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Classical Hodgkin Lymphoma (cHL) is a B cell-derived lymphoid malignancy, affecting 2.5–3/100,000 people per year. To date, in patients diagnosed with advanced cHL no reliable tool is able to—a priori—distinguish the subset of patients at high risk for relapse or refractory disease. Clinical risk indices for cHL, such as the International Prognostic Score (IPS), have not been successfully applied as a treatment decision tool in advanced stage cHL [1]. In this study we show, that a previously published gene expression-based predictor in advanced stage cHL patients treated with ABVD [2] does not prove prognostic in 401 BEACOPP-treated advanced stage cHL patients. Using transcriptome profiling, we identified however that three individual genes, PDGFRA, TNFRSF8 (encoding CD30) and CCL17 (encoding TARC), were significantly associated with progression-free survival (PFS) after multiple test correction in the BEACOPP-treated cohort, highlighting the potential of a modified gene expression profiling approach for pre-treatment risk assessment., Letter

Despite continuing improvements in the management of classical Hodgkin lymphoma (cHL), relapse remains associated with a risk of lymphoma-related mortality. The biological composition of relapse tumour biopsies shows interpatient variability, which can be leveraged to design prognostic biomarkers. Here, we validated the RHL30 assay, a previously reported gene expression model in an independent cohort of 41 patients with relapsed cHL. Patients classified as high-risk by the RHL30 assay had inferior failure-free survival (FFS) after autologous stem cell transplantation (2-year FFS 41% vs. 92%, P = 0·035). The RHL30 model is a robust biomarker that risk-stratifies patients considered for autologous stem cell transplantation.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and ‘eat-me’ signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation—a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Integrative analysis in patients with diffuse large B-cell lymphoma uncovers that biallelic mutations on TMEM30A are associated with a favorable outcome and enhanced sensitivity to CD47 blockade.

High-throughput sequencing has significantly contributed to revealing the molecular underpinnings of B-cell lymphomagenesis and disease progression. It is now a widely accepted concept that the diversity of clinical responses to front-line therapy and the development of relapsed/refractory disease are in part explained by 'inter-patient' genetic heterogeneity measurable by individual sets of somatic gene alterations in tumor genomes. Moreover, extensive 'intra-tumor' heterogeneity on the genotypic and phenotypic levels is the product of ongoing tumor evolution and adaptation to various selective pressures during cancer initiation, progression, and therapeutic intervention. As the management of disease progression remains one of the most significant clinical challenges, it is becoming increasingly important to delineate how B-cell lymphomas evolve over time and to develop progression-related biomarker assays. Toward this goal, recent investigations have moved from studying lymphoma biology at initial diagnosis to doing so at multiple time points during the disease course. Profiling progressed tumors, and in particular paired biopsies at initial diagnosis and disease progression of the same patients, has led to novel insights into clonal tumor evolution and tumor microenvironment dynamics. This review discusses the latest findings on genomic alterations and microenvironment biology associated with relapsed/refractory B-cell lymphomas, with a particular emphasis on alterations that are acquired or become more prevalent at disease progression. We also describe overarching tumor evolution patterns, and highlight emerging precision medicine methodologies that can aid in an improved understanding and management of relapsed/refractory disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

High-throughput sequencing has significantly contributed to revealing the molecular underpinnings of B-cell lymphomagenesis and disease progression. It is now a widely accepted concept that the diversity of clinical responses to front-line therapy and the development of relapsed/refractory disease are in part explained by 'inter-patient' genetic heterogeneity measurable by individual sets of somatic gene alterations in tumor genomes. Moreover, extensive 'intra-tumor' heterogeneity on the genotypic and phenotypic levels is the product of ongoing tumor evolution and adaptation to various selective pressures during cancer initiation, progression, and therapeutic intervention. As the management of disease progression remains one of the most significant clinical challenges, it is becoming increasingly important to delineate how B-cell lymphomas evolve over time and to develop progression-related biomarker assays. Toward this goal, recent investigations have moved from studying lymphoma biology at initial diagnosis to doing so at multiple time points during the disease course. Profiling progressed tumors, and in particular paired biopsies at initial diagnosis and disease progression of the same patients, has led to novel insights into clonal tumor evolution and tumor microenvironment dynamics. This review discusses the latest findings on genomic alterations and microenvironment biology associated with relapsed/refractory B-cell lymphomas, with a particular emphasis on alterations that are acquired or become more prevalent at disease progression. We also describe overarching tumor evolution patterns, and highlight emerging precision medicine methodologies that can aid in an improved understanding and management of relapsed/refractory disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Follicular lymphoma (FL) is an indolent disease but transforms in 2% to 3% of patients per year into aggressive, large cell lymphoma, a critical event in the course of the disease associated with increased lymphoma-related mortality. Early transformation cannot be accurately predicted at the time of FL diagnosis and the biology of transformed FL (TFL) is poorly understood. Here, we assembled a cohort of 126 diagnostic FL specimens including 40 patients experiencing transformation (

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

Effective treatment of diffuse large B-cell lymphoma (DLBCL) is plagued by heterogeneous responses to standard therapy, and molecular mechanisms underlying unfavorable outcomes in lymphoma patients remain elusive. Here, we profiled 148 genomes with 91 matching transcriptomes in a DLBCL cohort treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) to uncover molecular subgroups linked to treatment failure. Systematic integration of high-resolution genotyping arrays and RNA sequencing data revealed novel deletions in RCOR1 to be associated with unfavorable progression-free survival (P = .001). Integration of expression data from the clinical samples with data from RCOR1 knockdowns in the lymphoma cell lines KM-H2 and Raji yielded an RCOR1 loss-associated gene signature comprising 233 genes. This signature identified a subgroup of patients with unfavorable overall survival (P = .023). The prognostic significance of the 233-gene signature for overall survival was reproduced in an independent cohort comprising 195 R-CHOP-treated patients (P = .039). Additionally, we discovered that within the International Prognostic Index low-risk group, the gene signature provides additional prognostic value that was independent of the cell-of-origin phenotype. We present a novel and reproducible molecular subgroup of DLBCL that impacts risk-stratification of R-CHOP-treated DLBCL patients and reveals a possible new avenue for therapeutic intervention strategies.

