ALBERT

Description: Introduction: DLBCL is biological and clinically highly heterogeneous. Although different genetic aberrations, including recurrent somatic mutations, have been described in this tumor, their clinical impact remains to be clarified. The aim of the present study was to determine somatic mutations and copy number alterations of a selected group of genes in patients with DLBCL, in order to assess their prognostic importance and to identify potential personalized targeted drugs for these patients. Methods: 150 patients (78M/72F; median age, 66 years) diagnosed with de novo DLBCL no otherwise specified at Hospital Clínic and other institutions of the GELCAB, treated with immunochemotherapy, were included in the study. An independent series of 111 patients (54M/57F; median age, 63 years), diagnosed at different Japanese and Spanish institutions, was used to validate the significant findings. Targeted next generation sequencing (NGS) of 106 representative genes related with DLBCL and Copy Number Alterations (CNA) assessment were performed. Ten functional pathways were pre-defined, including NOTCH, tumor suppressor genes, JAK/STAT, epigenome/chromatic modifier, BCR signaling, PI3K-AKT-mTOR, MAP-kinase, B-cell differentiation, immune surveillance and cell cycle alterations. Cell of origin (COO) of the tumors was established using gene expression or the Lymph2Cx assay. Genomic-guided potential therapeutic opportunities for each patient were identified in silico by a Cancer Genome Interpreter platform. Results: A total of 765 potential driver mutations were identified in 89 of the 106 genes with a slightly higher number in germinal center B-cell like (GCB) than activated B-cell-like (ABC) DLBCL subtype. The most frequently mutated genes found in 〉15% of the cases were KMT2D (MLL2), MYD88, CREBBP and TP53, with other 27 genes being mutated in 〉5% of the cases. Several genes were differentially mutated in GCB DLBCL subtype (KMT2D, CREBBP, TNFRSF14, B2M, EZH2, GNA13, FOXO1, ACTB and SOCS1) or ABC subtype (MYD88, PIM1, CD79B and PRDM1). No relevant differences were observed in the clinical features according to individual mutations or CNA. No single gene mutation predicted response to therapy. Genetic alterations in KLHL6, ETV6, SGK1, L8q12.1, CD79B, PIM1 and TP53 predicted poor OS, whereas mutations of SOCS1 were associated with better outcome. Alterations in NOTCH pathway and tumor suppressor pathway were associated with poor outcome, whereas those of JAK/STAT pathway showed favorable prognosis (see table for detailed data). NOTCH pathway (HR 2.8; p=0.006) and tumor suppressor pathway (HR 2.4; p=0.005) maintained independent significance for OS along with R-IPI (H 4.0; p=0.006) in a multivariate analysis that also included COO and beta2-microglobulin. In addition, the prognostic value of NOTCH and tumor suppressor pathways was confirmed in the independent validation series. Finally, we identified 69 cases (46%) carrying at least one genomic alteration in 9 genes considered a biomarker of drug response supported by data of early clinical trials or pre-clinical assays; tumors of additional 26 patients (17%) had at least one gene alteration that could be exploited by a drug repurposing strategy. Conclusions: Integrating the deep sequencing analysis of a panel of selected genes and CNA, we have recognized novel target genes and defined the clinical relevance of alterations of NOTCH and tumor suppressor pathways in DLBCL. Using an in silico prescription pipeline we have also identified a number of candidate drugs with potential therapeutic interactions with driver oncogenic proteins. All these findings may orient future preclinical and clinical intervention strategies in DLBCL. Table Initial features, response to therapy and outcome according to pathways´ status Table. Initial features, response to therapy and outcome according to pathways´ status Disclosures Sancho: Celltrion, Inc: Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gonzalez Barca:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Gilead: Speakers Bureau. Ohshima:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau. Akashi:Sunitomo Dainippon Pharma: Consultancy; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Bristol Meyers Squibb: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Shionogi & Co., Ltd: Research Funding; Astellas Pharma: Research Funding.

