Abstract: Introduction. The majority of SMZLs display an indolent course, however the disease is still incurable and a significant proportion of patients (~25-30%) experience poor outcomes surviving 〈 5 years. Molecular alterations of SMZL are promising biomarkers, which might improve risk stratification of patients. The main objective of the study is to test the impact of molecular aspects on disease-specific survival prognostication in newly diagnosed SMZL. Methods. IELSG46 is a multicenter, international, retrospective, observational study in which already existing and coded health-related personal and biological material is used. The study included adults, who received a diagnosis of SMZL on spleen histology with a follow up 〉 5 years, and for whom tumor material collected before initiation of medical therapy was available. Mutation analysis was performed by CAPP-seq targeted deep next generation sequencing of tumor genomic DNA. A stringent bioinformatic pipeline was applied to suppress the background noise allowing to call variants with a sensitivity of 5x10-2 in FFPE derived DNA. Copy number variations (CNVs) were identified by using the sequencing reads-based GATK4-CNV algorithm. IGHV rearrangements were obtained by using LymphoTrack® IGH FR1 Assay Panel kit. Molecular clusters were identified by an iterative algorithm that maximizes genetic distinctiveness of subgroups by reassigning patients between clusters that are created a priori based on the co-occurrence of genetic lesions. Relative survival, defined as the ratio between actuarial survival observed in the SMZL cohort and expected survival of the general population matched to patients by geographical origin, sex, age and calendar year of diagnosis, was calculated using the Ederer II method. Results. The analysis included 303 patients with a SMZL diagnosis confirmed on spleen histology. The sample size allowed to identify 30% differences in survival for molecular subgroups comprising at least 5% of cases with a statistical power between 80-100%. Median follow-up was 9.2 years. At 10 years, 85% of patients were alive, consistent with the known indolent behavior of this lymphoma. Genes recurrently affected by non-synonymous somatic mutations in 〉 10% of SMZL included KLF2 (24%), NOTCH2 (19%), KMT2D (15%), TNFAIP3 (13%), EP300 and TP53 (10%). Deletion 7q was documented in 25% of cases and IGHV1-2*04 usage in 32%. By cluster analysis, three major molecular subgroups were identified, each of them characterized by a NOTCH pathway mutated gene (Fig. 1A). The first cluster was defined by NOTCH2 and/or KLF2 mutations and was enriched in TNFAIP3 mutations and IGHV1-2*04 gene usage (Fig. 1A). The second cluster was defined by SPEN mutations, and was enriched in KMT2D and other epigenetic gene mutations (Fig. 1A). The third cluster was enriched in NOTCH1 mutations (Fig. 1A). By relative survival analysis, the NOTCH2/KLF2 cluster showed a lower survival compared to the matched general population, indicating a significant impact of the disease on patients' expected survival (Fig. 1B). Conclusions. The large sample size and inclusion of SMZL confirmed by spleen histopathology review allowed for precise estimation of the prevalence of KLF2 and NOTCH2 mutations in this lymphoma. Three molecular clusters were identified in SMZL, each of them containing a NOTCH pathway gene, supporting the relevance of NOTCH signaling in the pathogenesis of SMZL. Patients belonging to the NOTCH2/KLF2 cluster had a lower relative survival compared to the matched general population. Disclosures Traverse-Glehen: Astra Zeneca: Other: Travel; Takeda: Research Funding. Gomes da Silva:Roche: Other: Institution's payment for consultancy, Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Other: Travelling support; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Ladetto:Celgene: Honoraria; Jannsen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria; Roche: Honoraria. Rambaldi:Pfizer: Consultancy; Novartis: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy; Celgene: Consultancy. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Zinzani:MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: 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, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Salles:Servier: Honoraria; Abbvie: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria. Zucca:Celltrion: Consultancy; AstraZeneca: Consultancy.