Non-Hodgkin lymphomas (NHLs) are the most common cancer to affect pet dogs. In contrast to the many genes whose mutation contributes to lymphomagenesis in humans, relatively little is known about the acquired genetic alterations that lead to canine B-cell lymphomas (cBCLs). We performed a survey of 84 canine NHL tumors to identify genes affected by somatic point mutations. We found mutations affecting TRAF3, which encodes a negative regulator of nuclear factor (NF)-κB, to be a common feature of cBCLs, with mutations observed in 44% of tumors including a combination of somatic and rare germ-line variants. Overall, 30% of the tumors contained ≥1 somatic TRAF3 mutation. The majority of mutations are predicted to cause loss of TRAF3 protein including those impacting reading frame and splicing. To determine whether TRAF3 loss might be relevant to human NHL, we also analyzed 148 human diffuse large B-cell lymphoma (DLBCL) tumors and identified loss of TRAF3 as a common event, affecting ∼9% of DLBCLs, and reduced expression of TRAF3 among deleted cases. This study implicates mutations affecting NF-κB activity as a novel genetic commonality between human and canine NHLs and supports the potential utility of cBCLs with mutated TRAF3 as a model of the more aggressive activated B-cell subgroup of DLBCL.

Summary Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and T cell/histiocyte rich large B cell lymphoma (THRLBCL) usually affect middle-aged men, show tumour cells with a B cell phenotype and a low tumour cell content. Whereas the clinical behaviour of NLPHL is indolent, THRLBCL presents with advanced stage disease and an aggressive behaviour. In the present study, array comparative genomic hybridization was performed in seven typical NLPHL, four THRLBCL-like NLPHL variants, six THRLBCL and four diffuse large B cell lymphomas (DLBCL) derived from NLPHL. The number of genomic aberrations was higher in THRLBCL compared with typical and THRLBCL-like variant of NLPHL. Gains of 2p16.1 and losses of 2p11.2 and 9p11.2 were commonly observed in typical and THRLBCL-like variants of NLPHL as well as THRLBCL. Gains of 2p16.1, affecting the REL locus were confirmed in an independent cohort. Expression of the REL protein was observed at similar frequencies in typical and THRLBCL-like variant of NLPHL as well as THRLBCL (33–38%). In conclusion, the present study reveals further similarities between NLPHL and THRLBCL on the genomic level, confirming that these entities are part of a pathobiological spectrum with common molecular features, but varying clinical presentations.

Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of diffuse large B cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somaticIL4Rmutations in 15 out of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8 leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain-of-function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantagein vivoThe pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establishIL4Rmutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

We recently found that FOXO1 repression contributes to the oncogenic program of classical Hodgkin lymphoma (cHL). Interestingly, FOXO3A, another member of the FOXO family, was reported to be expressed in the malignant Hodgkin and Reed-Sternberg cells of cHL at higher levels than in non-Hodgkin lymphoma subtypes. We thus aimed to investigate mechanisms responsible for the maintenance of FOXO3A as well as the potential role of FOXO3A in cHL. Here, we show that high FOXO3A levels in cHL reflect a B-cell-differentiation-specific pattern. In B cells, FOXO3A expression increases during the process of centroblast to plasma cell (PC) differentiation. FOXO3A levels in cHL were found higher than in germinal center B cells, but lower than in terminally differentiated PCs. This intermediate FOXO3A expression in cHL might manifest the "abortive PC differentiation" phenotype. This assumption was further corroborated by the finding that overexpression of FOXO3A in cHL cell lines induced activation of the master PC transcription factor PRDM1α. As factors attenuating FOXO3A expression in cHL, we identified MIR155 and constitutive activation of extracellular signal-regulated kinase. Finally, we demonstrate the importance of FOXO3A expression in cHL using an RNA interference approach. We conclude that tightly regulated expression of FOXO3A contributes to the oncogenic program and to the specific phenotype of cHL.

Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2 . Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation ( EZH2 Y641X ). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2 Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.

The ability to accurately model evolutionary dynamics in cancer would allow for prediction of progression and response to therapy. As a prelude to quantitative understanding of evolutionary dynamics, researchers must gather observations of in vivo tumor evolution. High-throughput genome sequencing now provides the means to profile the mutational content of evolving tumor clones from patient biopsies. Together with the development of models of tumor evolution, reconstructing evolutionary histories of individual tumors generates hypotheses about the dynamics of evolution that produced the observed clones. In this review, we provide a brief overview of the concepts involved in predicting evolutionary histories, and provide a workflow based on bulk and targeted-genome sequencing. We then describe the application of this workflow to time series data obtained for transformed and progressed follicular lymphomas (FL), and contrast the observed evolutionary dynamics between these two subtypes. We next describe results from a spatial sampling study of high-grade serous (HGS) ovarian cancer, propose mechanisms of disease spread based on the observed clonal mixtures, and provide examples of diversification through subclonal acquisition of driver mutations and convergent evolution. Finally, we state implications of the techniques discussed in this review as a necessary but insufficient step on the path to predictive modelling of disease dynamics.

Autism spectrum disorder (ASD) is a disorder with high levels of co-morbidities. The Developmental, Dimensional and Diagnostic Interview (3 Di) is a relatively new instrument designed to provide dimensional as well as categorical assessment of autistic behaviours among children with normal intelligence. Its sound psychometric properties and relatively short administration time make it a versatile instrument. The 3 Di was translated into Chinese (Cantonese) and its applicability among 194 clinic children was examined. Results found excellent reliability and validity, and achieved a sensitivity of 95% and specificity of 77%. It was able to capture the diagnosis of ASD among children presenting with attention deficit hyperactivity disorder. However, although the disorder of ASD is considered universal, the use of a western instrument in a Chinese context should also take note of cultural influences that may impact on the manifestation of its symptoms.