Description: Abstract 1647 Mantle cell lymphoma (MCL) is an incurable B-lymphoid neoplasm harboring the t(11;14)(q13;q32) translocation which leads to the overexpression of cyclin D1, with the consequent cell cycle deregulation. Typically, MCL is characterized by bad prognosis and an aggressive course of the disease. Unfortunately, current therapies have shown limited efficacy and relapses occur early, thus our purpose was to evaluate the antitumoral properties of the multikinase inhibitor sorafenib in MCL. Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. We analyzed the sensitivity to sorafenib in 9 MCL cell lines and 17 primary MCL cells by flow cytometry after annexin V staining. Sorafenib induced apoptosis in MCL cell lines with a mean LD50 of 11.5 ± 5.0 μM at 24 hours, while at 48 hours decreased to 7.1 ± 2.7 μM. In primary MCL cells, the mean LD50 was 13.0 ± 3.6 μM at 24 hours, while at 48 hours it notably decreased to 9.4 ± 3.4 μM. These data indicated that sorafenib exerted a time- and dose-dependent cytotoxic effect in MCL cells. Both in cell lines and primary MCL cells, sorafenib induces rapid dephosphorylation of the BCR (B-Cell Receptor)-associated tyrosine kinases, SYK and LYN, as well as of FAK, a downstream SRC target involved in focal adhesion. In line with this, we demonstrate a strong synergy when combining sorafenib with the SYK inhibitor, R406. In parallel, we show that sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an unbalance between pro- and anti-apoptotic proteins and facilitates the release of Bax from cyclin D1. This process leads to the induction of the mitochondrial apoptotic pathway and caspase-dependent and independent mechanisms. Moreover, sorafenib inhibits MCL cell migration as well as actin polymerization in response to CXCL12. FAK siRNA-mediated knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target for the action of sorafenib in MCL cells. Importantly, this compound resensitizes MCL cells cocultured with bone marrow-derived stromal and follicular dendritic-like cells to bortezomib-induced apoptosis indicating that sorafenib was able to antagonize stroma-mediated resistance in MCL. In conclusion, we provide first evidence on the molecular mechanism of action of the multikinase inhibitor sorafenib in MCL. We propose that this compound inhibits cell migration and stroma-mediated bortezomib resistance by interfering BCR signaling and protein translation. All these results suggest that sorafenib, alone or in combination with bortezomib-based therapies, may represent a promising approach for the treatment of MCL patients. Research funding This work was supported by grants from Ministerio de Ciencia e Innovación (SAF 09/9503) and Redes Temáticas de Investigación Cooperativa de Cáncer from the Instituto de Salud Carlos III RED 2006-20-014 (D.C.). S.X-T. is a recipient of predoctoral fellowship from Ministerio de Ciencia e Innovación (FPU) and M.L-G. holds a contract from Fundación Científica de la Asociación Española contra el Cáncer. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1538 According to the WHO 2008 Classification, the cellular origin of mantle cell lymphoma (MCL) is traced to a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. This notion, however, is seriously challenged by both the remarkable restrictions of the immunoglobulin gene repertoire in MCL and, furthermore, by the fact that the great majority of cases exhibit imprints of somatic hypermutation (SHM) in rearranged IGHV genes, ranging from (mostly) minimal to pronounced. These findings support an antigen-driven origin for MCL, at least for a substantial fraction of the entire cohort. Activation-induced cytidine deaminase (AID) is induced in B cells following contact with antigen and is critically implicated in both somatic hypermutation (SHM) and class switch recombination (CSR). Although the available information about AID expression and in vivo CSR in MCL is limited and contradictory, at least some MCL cases have been reported to express AID and undergo ongoing CSR. With this in mind, here we investigated AID-mRNA isoform expression and isotype switch events in a large series of MCL cases and explored possible associations with IGHV gene repertoire and SHM status. Overall, 107 cases were included in the study and tumor-involved diagnostic tissue samples of different types were evaluated, including: fresh-frozen lymph nodes (LN, n=53), peripheral blood (PB, n=42), spleen (n=5), bone marrow biopsies (n=3) and other (n=4). The neoplastic lymphocytic infiltration ranged from 52–98% (median 80%). Thirty-five of 107 cases (32.7%) carried IGHV genes with 100% identity to the germline (GI) whereas the remaining 72 cases bore some imprint of SHM: in particular, 48/107 cases (44.9%) carried IGHV genes with 97–99.9% GI and, finally, 24/107 cases (22.4%) carried IGHV genes with