Abstract: Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Abstract: Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models. Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism ( & lt;0.9), additive (0.9-1.1), antagonism/no benefit ( & gt; 1.1) after 72 hr treatments. Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P & lt; 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s & gt; 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM. A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h. In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P & lt; 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P & lt; 0.001). Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines. Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies. Citation Format: Eugenio Gaudio, Filippo Spriano, Chiara Tarantelli, Matilde Guala, Eugenia Riveiro, Gaetanina Golino, Antonio Lupia, Giosuè Costa, Roberta Rocca, Luciano Cascione, Silvia Jenni, Yi-Chien Tsai, Beat Bornhauser, Stefano Alcaro, Francesco Paduano, Francesco Trapasso, Emanuele Zucca, Anastasios Stathis, Natalina Pazzi, Franco Cavalli, Francesco Bertoni. EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4796.

Abstract: Classic hairy cell leukemia (HCL) is a tumor of mature clonal B cells with unique genetic, morphologic, and phenotypic features. DNA methylation profiling has provided a new tier of investigation to gain insight into the origin and behavior of B-cell malignancies; however, the methylation profile of HCL has not been specifically investigated. DNA methylation profiling was analyzed with the Infinium HumanMethylation27 array in 41 mature B-cell tumors, including 11 HCL, 7 splenic marginal zone lymphomas (SMZLs), and chronic lymphocytic leukemia with an unmutated (n = 7) or mutated (n = 6) immunoglobulin gene heavy chain variable (IGHV) region or using IGHV3-21 (n = 10). Methylation profiles of nontumor B-cell subsets and gene expression profiling data were obtained from public databases. HCL had a methylation signature distinct from each B-cell tumor entity, including the closest entity, SMZL. Comparison with normal B-cell subsets revealed the strongest similarity with postgerminal center (GC) B cells and a clear separation from pre-GC and GC cellular programs. Comparison of the integrated analysis with post-GC B cells revealed significant hypomethylation and overexpression of BCR–TLR–NF-κB and BRAF-MAPK signaling pathways and cell adhesion, as well as hypermethylation and underexpression of cell-differentiation markers and methylated genes in cancer, suggesting regulation of the transformed hairy cells through specific components of the B-cell receptor and the BRAF signaling pathways. Our data identify a specific methylation profile of HCL, which may help to distinguish it from other mature B-cell tumors.