The pathogenesis of primary mediastinal large B-cell lymphoma (PMBCL) is incompletely understood. Recently, specific genotypic and phenotypic features have been linked to tumor cell immune escape mechanisms in PMBCL. We studied 571 B-cell lymphomas with a focus on PMBCL. Using fluorescence in situ hybridization here, we report that the programmed death ligand (PDL) locus (9p24.1) is frequently and specifically rearranged in PMBCL (20%) as compared with diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma. Rearrangement was significantly correlated with overexpression of PDL transcripts. Utilizing high-throughput sequencing techniques, we characterized novel translocations and chimeric fusion transcripts involving PDLs at base-pair resolution. Our data suggest that recurrent genomic rearrangement events underlie an immune privilege phenotype in a subset of B-cell lymphomas.

Classical Hodgkin lymphoma and primary mediastinal B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by whole-genome and whole-transcriptome sequencing recurrent somatic coding-sequence mutations in the PTPN1 gene. Mutations were found in 6 of 30 (20%) Hodgkin lymphoma cases, in 6 of 9 (67%) Hodgkin lymphoma-derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in Hodgkin lymphoma cell line KM-H2 resulted in hyperphosphorylation and overexpression of downstream oncogenic targets. Our data establish PTPN1 mutations as new drivers in lymphomagenesis.

The clinical significance of

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and “eat-me” signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of “hot” and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general. Citation Format: Shannon Healy, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W. Woolcock, Elena Vigano, Libin Abraham, Michael Gold, Adele Telenius, Pedro Farinha, Graham Slack, Susana Ben-Neriah, Daniel Lai, Allen W. Zhang, Sohrab Salehi, Hennady P. Shulha, Derek S. Chiu, Sara Mostafavi, Alina S. Gerrie, Diego Villa, Laurie H. Sehn, Kerry J. J. Savage, Andrew J. J. Mungall, Andrew P. Weng, Marcel Bally, Ryan D. Morin, Gabriela V. Cohen Freue, Joseph M. Connors, Marco A. Marra, Sohrab P. Shah, Randy D. Gascoyne1, David W. Scott, Christian Steidl, Ulrich Steidl. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell 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 3480.

Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2. Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.

Here we describe DataBiNS-Viz – a visualization and exploration environment for non-synonymous coding single nucleotide polymorphisms (nsSNPs) data gathered by the BioMoby-based DataBiNS workflow. DataBiNSViz enables execution of the DataBiNS workflow on pr oteins described by KEGG , PubMed, or OMIM identifiers, followed by manual exploration of the integrated structure/function and pathway data for those proteins, with a particular focus on nsSNP data in-context. The tool can be freely accessed at http://bioinfo.icapture.ubc.ca:8090/ DataBiNS (please use the Firefox or Safari web browsers). Examples of the retrieved data are given under the “Help on inputs” option. Detailed documentation can be accessed at http://bioinf o.icapture.ubc.ca/mywiki/ DataBiNS.

We present a novel hierarchical Bayes statistical model, xseq, to systematically quantify the impact of somatic mutations on expression profiles. We establish the theoretical framework and robust inference characteristics of the method using computational benchmarking. We then use xseq to analyse thousands of tumour data sets available through The Cancer Genome Atlas, to systematically quantify somatic mutations impacting expression profiles. We identify 30 novel cis-effect tumour suppressor gene candidates, enriched in loss-of-function mutations and biallelic inactivation. Analysis of trans-effects of mutations and copy number alterations with xseq identifies mutations in 150 genes impacting expression networks, with 89 novel predictions. We reveal two important novel characteristics of mutation impact on expression: (1) patients harbouring known driver mutations exhibit different downstream gene expression consequences; (2) expression patterns for some mutations are stable across tumour types. These results have critical implications for identification and interpretation of mutations with consequent impact on transcription in cancer., Assessing functional impact of mutations in cancer on gene expression can improve our understanding of cancer biology and may identify potential therapeutic targets. Here, Ding et al. describe a novel statistical model named xseq for a systematic survey of how mutations impact transcriptome landscapes across 12 different tumour types.

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

The survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-κB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation. Here we show that FOXO1 directly upregulates the full-length isoform PRDM1α in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1α acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1α downregulation in cHL and identify PRDM1α as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation.

The mutational landscape of Hodgkin lymphoma cell lines determined by whole-exome sequencing