Description: Introduction Although CLL predominates in the elderly, there are few studies focusing on the characteristics, treatment, and prognosis of elderly patients with CLL. Patients and Methods Retrospective single center study of 949 patients diagnosed with CLL between 1990 and 2012. We defined two age-groups:

Description: A remarkable feature of chronic lymphocytic leukemia (CLL) is the existence of quasi-identical, stereotyped B-cell receptor immunoglobulins (BcR IGs), strongly supporting an antigen-driven pathway to CLL development. Subsets of cases with distinct stereotyped BcRs collectively account for almost one-third of all CLL. Furthermore, just a handful of major stereotyped subsets represent a substantial fraction of the entire cohort and, perhaps more importantly, an even larger fraction of clinically aggressive CLL. In several major subsets, stereotypy extends from shared primary IG sequences to shared clinical and biological features, including immune signaling, mRNA and miRNA expression, DNA methylation, and genomic aberrations. Regarding the latter, recent evidence indicates that different subsets display distinct profiles of recurrent gene mutations, even when limiting the analysis to subsets with similar IGHV gene mutational status. However, it should be emphasized that even the largest subsets account for only ∼3% of the cases with available IGHV-D-J sequence information, indicating that for meaningful conclusions to be reached, large patient cohorts are essential. Here, taking advantage of a series of 2482 CLL cases consolidated in the context of a multicenter collaboration coordinated by ERIC, the European Research Initiative on CLL, we systematically explored the genetic background of stereotyped subsets. Our main focus was on recurrent mutations in the NOTCH1 (entire exon 34 or targeted analysis for del7544-45), TP53 (exons 4-9), SF3B1 (exons 14-16), BIRC3 (exons 6-9) and MYD88 (exon 5) genes. Overall, 1313 cases (52.9%) carried mutated IGHV genes (M-CLL), whereas the remaining 1169 cases (47.1%) carried unmutated IGHV genes (U-CLL). Cases were sub-classified into the following major subsets: (i) U-CLL: #1, n=72; #3, n=25; #5, n=11; #6, n=22; #7, n=37; #8, n=20; (ii) M-CLL: #4, n=32; #77, n=12; #148, n=20; and, (iii) subset #2 (IGHV3-21, variable mutational status), n=57. Mutations in the MYD88 and BIRC3 genes were relatively rare, with no clear bias to any subset. With regards to the other three genes, only a single mutation in the TP53 gene was identified in a total of 80 M-CLL subset cases. Among U-CLL subsets and clinically aggressive subset #2, we noted asymmetric mutation frequencies, summarized as follows. (1) TP53 mutations were (a) enriched in subsets #3 and #7 (frequency 〉10%) and, in contrast, absent in subsets #5 and #6, though all these subsets utilize the IGHV1-69 gene; (b) enriched in subset #1 (9%) and, interestingly, subset #99, a less populated subset that is highly similar to subset #1 (2/4 cases positive for TP53 mutations); (c) absent in subset #2; and, (d) relatively infrequent in subset #8 (5%), the latter known to display the highest risk for Richter’s transformation among all CLL. Differences between these subsets showed a trend for statistical significance (p=0.09). (2) NOTCH1 mutations exhibited (a) increased frequency in subsets #1 (28%) and #8 (25%); (b) among IGHV1-69 expressing subsets, lower frequencies in subsets #3 (8%), #5 (10%) and #7 (3%) compared to subset #6 (25%); and (c) intermediate frequency (9%) in subset #2 (p=0.0078 for comparison between subsets). (3) SF3B1 mutations were (a) significantly (p