Abstract: SMZL usually has an indolent clinical course. Transformation to aggressive lymphoma occurs in only 4-5% of cases. Tools to identify patients facing a more aggressive clinical course and targeted therapies are needed. In the last years, we have characterized SMZL by DNA profiling (Blood 2011) and RNA/miRNA expression profiling (Mod Path 2013), and shown that somatic mutations of NOTCH2 and other genes involved in the pathway are frequent and mutually exclusive with mutations affecting NKFB pathway (JEM 2012). Here, we report genome-wide DNA promoter methylation profiling of 137 SMZL samples with its clinical and biologic implications. Methods A test series of 101 samples, including 3 SMZL cell lines (Karpas1718, VL51, SSK41) and 98 clinical SMZL cases from 10 Centers and, a validation set of 36 SMZL cases from 2 additional Centers were analyzed with the Illumina Infinium HumanMethylation27 arrays, as described (BJH 2013). Gene GEP was available for a subset of 11/101 cases. Methylation profiles were correlated with the presence of the copy number aberrations (CNAs) and somatic mutations recurrent in SMZL, IGHV mutational status, HCV infection and clinical data. Probes mapped outside CpG islands were discarded. Supervised analyses were performed using a t-test on the continuous beta values, followed by multiple test correction. For Fisher’s test, probes were classified into unmethylated (beta-values 〈 0.3) or methylated (beta-value 〉 0.3). Results Unsupervised clustering analysis of 101 SMZL samples identified 2 clusters. Cluster one (23/101, 23%) comprised the 3 cell lines and included cases characterized by a generalized higher degree of promoter methylation (High-M), and had a significantly poorer overall survival (OS) than the remaining 79/101 (77%) cases (Low-M) (P=0.048; HR 2.4; 95% CI, 1-5.8). To better understand the biology underlying this clustering, supervised analysis comparing High-M to Low-M cases was performed. By combining t- and Fisher's tests, 3200 gene probes showed differential methylation status between High-M and Low-M lymphomas. This probes signature was significantly enriched of promoters of promoter regions of PRC2-complex targets, genes involved in chromatin remodeling, and HOX and SOX family genes, and genes down-regulated by CDH1. Genes coding for subunits of the PRC2-complex (EZH2, EED, SUZ12) appeared unmethylated in their promoter regions and over-expressed by GEP in the High-M set, while EZH2-target genes and genes harboring the H3K27me3 mark had methylated promoters and down-regulated expression. A number of reported tumor-suppressor genes were highly methylated in High-M cases, including KLF4, DAPK1, CDKN2A/B, CDKN1C, CDH1, CDH2 and TIMP3. Known pro-survival lymphoma genes, such as SYK, MCL1, CARD11, BIRC5, BTK, BCL10, FOXP1, CD40, TACI and TCL1A/B, were un-methylated and over-expressed in High-M cases. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set. An unsupervised clustering using the top-100 differentially methylated probes obtained by t-test in the test series identified two clusters with one (11/36, 30%) bearing methylation profile similar to High-M and a poorer OS (P=0.038). All the samples were then pooled to improve the statistical power of further analyses regarding the impact of methylation on the clinical course and on the association with biologic variables. High-M was significantly associated with poorer OS (P=0.008; HR 2.58; 95% CI, 1.2-5.4), transformation into aggressive lymphoma (33% vs 0.02%, P 〈 0.001) and with 7q31-33 loss (38% vs 16%, P=0.026). In addition, High-M showed a higher frequency of NOTCH-pathway somatic mutations (39% vs 16%, P=0.063). HCV was more frequent in Low-M (20% vs 4%, P=0.058). In a Cox regression model adjusted for the variables significantly associated with outcome at univariate analysis [High-M, age 〉 60, Interguppo Italiano Linfomi (IIL) SMZL score, HCV infection], High-M (HR 16; 95% CI, 1.3-201) and I IL SMZL score (HR 4.5; 95% CI, 1.3-15) retained their prognostic significance on OS (P 〈 0.001). Conclusions Genome-wide promoter-methylation profiling in splenic MZL identified a subset of patients with poorer OS, characterized by a high promoter methylation and by specific genetic and biologic features. These data may provide the rational to explore epigenetic drugs in SMZL. *, AJA and AR equally contributed. Disclosures: No relevant conflicts of interest to declare.

Abstract: Methylation profiling identifies subgroups of SMZL with distinct biological features. Demethylating agents can reverse some of the adverse epigenetic alterations.

Abstract: Copanlisib is a pan–class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Kα and PI3Kδ. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18 phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas.

Abstract: Bromodomain and extra-terminal domain (BET) proteins, cyclic adenosine monophosphate response element-binding protein (CBP), and the E1A-binding protein of p300 (EP300) are important players in histone acetylation. Preclinical evidence supports the notion that small molecules targeting these proteins individually or in combination can elicit antitumor activity. Here, we characterize the antitumor activity of the pan BET/CBP/EP300 inhibitor NEO2734 and provide insights into its mechanism of action through bromodomain-binding assays, in vitro and in vivo treatments of cancer cell lines, immunoblotting, and transcriptome analyses. In a panel of 60 models derived from different tumor types, NEO2734 exhibited antiproliferative activity in multiple cell lines, with the most potent activity observed in hematologic and prostate cancers. Focusing on lymphoma cell lines, NEO2374 exhibited a pattern of response and transcriptional changes similar to lymphoma cells exposed to either BET or CBP/EP300 inhibitors alone. However, NEO2734 was more potent than single-agent BET or CBP/EP300 inhibitors alone. In conclusion, NEO2734 is a novel antitumor compound that shows preferential activity in lymphomas, leukemias, and prostate cancers.