Introduction: Disease progression remains a significant clinical burden in classical Hodgkin lymphoma (CHL) with 25-30% of patients relapsing after first-line treatment. The current standard of care for relapsed CHL is high dose chemotherapy followed by autologous stem cell transplantation (ASCT). This secondary therapy only cures approximately 50% of patients, with virtually no reliable biomarkers to identify the patients in which this salvage treatment regimen fails. The specific aim of this study was to establish the extent of changes in tumor microenvironment (TME) composition between matched primary and relapse samples, and to build and validate a prognostic model for post-ASCT outcomes using relapse samples. Materials & Methods:NanoString digital gene expression profiling was used to ascertain the gene expression of 784 genes of interest from 245 biopsies sampled from 174 CHL patients. This cohort included 90 patients with single biopsies performed at first diagnosis (primary), 13 patients with single biopsies taken at relapse, and 71 patients with paired biopsies taken at first diagnosis and at relapse. All patients received ABVD as first-line treatment, and 151 patients went on to receive ASCT. The 784 genes of interest were selected based on previously reported associations with outcome in CHL and/or components of the TME. Spearman statistics were used for pairwise correlations and log-rank tests were used to assess survival differences. Bootstrap aggregation with concordance statistics (C-stat) was used to calculate the post-ASCT prognostic properties and a two-sample t-test was used to compare primary and relapse samples. Penalized elastic-net multivariate cox regression was used for model construction. An independent, similarly treated, cohort of 31 relapse biopsies was used for model validation. Results: Comparative gene expression analysis revealed that 17 of the 71 patients (24%) exhibited poor correlations between their paired primary and relapse samples (r2 < 0.75) - indicative of significant differences in their TME compositions. Amongst these differences was a striking inverse correlation between macrophage and B-cell gene expression pattern changes (r2 = -0.809). We validated these findings by using CD20 and CD163 immunohistochemistry confirming this inverse correlation of relative changes in macrophage and B cell content in the TME (r2= -0.645). Patients who exhibited poor gene expression correlations had inferior post-ASCT failure-free survival (FFS) (3-year: 38.5%) compared to patients with high gene expression correlations (3-year: 77%; P = 0.005). A comparative C-stat analysis of prognostic properties between primary and relapse samples demonstrated that relapse samples contain superior prognostic features for prediction of post-ASCT outcomes (relapse C-stat 0.785 ± 0.073 vs. primary C-stat 0.594 ± 0.079, P < 0.001). To this end, we developed a gene expression prognostic model using penalized Cox regression that was based on gene expression measurements in relapse samples (RHL30). RHL30 was able to risk stratify patients according to post-ASCT outcomes in the training cohort (5-year post-ASCT FFS: 23.8% in high-risk vs. 77.5% in low-risk; 5-year post-ASCT overall survival [OS]: 30.9% in high-risk vs. 85.4% in low-risk). This was validated in an independent cohort of 31 patients with relapsed CHL with a 5-year post-ASCT FFS of 37.5% in the high-risk vs. 70.1% in the low-risk groups (P = 0.017) and 5-year post-ASCT OS of 37.5% in the high-risk vs. 71.6% in the low-risk groups (P = 0.006). Conclusions: The TME gene expression profile differs significantly between matched primary and relapse CHL samples in a subset of patients with relapsed CHL. Gene expression measurements derived from relapse samples contain superior predictive properties for response to ASCT. To this end, we have developed a novel clinically applicable prognostic model (RHL30), derived from relapse samples, that identifies patients who have a high likelihood to benefit from ASCT (low-risk), and conversely a subgroup of patients who may benefit from additional or alternative therapeutic approaches (high-risk). Disclosures Connors: Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; Millennium Takeda: Research Funding; NanoString Technologies: Research Funding; F Hoffmann-La Roche: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Introduction: Follicular lymphoma (FL) remains a significant clinical burden as it is an incurable disease and most patients will eventually suffer from disease progression. Two clinical events are associated with poor outcomes for patients with FL: (1) histological transformation (TFL) of their original FL into a high-grade, aggressive lymphoma subtype (2-3% of patients per year) and (2) early disease progression (PFL) where patients experience treatment failure within 2 years of receiving therapy (20% of patients). Despite recent high-throughput sequencing studies, the nature of tumor clonal dynamics leading to TFL or PFL is poorly understood and it is unknown if similar, or contrasting, modes of selection underpin these FL clinical events. Materials & Methods:We assembled a study cohort consisting of 21 patients: 15 experiencing TFL and 6 PFL. For each TFL and PFL patient, we obtained primary biopsies (T1; taken at the time of the initial FL diagnosis), biopsies at transformation/progression (T2) and matched normal samples. We performed whole genome sequencing on each specimen and identified single point mutations and copy number alterations using MutationSeq and TITAN, respectively. We compared T1 to T2 somatic mutation profiles and identified mutations associated with extinction of T1 clones and expansion of T2 clones. To validate these patterns, we selected 192 positions from each patient for deep-targeted sequencing validation (~10733X) in their T1, T2, and normal samples. We applied a statistical model (PyClone) to estimate cancer cell fraction (CCF) of each validated mutation. These CCF estimates were used to construct clonal phylogenies (Citup) and infer clonal dynamic patterns during their evolutionary histories. The Wright-Fisher model of genetic drift was used to model tumor evolution. Results: Temporal analysis of mutational burden revealed that mutational burden was significantly higher in T2 (8162 mutations ± 2146) than in T1 (6373 ± 2630) tumors for both TFL and PFL patients (Wilcox P < 0.001). This was independent of time interval between sampling (Spearman R2 = 0.029, P = 0.456). Mutation variant allelic fraction (VAF) distributions revealed that all distributions showed evidence of shared clonal ancestry between T1 and T2 tumors accompanied by substantial numbers of T1 and T2-specific mutations. We selected ≥ 192 mutations per patient from these distributions and performed deep-targeted amplicon sequencing, validating 96.3% of mutations and acquiring precise VAFs to infer clonal dynamics. In 13 of 15 TFL patients (87%), we observed dramatic clonal dynamics, characteristic of T2 tumors dominated by clones (or phylogenetic lineages) that were absent or extremely rare in T1 tumors (< 1% CCF). Digital droplet PCR was used to confirm the existence of both scenarios (confirming a clone as rare as 2 out of approximately 105 cells). Tumor evolution modeling demonstrated that this mode of evolution was driven through positive selection for mutations that confer fitness advantages and not by genetic drift. In contrast, PFL patients exhibited markedly different patterns of clonal dynamics compared to TFL patients. 4 of 6 PFL patients (67%) harbored readily detectable clones at T1, which expanded to full clonal prevalence during treatment with immuno-chemotherapy. Tumor evolution modeling demonstrated that this mode of evolution could be explained under neutral evolutionary dynamics (drift). Conclusions: We have shown that histological transformation and early progression manifest through divergent modes of tumor evolution. As the transformation phenotype may arise after diagnosis, more frequent monitoring of these patients will be required to determine the exact timing of the evolutionary inflection point that elicits transformation. In comparison, prediction of early treatment resistance should be achievable through comprehensive characterization of the genetic and clonal composition at diagnosis; this would ultimately identify patients who may benefit from upfront alternative therapies without the need to first endure predictable early treatment failure. Disclosures Sehn: roche/genentech: Consultancy, Honoraria; amgen: Consultancy, Honoraria; seattle genetics: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; celgene: Consultancy, Honoraria; lundbeck: Consultancy, Honoraria; janssen: Consultancy, Honoraria. Connors:Millennium Takeda: Research Funding; Seattle Genetics: Research Funding; F Hoffmann-La Roche: Research Funding; Bristol Myers Squib: Research Funding; NanoString Technologies: Research Funding. Scott:Janssen: Consultancy; Celgene: Consultancy; Roche: Honoraria; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies.

