardin et al., BJH 2009), 6q25-q26, CDK4, and the 13q14 locus were analyzed by multiplex ligation-dependent probe amplification (MLPA) (MRC-Holland CLL kit), and MYC, CDKN2A, ATM, RB1 and TP53 were assessed by both methods. The analyses of the prognostic value of GCNA was adjusted for clinical prognostic factors summarized in the quantitative MIPI score (age, performance status, LDH, and WBC). The rate of proliferating tumor cells (Ki-67 index) was centrally assessed by the reference pathologists of the European MCL Pathology Panel according to published guidelines (Klapper et al., J Hematopathol 2009). Outcome variables were TTF from treatment start to stable disease, progression, or death from any cause, and OS from trial registration to death from any cause. Results Of 135 patients fulfilling the inclusion criteria (median age 56 years), 49%, 26%, and 25% of patients were of low, intermediate, and high MIPI risk . The most frequent amplification involved MYC (18%), whereas the most frequent deletion involved the 13q14 locus (36%), including RB1 in 26%. As expected, CDKN2A and TP53 deletions were frequently found (25% and 22%, respectively). ATM alterations mostly consist of deletion (25%), but amplification was found in 3 of 129patients. The frequencies of GCNA did not differ according to the type of sample analyzed i.e. tumor biopsies (n=79) vs. high tumor load peripheral blood or bone marrow samples (n=56). The Ki-67 index was higher in patients with CDKN2A or RB1 deletion compared to patients without, but was not different between patients with or without TP53 deletion. Only TP53 gene status was associated with MCL cytology, with more frequent deletion in blastoid forms (4/8) than in classic MCL (11/81, 14%). In univariable analyses, deletions of CDKN2A, 13q14, RB1, CDKN1B, and TP53 were associated with shorter TTF and OS, whereas GCNA of 6q25-q26, MYC, ATM, CDK2, CDK4, and MDM2 were not prognostic. In multivariable analyses, adjusting for MIPI score, CDKN2A and TP53 deletions showed independent prognostic impact with hazard ratios of 2.4 (p=0.001) and 2.3 (p=0.004) for TTF and 2.3 (p=0.007) and 2.4 (p=0.007) for OS. This effect was observed in both treatment arms (Figure 1). In addition, there was an interacting effect of CDKN2A (p16) deletion and TP53 deletion on TTF (p=0.004). Conclusions The introduction of high-dose cytarabine in first-line treatment of younger MCL patients did not erase the adverse prognostic value of TP53 and CDKN2A deletions observed with previous regimens. Moreover, our study identified a small patient group of very bad prognosis which could benefit of more aggressive regimens or new targeted drugs combination. Figure 1: TTF (left) and OS (right) according to CDKN2A/TP53 deletion status in patients of the R-CHOP/R-DHAP study arm; nom=not deleted, del=deleted Figure 1:. TTF (left) and OS (right) according to CDKN2A/TP53 deletion status in patients of the R-CHOP/R-DHAP study arm; nom=not deleted, del=deleted Figure 2 Figure 2. Disclosures Feugier: Roche: Honoraria. Haioun:Roche, Celgene, Takeda, Pfizer, Janssen,: Honoraria.

Description: 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.

Description: Abstract 2632 Hans algorithm using immunohistochemistry correlates well with gene expression data in Diffuse large B-cell lymphomas (DLBCL) (Meyer PN, 2011) and has demonstrated in some studies clear survival differences in favor of germinal-centre (GC) vs non-germinal centre (n-GC) B-cell among DLBCL treated with R-CHOP. We undertook an immunohistochemical study among patients aged 18 to 59 years with aaIPI 1 included in the GELA trial LNH 03-2B that compared R-ACVBP intensified immunochemotherapy to standard R-CHOP. This trial demonstrated an improvement of EFS, PFS and overall survival (OS) of patients treated with R-ACVBP (C Recher et al, in press). Our goal was to evaluate survival of patients with GC and n-GC DLBCL according to treatment regimens. We analyzed by immunohistochemistry the expression of CD10, BCL6 and MUM1 and classified patients as GC or n-GC according to the Hans algorithm. Among the 380 patients enrolled in this study, 229 patients were available for Hans algorithm classification. There was no differences considering clinical characteristics of these 229 patients (age, sex, B symptoms, PS, Stage, LDH, number of extranodal sites, bulky mass, bone marrow involvement) compared to the whole LNH03-2B population. 175 DLBCL cases were present on a tissue microarray (TMA) and 54 other cases were analyzed using unstained slides. 101 patients were classified as GC and 128 as n-GC. 107 patients were treated by R-ACVBP and 122 by R-CHOP. EFS, PFS and OS were not different between the GC and n-GC profile among the whole population (P=.82, P=.90, P=.68, respectively). There was no statistical difference in EFS, PFS and OS between R-ACVBP and R-CHOP in GC patients (P=.78; P=.84, P=.33, respectively). Interestingly, EFS, PFS and OS were significantly much longer among n-GC patients treated by R-ACVBP compared to R-CHOP (P=.02; P=.007, P=.007, respectively). Results were similar considering only TMA population (P=.02, P=.001, P=.001, respectively). This subgroup analysis suggests that the survival benefit related to R-ACVBP over R-CHOP in the LNH 03-2B is in large part linked to a survival improvement in the n-GC population. This algorithm, easy to apply on routine paraffin-embedded tissue, might be useful in the future to select patients that can primarily benefit from this intensive regimen. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Peripheral T-cell lymphoma (PTCL) consists of an uncommon and heterogeneous group of lymphomas that are often challenging to diagnose and classify. Since most patients also have a poor survival with standard multiagent chemotherapy, more effective therapeutic approaches are needed to improve patient outcome. Table1: Pathological diagnosis Number of cases profiled AITL 36 ALK(+)ALCL 19 ALK (−)ALCL 08 ATLL 12 T/NK 14 PTCLU 44 Other rare entities 10 Methods: A mRNA profiling study using Affymetrix HGU133+2 arrays on 143 cases of PTCL and NK-cell lymphoma (NKCL) from the International Peripheral T-cell Lymphoma Project, was conducted on pre-treatment biopsies. These included the following pathologically classified cases (Table 1). In addition, we also profiled nine NK cell lines, seven T cell lines, normal resting and activated CD4+ and CD8+ T cells and resting and IL2- activated NK cells from healthy individuals. BRB-ArrayTools was used to develop gene classifiers for the major PTCL entities and survival predictors for AITL based on gene expression data. Results: We have identified key molecular signatures for PTCL and NKCL that have allowed us to construct a robust classifier for AITL (207 transcripts), ALK+ ALCL (94), ATLL (225) and NKCL (127). PTCL-U group may have 3 or 4 molecular subgroups and additional studies with more cases, are necessary to further define this group. Misclassified cases were identified and re-assigned to the molecularly defined entities, including re-assigning of 9/44 PTCL-U to AITL. We have confirmed the enriched expression of genes identified in follicular helper T-cells in AITL, suggesting that AITL is derived from this T-cell subset. A number of oncogenic pathways (e.g. NF-κB, HIF-a,VEGF, IL6) and tumor/host interactions that contributed to local tumor-induced immunosuppression (e.g. TGF-b), were identified in AITL. A molecular predictor of outcome was developed for AITL and validated by leave one-out-cross validation. Since PTCL is an uncommon disease, future studies will require the collaboration of multiple large clinical groups with tissue resources for both discovery and validation. Conclusion: This study has demonstrated that GEP will allow the construction of robust and biologically-meaningful classifiers for PTCL, and prognosticators can be derived for well-defined entities with a sufficient number of cases. GEP will also allow us to identify therapeutically-relevant oncogenic pathways and tumor/host interactions that may lead to improvement in the therapy and outcome of patients with PTCL and NKCL. (This study is a part of the International T-cell Lymphoma Project)