Description: Introduction CLL, SLL and cMBL are considered to be part of the same spectrum of clonal expansions of CD5+ B cells. Clinically, the transition from one to another of these forms over time is a well recognized event. Diagnostic criteria to separate these disorders have been proposed (IWCLL, 2008; WHO, 2008). Aim To compare presenting and evolving features of three groups of patients with cMBL, Rai 0 CLL or SLL and to ascertain the usefulness of current diagnostic criteria for these disorders. Patients and Methods Retrospective study of clinical, biologic and evolving characteristics of patients diagnosed with cMBL, Rai 0 CLL or SLL according to current criteria (CLL: ≥5x109 clonal B cells/L in peripheral blood; SLL

Description: Introduction: MCL is a mature B-cell neoplasm characterized by t(11;14) (q13;q32) and cyclin D1 (CCND1) overexpression. Molecular studies have revealed other alterations in cell-cycle regulation, DNA damage response and cell survival pathways, with a landscape of somatic mutations being recently identified. CNS involvement is a well known complication, occurring in 4-26% of MCL at five years, with an ominous significance. Although different clinical variables have been identified as risk factors for CNS infiltration, the biological parameters related to this complication have not been extensively studied. The aim of the study was to explore the biological parameters associated with CNS involvement in a multicentre and retrospective series of MCL patients. Patients and Methods: 285 patients (M:74%; 64 yr) diagnosed of MCL between 1990-2014 (median survival of 4 years) were analysed. In addition to standard clinico-biological variables, IGHV mutational analysis, chromosomal alteration studies and Sanger sequencing of NOTCH1, NOTCH2, TP53, BIRC3, WHSC1, MEF2B, MLL2, TLR2 and PRDM1 were performed. Results: CNS involvement was observed in 15/285 MCL patients (5.2%), with a 5-yr risk of 9.1% (95%CI: 4.6-13.6), one patient at diagnosis, and at first or second/ulterior progressions in 7 cases each. The clinical, pathological and molecular risk factors identified are detailed in the Table. In addition to what has been already described, CNS involvement was usually observed in MCL cases with a clinical nodal presentation (p=0.05). In fact, no indolent MCL with a non-nodal presentation developed this complication during the follow-up period. No differences were observed in the risk of CNS involvement between patients treated in first-line with conventional or high-dose intense treatment (5-yr risk: 6.1%+/-6% vs. 10.7%+/-10.6%, p=ns). Regarding the biological features, no differences in terms of the IGHV mutational status were observed in cases developing CNS involvement compared to the others (75% vs. 68.7%, using 97% identity cut-off). Similarly, the IGHV gene usage of CNS involved cases corresponded to the more frequent IGHV genes observed in MCL (usually IGHV1-18, IGHV3-23, IGHV4-34, IGHV4-59). Although not significant, a predominance of high number copy number alterations (CNA) (〉4) could be observed in the genetic study of MCL cases with CNS involvement as could be expected for the enrichment in blastoid variants (up to 50% of these cases). In fact, we did not observe any case with CNS involvement among those cases with 3 or less CNA. CNS involvement was not related to common poor prognosis genetic alterations such as 9p, 11p and 17p losses, but the presence of 8q gains was associated with a higher risk of CNS involvement (p=0.05). We did not find any significant association between CNS involvement and the large number of oncogenic mutations studied. Conclusions: CNS involvement in MCL is associated with initial aggressive clinico-biological characteristics. Non-nodal MCL cases with a low number of genetic alterations did not present CNS involvement. Finally, the presence of 8q gains was associated with a higher risk of CNS infiltration. Table Initial Clinical Features Category N 5 yr-CNS involvement (%, 95%CI) HR p Performance status (ECOG) 〉 1 8/51 41.5 (+/-28) 4.2 .003 ≤ 1 7/128 9.4 (+/-5.5) Nodal disease Yes 14/185 13.3 (+/-7.6) 6.1 .05 No 1/77 1.4 (+/-2.7) Hemoglobin (g/L) 〈 105 12/93 24.7 (+/-14.7) 3.2 .05 ≥ 105 3/78 5.3 (+/-6.3) LDH 〉 ULN 4/89 27.1(+/-19.4) 6.7 ULN 11/114 21.6 (+/-14.9) 3.5 .04 〈 ULN 3/66 8.7(+/-10) Molecular & Pathological data Histological variant Blastoid 6/58 17.3 (+/-13.7) 3.5 .02 Others 8/156 1.3 (+/-7.2) Ki-67 〉 30% 5/44 17.5 (+/-14.9) 3.6 .06 ≤ 30% 3/61 6.7 (+/-9.4) SOX11 Positive 8/153 2.9 (+/- 5.7) 2.6 ns Negative 1/42 2.1 (+/-2.4) IGHV ≥97% 6/109 9.5 (+/-9.6) 1.9 ns 4 2/87 3.9 (+/-5.7) 1.1 ns ≤ 4 1/44 2.3 (+/-4.3) Chromotripsis Yes 1/17 12.5 (+/-22) 3.2 ns No 2/106 1.9 (+/-2.7) 8q gain Yes 2/30 13.1(+/-19) 7.5 .05 No 2/97 1 (+/-1.96) CNA: copy number alteration; IGHV: immunoglobulin heavy chain; LDH: Lactate dehydrogenase Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Mature normal and tumor B cells express a unique rearranged immunoglobulin (IG) gene that can be used as a marker of the clonal expansion of the cell. Somatic hypermutation (SHM) in the V(D)J region of IG genes are acquired in the germinal center and are a surrogate imprint of the cell of origin of lymphoid neoplasms. In chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the identification of SHM distinguishes subtypes of tumors with different clinical and biological behavior. Although still less studied, cases carrying highly similar IG sequences (i.e. stereotyped IG), specific light chain (LC) rearrangements, and the presence of class switch recombination (CSR) of the constant region of the heavy chains further sub-classify patients into potentially distinct clinico-biological subgroups. The analysis of the rearranged IG gene is currently performed by specific Sanger sequencing (SSeq) or next-generation sequencing protocols. Whole-genome sequencing (WGS) of B-cell neoplasms should store the information to reconstruct the entire rearranged IG gene [heavy (IGH) and kappa or lambda (IGK, IGL) LC]. However, the high genomic complexity and homology of these regions have prevented the analysis of the rearranged IG genes in WGS using standard bioinformatics pipelines. Aim: To assess the use of WGS data to fully characterize the rearranged IG gene in B-cell neoplasms. Methods: We developed IgCaller, a fast, easy-to-run program that uses already aligned WGS data to dissect the rearranged IGH V(D)J genes, IGK and IGL VJ genes, and the presence of constant heavy chain CSR. IgCaller also determines the homology of the rearranged sequences compared to the patient's germ line or reference genome. We demonstrated the accuracy of IgCaller using WGS data of 331 B-cell neoplasms [240 CLL (152 cohort 1 (C1)-CLL; 88 independent cohort 2 (C2)-CLL), 61 MCL, and 30 multiple myeloma (MM)] and compared with SSeq of the IGH V(D)J and/or LC and isotype expression. Results: IgCaller identified a complete IGH productive rearrangement [V(D)J] in 133 (88%) C1-CLL, 80 (91%) C2-CLL, 61 (100%) MCL, and 21 (70%) MM. A partial (VJ) rearrangement was detected in 8 (5%) C1-CLL and 1 (3%) MM. Available SSeq of the V(D)J or at least V gene for 131 C1-CLL, 10 C2-CLL, and 60 MCL successfully characterized by WGS highlighted only one discordant V(D)J rearrangement. Small discrepancies (only J or V disagreement) were found when the J (n=4) or V (n=1) gene identified by SSeq based on homology (IMGT/V-QUEST tool) did not correspond to the rearranged gene detected by WGS, which was the second scoring gene in IMGT/V-QUEST suggesting that our non-homology WGS-based approach might be more accurate. Of note, IgCaller identified the presence of two distinct IGH subclones in 1 case. Next, the comparison of the percentage of homology of the rearranged sequence to the germ line in 131 C1-CLL and 60 MCL with complete V gene both by SSeq and WGS showed a high correlation and concordance in both cohorts [R〉0.95, p90% B-cell neoplasms studied. The complete characterization of the rearranged IG gene based on WGS data, when available, could facilitate the analysis of LC rearrangements and CSR, and replace the traditional SSeq of the IG loci both in research and clinical settings. Disclosures No relevant conflicts of interest to declare.

Description: Key Points This report describes a multidisciplinary study characterizing the largest series of cyclin D1− MCL patients. CCND2 translocations are the most frequent genetic event (55%) in cyclin D1− MCL.

Description: Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1− MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1−/D2− MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1−/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1− MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1− MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1− MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1−/D2−/D3− MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1− MCL.