Introduction : Follicular lymphoma (FL) remains an incurable malignancy as patients eventually experience progressive disease. A subset of patients is at risk of early lymphoma-related mortality due to histological transformation (TFL) to aggressive lymphoma (2-3% of patients per year) or early progression after immuno-chemotherapy, each of which leads to shortened survival. Mutations associated with transformation and/or early progression have been reported in the literature (e.g. CDKN2A, TP53, B2M) and the m7-FLIPI clinico-genetic risk model defines a high-risk patient group with poor failure-free survival after first-line treatment. However, the current knowledge of genetic alterations underlying transformation and/or early progression is inadequate to explain the majority of transformed cases and accurately predict early progressive disease. Materials and Methods: We performed targeted capture-based sequencing of 94 genes in a large cohort of fresh-frozen and formalin-fixed and paraffin-embedded patient specimens (402 samples in total). This cohort comprised 277 samples from 159 patients that experienced transformation (including 128 samples at T1 (the time of FL) and 149 samples at T2 (the time of transformation) with 118 paired biopsies) and 125 samples from 125 patients (pre-treatment samples only) presenting with either early progression within 2.5 years after starting immunochemotherapy (n=41) or late or never progression for at least 5 years after starting immunochemotherapy or observation (n=84). Mutations were called using MutationSeq and putative single nucleotide polymorphisms were filtered out using either matching germline samples (n=84) or dbSNP (v147). Bayesian proportion tests were used to compare the prevalence of gene mutations between groups. Results : We first compared T1 (n=128) and T2 (n=149) samples from 159 patients experiencing transformation. Eleven genes were more commonly altered in transformed lymphoma. These included genes that had previously been associated with transformation, such as TP53, B2M, MYC and EBF1, as well as genes that have not yet been implicated as contributing to this process, for example EZH2, CCND3, PIM1 and ITPKB. Moreover, mutations in GNA13, S1PR2 and P2RY8, that have been implicated in dissemination of germinal centre B cells, were enriched in T2 samples. In addition, cell-of-origin classification was available for 108 of the TFL cases with DLBCL histology, 18 and 90 of which were of the ABC and GCB subtype, respectively. Although the number of ABC-TFL cases was small, we observed higher percentages of BCL10 (16% versus 1%, Fisher P=0.004), CD79B (22% versus 4%, Fisher P=0.005) and MYD88 mutations (28% versus 9%, P=0.006) in ABC-TFL than in GCB-TFL, suggesting that B-cell receptor signaling and NF-κB signaling are important contributors to the ABC phenotype in TFL. Next, we assessed the association of gene mutations with patient outcomes contrasting early progressers (n=41) and late or non-progressers (n=84). Eleven genes were mutated more commonly in early progressers than in late progressers, including KMT2C, TP53, BTG1, MKI67, XBP1 and SOCS1. Overall, 32 out of 41 early progressers (78%) had mutations in at least one of the 11 early progression-associated genes, but none of the individual early progression-associated genes were mutated at a frequency > 22%. Thus, early progression appears to be related to relatively infrequent genetic alterations. None of the early progression-associated gene mutations form part of the m7-FLIPI outcome predictor. Furthermore, in our cohort that was enriched for clinical extremes, the m7-FLIPI was similarly associated with early progression when compared to the FLIPI but it was not superior, having higher specificity (89% versus 76%), but lower sensitivity (36% versus 63%). Conclusions: We found novel associations of gene mutations with transformation and showed that TFL is molecularly heterogeneous, with the ABC subtype being characterized by differential gene mutation when compared to the GCB subtype. With regards to progressive disease after immuno-chemotherapy, our approach identified early progression as a distinct clinico-genetic disease category that is imperfectly captured by traditional prognostic tools. Disclosures Connors: Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Millennium Takeda: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.

In classical Hodgkin lymphoma (CHL), 20%-30% of patients experience relapse or progressive disease after initial treatment. The pathogenesis and biology of treatment failure are still poorly understood, in part because the molecular phenotype of the rare malignant Hodgkin Reed-Sternberg (HRS) cells is difficult to study. Here we examined microdissected HRS cells from 29 CHL patients and 5 CHL-derived cell lines by gene expression profiling. We found significant overlap of HL-specific gene expression in primary HRS cells and HL cell lines, but also differences, including surface receptor signaling pathways. Using integrative analysis tools, we identified target genes with expression levels that significantly correlated with genomic copy-number changes in primary HRS cells. Furthermore, we found a macrophage-like signature in HRS cells that significantly correlated with treatment failure. CSF1R is a representative of this signature, and its expression was significantly associated with progression-free and overall survival in an independent set of 132 patients assessed by mRNA in situ hybridization. A combined score of CSF1R in situ hybridization and CD68 immunohistochemistry was an independent predictor for progression-free survival in multivariate analysis. In summary, our data reveal novel insights into the pathobiology of treatment failure and suggest CSF1R as a drug target of at-risk CHL.