Abstract: Background. Mantle cell lymphoma (MCL), which is characterized by the deregulation of cyclin D1 (CCND1), is one of the most common lymphoma subtypes, accounting for 5-10% of all cases. MCL prognosis is often very poor. Several novel non-chemotherapeutic agents have shown promising activity in MCL, but novel agents and in particular new drug combinations are needed to improve patients' outcome. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA contribute to MCL pathogenesis and represent potential therapeutic targets. Bromodomain and extra-terminal (BET) proteins are epigenetic readers contributing to gene transcription. OTX015 is a bromodomain (BRD) inhibitor that has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) as well as promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). Here, we present preclinical evidence of OTX015 activity in combination with other targeted drugs in MCL. Material and methods. MCL cell lines (REC1, MAVER1, UPN1, JeKo1, SP53, Mino, Granta519) were exposed to increasing doses of OTX015 alone or in combination with increasing doses of other drugs, including everolimus, BEZ235, ibrutinib, carfilzomib, pomalidomide, 5-AZA, vorinostat and dexamethasone. The MTT assay was performed after 72h exposure. Real-time PCR and Western blotting were used for RNA and protein expression analyses. Synergy was assessed by the Chou-Talalay combination index (CI) with the Synergy R package: CI 〈 0.3, strong synergy; 0.3-0.9, synergy; 0.9-1.1, additive effect. Results. OTX015 showed antiproliferative activity as a single agent in 6/7 MCL cell lines with IC50s 〈 500 nM. Four MCL cell lines were exposed to DMSO or OTX015 (500 nM and IC50) for 4 and 24h to evaluate expression of CCND1 and other members of signaling pathways known to be impacted by BRD inhibitors. No reduction in CCND1 was observed at the RNA or protein levels after OTX015 exposure. However, MYC was downregulated and the transcriptional regulator HEXIM1 and histone-coding genes were upregulated. In light of reported strong synergy between OTX015 and the mTOR inhibitor everolimus in diffuse large B-cell lymphoma, we evaluated OTX015 combined with everolimus or the dual PI3K/mTOR inhibitor BEZ235 in 6 MCL cell lines (REC1, MAVER1, UPN1, JeKo1, SP53, Mino). In addition, combinations of OTX015 with the BTK-inhibitor ibrutinib, the proteasome inhibitor carfilzomib, the immunomodulator pomalidomide, the demethylating agent 5-AZA, the HDAC-inhibitor vorinostat, and the glucocorticoid dexamethasone were assessed in REC1 and MAVER1. Combinations of OTX015 with everolimus, pomalidomide, dexamethasone, and ibrutinib showed the strongest activity (Fig 1). Strong synergy between BEZ235 and OTX015 was only seen in the ibrutinib-resistant cell line MAVER1. Conclusions. OTX015 showed preclinical activity as a single agent in MCL cells. The mechanism of action does not appear to involve CCDN1 down-regulation and gene expression profiling studies will be needed to identify the involved pathways. OTX015 had additive or synergistic activity with several targeted compounds in multiple MCL cell lines, identifying combinations that may merit further investigation in the preclinical and clinical settings. Fig. 1. Chou-Talalay analysis of OTX015 combinations in MCL cell lines. Y-axis: CI 〈 0.3, strong synergy; 0.3-0.9, synergy; 0.9-1.1, additive effect; 〉 2.25, antagonism. Outliers were excluded. C.I., Combination Index. Fig. 1. Chou-Talalay analysis of OTX015 combinations in MCL cell lines. Y-axis: CI 〈 0.3, strong synergy; 0.3-0.9, synergy; 0.9-1.1, additive effect; 〉 2.25, antagonism. Outliers were excluded. C.I., Combination Index. Disclosures Stathis: Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.