Description: 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.

Description: Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1607 Background: Lymphoid malignancies derived from T and NK cells (PTCLs) constitute a heterogeneous group of uncommon disease entities, with marked geographic variation in their distribution. The most recent data from the International T-cell Lymphoma Project based on a retrospective analysis of PTCLs diagnosed between 1990 and 2002 (Blood. 2011;117(25): 6756–67) indicate that PTCL, not otherwise specified (PTCL,NOS) represents the most common PTCL worldwide (25,9%), followed by angioimmunoblastic T-cell lymphoma (AITL) (18,5%). Over the last few years, a better characterization of the cellular origin and pathophysiology of PTCLs has led to the development of new diagnostic markers. Aim of the study: To characterize the epidemiology of mature T/NK-cell malignancies in Western Europe (France and bordering countries) and to examine whether the availability of new tools/antibodies and changing concepts over the past years might have translated into an apparent change in the relative distribution of PTCL entities. Materials and methods: The histopathological diagnosis of PTCL entities according to the 2008 WHO classification were collected through two independent sets of PTCLs in France and bordering countries. Results: Over the past two years (2010–2011), 933 newly diagnosed non-cutaneous PTCLs were reviewed in reference centers through the prospective network “Lymphopath” aiming to review any newly diagnosed lymphoma in France. According to the 2008 WHO classification, the 933 PTCLs comprised: 314 AITL (33,6%), 239 PTCL,NOS (25,6%), 78 ALK-positive anaplastic large cell lymphoma (ALCL) (8,3%), 72 ALK-negative ALCL (7,7%), 59 extranodal NK/T-cell lymphomas (ENKTL 6%), 33 enteropathy-associated T-cell lymphoma (4%), 32 HTLV1+ adult T leukemia/lymphoma (3%), 7 hepatosplenic T-cell lymphoma (1%) and 99 cases of other entities or unclassifiable (11%). A high prevalence of AITL was also found in an independent set of PTCLs retrospectively collected in the framework of a multicentric T-cell lymphoma research consortium “Tenomic” where non-cutaneous PTCL with frozen material available (n=623) from 1999 to 2009 in France and Belgium were retrieved and collegially reviewed for consensus diagnosis. In this collection, AITL (n=288, 46%) also outnumbered PTCL, NOS (n=215, 35%). Of the 196 AITL cases extensively investigated for CD10, TFH markers (PD1, CXCL13), CD21/CD23 follicular dendritic cells (FDC) and EBV, the initial diagnosis was recorded in 178 cases as: AITL in 155 cases (87%), PTCL, NOS in 21 cases (12%), and intermediate between PTCL,NOS and AITL (PTCL,NOS/AITL) in 2 cases, indicating the impact of additional stainings for the diagnosis of AITL. The 107 PTCL,NOS cases also extensively immunostained included 9 follicular variant of PTCL,NOS, 8 PTCL,NOS/AITL cases, 5 cases intermediate between PTCL,NOS and ALK-negative ALCL (of which 2 had been diagnosed as such and two as PTCL,NOS), and 85 remaining cases truly unspecified. Of these, 60 had been initially diagnosed as PTCL,NOS; 2 as PTCL,NOS/AITL; 4 as ALK-negative ALCLs and 1 as ENKTL. Conclusion: This study based on two independent large cohorts of non-cutaneous PTCLs highlights AITL as the most prevalent entity in Western Europe. It shows that extensive studies including investigation for CD10, TFH markers, FDC and EBV can at least partly contribute to the reclassification of some PTCL, NOS into the AITL spectrum category. Disclosures: No relevant conflicts of interest to declare.