Background: MYC and BCL2 are critical driver genes for non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). However, the clinical impact of MYC and BCL2 genetic alterations, apart from translocations, has not been comprehensively investigated using high-resolution genetic assays, such as next generation sequencing and high-resolution SNP arrays. Moreover, correlations with cell of origin (COO) subtype, determined by gene expression profiling, have not been widely studied in a large homogeneously treated cohort. We determined the frequency and clinical impact of MYC and BCL2 genetic aberrations in DLBCL in a large population-based cohort uniformly treated with R-CHOP. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were treated with R-CHOP in BC. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of MYC and BCL2 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Dual positivity for MYC (cut off; 40%) and BCL2 (cut off; 50%) proteins identified a dual protein expresser (DPE) phenotype. COO classification was achieved using the Lymph2Cx assay based on NanoString technology in 299 patients and the Hans algorithm in 32 cases with low tumor content ( Results: COO determination revealed 193 cases to be GCB subtype, 107 cases ABC/non-GCB and 30 were unclassifiable. Using next generation sequencing, 310 SNVs were detected in MYC (29/347; 8% of cases) and BCL2 (88/347; 25% of cases), with mean redundant coverage depths of 633-fold. All MYC and 98% of BCL2 mutations were misssense mutations. Analysis of copy number alterations using GISTIC 2.0 revealed significant focal gain/amplification (gain/amp) peaks affecting 8q24, including the MYC locus (68/341; 20% of cases) and 18q21, including the BCL2 locus (82/341; 24% of cases). MYC and BCL2 translocations were detected in 38/283 (13%) and 90/300 (30%) of tumors, respectively. MYC gain/amp, BCL2 mutation, BCL2 translocations and double MYC/BCL2 translocation (DHIT) were seen significantly more often in GCB-DLBCL (26% vs 10%, p=0.001; 35% vs 11%, p With a median follow up of 6.5 years for living patients, the presence of MYC translocation, BCL2 gain/amp and BCL2 mutation were associated with an inferior 5y-time to progression (TTP, 53% vs 30%, p=0.019; 60% vs 32%, p=0.009 and 52% vs 17%, p=0.026, respectively) in ABC subtype. In GCB subtype, BCL2 translocation, MYC gain/amp and MYC translocation were associated with an inferior 5y-TTP (85% vs 62%, p=0.001; 80% vs 63%, p=0.011; and 79% vs 61%, p=0.013, respectively). In a multivariate Cox model of TTP including IPI and DPE, BCL2 gain/amp remained prognostic (HR=2.4 [1.3-4.5], p=0.008) independent of IPI and DPE in ABC-DLBCL. In GCB-DLBCL, BCL2 translocation and/or MYC gain/amp showed strong prognostic value (HR=3.0 [1.4-6.4], p=0.006) independent of IPI and DPE. In the IPI high risk group (IPI=3-5), the presence of a BCL2 translocation and/or MYC gain/amp defined a remarkably poor outcome group in GCB-DLBCL (5y-TTP; 29%). Similar poor outcome was observed in ABC-DLBCL cases that harbored BCL2 gain/amp (5y-TTP; 22%). Conclusions: The DHIT genotype was only seen in GCB-DLBCL. High-resolution genomic assays identified extremely poor prognostic groups within each COO subtype on the basis of MYC and BCL2 genetic status in a large uniformly R-CHOP-treated population-based cohort of DLBCL. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T-cell progenitors, characterized by activating NOTCH1 mutations in over 50% of children and adult cases. Although intensive multiagent chemotherapy achieves cure in most pediatric patients, the majority of adults succumb quickly to their disease. The basis for this divergence is likely multifactorial, but we sought in this study to investigate whether cell intrinsic features might contribute to the disparate biologies in pediatric and adult patients. In our prior abstract, we modeled pediatric and adult leukemias by transduction of hematopoietic stem/progenitor cells (HSPC) derived from mouse fetal liver (FL) and adult bone marrow (ABM) with activated NOTCH1 virus followed by transplantation into histocompatible recipient animals. We observed that whereas FL- and ABM-derived HSPC generate similar primary acute T-cell leukemias in terms of penetrance, latency, disease burden/distribution, and immunophenotype, FL leukemias exhibit much greater cycling activity than ABM leukemias, yet are dramatically impaired in their ability to propagate disease in secondary and tertiary recipients compared to ABM leukemias. Using a combination of gene expression profiling and in vitro culture assays, we attributed this differential behavior to NOTCH1-induced autocrine IGF signaling that is operative in FL, but not ABM-derived HSPC. Here we report that NOTCH1 mediates its effects on IGF1 in FL-derived HSPC directly by physical occupancy over the IGF1 promoter in a dimerization-dependent fashion. As well, increased NOTCH1 occupancy at the IGF1 promoter region in FL tissues is associated with reduced histone H3K27 trimethylation (a mark of transcriptionally silent chromatin), yet there is equivalent histone H3K4 trimethylation (a mark identifying transcriptionally active promoters) in both FL and ABM tissues, suggesting that NOTCH1 may be responsible for interconverting the IGF1 locus between active and inactive, but poised chromatin states. NOTCH1 occupancy is also associated with enhanced physical interactions between the IGF1 promoter region and distant genomic loci as revealed by circularized chromosome conformation capture (4C) assay and confirmed by chromosome conformation capture (3C) assay, including sites with H3K4 monomethylation (a mark of transcriptional enhancers) suggesting that NOTCH1 promotes "looping in" of distant enhancer elements that drive IGF1 expression in FL tissues. We conclude from these studies that NOTCH1 enacts differential, developmental stage-specific transcriptional programs by a combination of local epigenetic patterning and long-range genomic interactions. These findings support the notion that pediatric and adult T-ALL may potentially be regarded as related, but biologically distinct diseases, and that novel, age-specific therapies that exploit these differences may improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

Background: Diffuse large B cell lymphoma (DLBCL) is divided into two distinct molecular subtypes, germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. Genetic landscape studies of DLBCL have revealed several GCB-DLBCL specific mutations, including CREBBP, GNA13, EZH2, TNFRSF14, BCL2 and MEF2B. Functional studies have recently shown that the inactivation of Gα13 signaling pathway genes, including GNA13, together with BCL2 over-expression, allows GC B-cells to escape the germinal center niche and widely disseminate. Although these findings revealed a critical role of genetic alterations of Gα13 signaling pathway in GC-driven mouse models of lymphomagenesis, clinical correlation is lacking. Here we analyzed the clinical impact of genetic alterations of Gα13 signaling pathway in a large population-based DLBCL cohort. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of GNA13, P2RY8, ARHGEF1, S1PR2 and RHOA was performed using a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Cell-of-origin classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014; 123) in 299 patients and the Hans algorithm (Hans, Blood 2004; 103) in 32 cases with low tumor content ( Results: Using next generation sequencing, 225 SNVs and 5 Indels were detected in GNA13 (16%), P2RY8 (18%), ARHGEF1 (6%), S1PR2 (3%) and RHOA (6%). SNP 6.0 microarrays revealed heterozygous deletions in GNA13 (2%), ARHGEF1 (1%), S1PR2 (4%) and RHOA (8%), but homozygous deletion was not found in any of these five loci. GNA13, P2RY8 and ARHGEF1 mutations were significantly more frequent in the GCB subtype than ABC subtype (26% vs. 6%; p In the cases with mutations of any of the five Gα13 signaling pathway genes, BCL2 over-expression (cut off; 50%) and translocation was associated with increasing stage (p=.018 and p=.005, respectively), but not in wt cases (p=.53 and p=.63, respectively). Specifically, in the cases with GNA13 and P2RY8 mutations individually, BCL2 over-expression was associated with advanced stage (stage III/IV, p=.018 and p=.037, respectively), but not in wild type (wt) cases. Importantly, BCL2 over-expression in the cases harboring Gα13 pathway mutations was not significantly associated with other poor risk features, including any other IPI factors or bone marrow involvement, indicating that genetic alterations in Gα13 signaling pathway accompanied by BCL2 over-expression might promote lymphoma dissemination into lymph nodes but not extranodal sites. With a median follow up of 6.5 years for living patients, there was no prognostic impact of harboring any isolated Gα13 pathway mutation in GCB-DLBCL patients. However, in cases with any Gα13 pathway mutations, BCL2 over-expression was significantly associated with an inferior 5y-time to progression (TTP; 90% vs 62%, p=.003) and disease-specific survival (DSS; 90% vs 71%, p=.042), but not in wt cases (Fig 1). In a Cox model of TTP including the IPI, BCL2 over-expression remained prognostic in the cases harboring any Gα13 pathway mutations (HR=4.13 [1.42-12.01], p=.009), but not in wt cases (HR=1.70 [0.62-4.68], p=.31). In cases with any Gα13 pathway alterations including copy number loss, BCL2 over-expression was also significantly associated with an inferior TTP (HR=3.64 [1.39-9.57], p=.009) independent of IPI, but not in the cases without genetic alterations (HR=1.75 [0.57-5.34], p=.33). Conclusions: Genetic alterations in Gα13 signaling pathway genes cooperate with BCL2 over-expression to promotes lymphoma dissemination to nodal sites and is associated with the poor outcome in GCB-DLBCL Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Introduction: Hodgkin lymphoma (HL) is a common malignancy of children and adolescents and is highly curable with a 5-year overall survival (OS) rate of > 97%, yet dose-intensified chemotherapy regimens in combination with radiation therapy come with a high cost in form of long-term toxicity and morbidity (Castellino et al., Blood 2011). This major clinical challenge has resulted in the evaluation of risk-adapted treatment regimens in clinical trials aiming to achieve the optimal equilibrium between high survival rates and prevention of treatment-related toxicity. However, risk stratification is currently limited to the use of clinical factors as there are no validated integral biomarkers that can be employed to either improve risk stratification or as surrogate markers of treatment outcome in pediatric HL. The aim of our study was to perform gene expression profiling (GEP) to uncover disease biology underlying treatment response and develop a prognostic model to tailor first-line therapy in pediatric HL. Methods: We selected 203 formalin-fixed, paraffin-embedded tissue (FFPET) specimens from patients enrolled in a randomized phase 3 clinical trial (AHOD0031) of the Children's Oncology Group (COG) based on the availability of archived FFPET blocks. That trial was designed to assess the value of early chemotherapy response for tailoring subsequent therapy in intermediate-risk pediatric HL. We performed GEP on RNA extracted from pre-treatment FFPET biopsies using NanoString technology and a customized codeset encompassing probes for 784 genes. These genes were either previously reported to be associated with prognosis and outcome in HL or represent the cellular diversity of the tumor microenvironment. Event free survival (EFS) and OS were estimated using the Kaplan-Meier method. Gene expression data were used to develop a predictive model for EFS using penalized Cox regression with parameters trained using leave-one-out cross-validation. Results: Of the 203 tissue samples obtained from the Biopathology Center at the Cooperative Human Tissue Network, 182 (89.7%) passed quality assurance testing, similar to the pass rate achieved for adult HL samples obtained from the Eastern Cooperative Oncology Group trial E2496 (Scott et al., JCO 2013). We applied our previously published 23-gene predictor for OS (Scott et al., JCO 2013), developed using biopsies from adult HL patients to our pediatric cohort. After calibrating the new codeset, 53 patients were classified as "high-risk" and 129 as "low-risk". Importantly, the model failed to predict inferior outcomes in the "high-risk" group (5-year OS 100% vs 95%, log-rank P = 0.125; 5-year EFS 82% vs 70%, log-rank P = 0.159), with patients in the "high risk" group trending to have superior outcomes than the "low risk" patients. Moreover, only 2 genes from this model, IFNG and CXCL11, were significantly associated with EFS in univariate Cox regression analysis (P = 0.003 and 0.048, respectively) but with inverse hazard ratios in the pediatric group compared to adult patients. Therefore, we sought to develop a novel EFS predictive model for pediatric patients treated in the AHOD0031 trial. Using univariate Cox regression we identified 79 genes significantly associated with EFS (raw P < 0.05). Using the expression of these 79 genes as the input to penalized Cox regression, we developed a 16-gene model to predict EFS in our training cohort. Using an optimized cut-off for the model score, 31% of patients were designated high-risk and had significantly inferior post-treatment outcome (5-year EFS 38% vs 89%, log-rank P < 0.0001). When multivariate analyses were performed including our EFS-model score, disease stage and initial treatment response as variables, only the model score was significantly associated with EFS (P < 0.0001, HR 11.3 (95% CI 5.5-23.4)). Conclusions: Failure of the GEP-based model developed in adult HL suggests distinct biology underlies treatment failure in the pediatric age group. We describe the development of a novel predictive model for EFS in intermediate-risk pediatric HL patients that will be validated in an independent cohort of patients treated in the AHOD0031 trial. Successful validation of the model may provide a clinically relevant biomarker for pediatric and adolescent HL patients allowing refinement of risk stratification and the combination of molecular and clinical risk factors at diagnosis. Disclosures Scott: Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Background: Although R-CHOP has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL), 40% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Recent studies using next-generation sequencing strategies have described the landscape of recurrent mutations in DLBCL. However, with the exception of TP53 and FOXO1, little is known about the clinical relevance of recurrent mutations and importantly, the interactions of these genetic alterations in DLBCL. Moreover, an integrated analysis of copy number alterations and recurrent mutations annotated across cell-of-origin (COO) distinctions for a large cohort of DLBCL cases who have received uniform therapy is lacking. The present study examined the frequency and clinical impact of recurrent genetic aberrations of DLBCL using high-resolution technologies in a large population-based DLBCL cohort. Methods: We analyzed 348 newly diagnosed DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency (Vancouver) with available DNA extracted from fresh frozen biopsy material (tumor content >30%). Matched germ line DNA was available for 67 patients. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Targeted re-sequencing of the coding exons of 56 genes was performed using a Truseq Custom Amplicon assay. Gene selection was based on mutational frequencies that have been previously described in DLBCL mutational landscape publications. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Tissue microarrays were constructed using duplicate 0.6mm cores from 332 cases, and breakapart FISH assays for MYC, BCL2 and BCL6 and IHC staining for MYC, BCL2 and cell of origin proteins were performed. COO classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014;123) in 299 patients as well as Hans algorithm (Hans, Blood 2004;103) in 32 cases with low tumor content. 194 cases were assigned to GCB subtype, 107 cases, ABC/non-GCB and 30 were unclassifiable. Results: In the mutation analysis, we identified 2,757 SNVs and 245 small indels. The mean depth of coverage was 634. Recurrent mutation frequencies varied between 0 and 58, with a mean of 8.25 per case. 98% of cases harbored at least one mutation and 95% of cases multiple mutations. 10 mutated genes were detected significantly more frequently in the GCB subtype including CREBBP, GNA13, EZH2, TNFRSF14, IRF8,STAT3, BCL2, SGK1, MEF2B and CD83, and 4 mutated genes, MYD88, CD79B, PRDM1 and PIM1, in the ABC subtype. In the copy number analysis, 45 significant amplification peaks and 57 deletion peaks were revealed by the GISTIC algorithm. As previously reported, 9p21.3, including CDKN2A,were more frequently detected in the ABC subtypes. With a median follow up of 6.5 years for living patients, the 5 y disease specific survival (DSS) and time to progression (TTP) of all patients were 72% and 64%, respectively. The clinical cohort was representative of registry data from BC based on a comparison of patient characteristics and survival outcomes with 1,194 control DLBCL R-CHOP patients. The ABC subtype was significantly associated with an inferior DSS and TTP (both p Conclusions: Our approach using next generation sequencing and high resolution SNP array provides an accurate estimation of frequency and clinical significance of recurrent genetic alterations of DLBCL in a uniformly R-CHOP-treated large population-based cohort of patients. Disclosures No relevant conflicts of interest to declare.

Introduction : Classical Hodgkin lymphoma (cHL) is the most common lymphoma affecting individuals under the age of 30. Despite improvements in standard treatment, 25-30% of patients still relapse after first-line treatment with ABVD. Relapsed patients are then typically treated by high dose chemotherapy and autologous stem cell transplantation (HDC/aSCT). However, this salvage therapy only cures approximately 50% of relapsed patients, and the mechanisms of second-line treatment failure are unclear. Moreover, currently no reliable biological markers are available to predict the outcome of HDC/aSCT at the time of relapse. The specific aim of this study is to compare diagnostic and relapse biopsies along with integrating clinical outcome data to uncover biology associated with resistance to first-line therapy, and identify prognostic markers for HDC/aSCT. Materials & Methods: NanoString Technologies digital gene expression profiling was used to ascertain the gene expression of 785 genes in formalin-fixed paraffin embedded tissue of 245 biopsies sampled from 174 cHL patients. This cohort included 90 patients with single biopsies performed at first diagnosis, 13 patients with single biopsies taken at relapse, and 71 patients with paired biopsies taken at both first diagnosis and at relapse. All 174 patients relapsed following uniform first line treatment with ABVD. Of these, 151 patients went on to receive salvage treatment that included HDC/aSCT. The 785 genes reflect biological signatures representative of cell types typically found in the microenvironment of cHL, and those previously reported to be associated with outcome in cHL. A tissue microarray was constructed containing all biopsies for immunohistochemistry (IHC) using CD68 antibodies (clone KP1). Spearman correlation tests were used for comparative gene expression analyses and IHC correlations. Differential expression analyses were performed using a non-parametric Wilcoxon test. Gaussian mixture models were used to cluster patients into poorly and well-correlated biopsy pairs. Outcome correlations were performed using the log-rank test and Cox regression analysis. Results : 24% of patients had a histological subtype transition between diagnostic and relapse biopsies with the most common transition (46%) from mixed cellularity (at diagnosis) to nodular sclerosis (at relapse). Comparative gene expression analysis revealed that 17 of the 71 patients (24%) had poorly correlated biopsy pairs (R2 < 0.75). Specifically, genes associated with macrophage differentiation (e.g. CD68, MARCO; FDR < 0.1) were found to be more highly expressed in the relapse biopsies compared to the matching samples at first diagnosis. CD68 IHC staining was well correlated with mRNA expression (p < 0.001) and confirmed that CD68+ cells were significantly more frequent in the relapse biopsies (p = 0.023). Patients with poorly correlated biopsy pairs had an inferior post-HDC/aSCT failure-free survival (5-y FFS: poorly correlated 39% vs. well correlated 68%; log-rank p = 0.005). Additionally, we applied our previously published 23-gene predictor (Scott, JCO, 2013), that was originally developed for pre-treatment biopsies, to the relapse biopsies. Using the published thresholds, 19% of patients were designated high-risk and had significantly inferior outcomes post-HDC/aSCT (5 year post-HDC/aSCT overall survival 40% vs. 82%, log-rank p = 0.001; 5 year post-HDC/aSCT failure-free survival 39% vs. 72%, log-rank p = 0.004). Conclusions : Our comparative analysis of cHL pre-treatment and relapse biopsies reveals differences at both the histopathological and molecular levels. We have identified novel factors, such as a poor correlation between paired biopsies and the 23-gene predictor, that are predictive of an inferior post-HDC/aSCT outcome. These findings suggest that re-biopsying patients at relapse will yield important biological insight and superior predictive power for second-line treatment failure. Disclosures Gerrie: F Hoffmann-La Roche: Other. Savage:F Hoffmann-La Roche: Other.

Introduction: Constitutive MHC class II expression is a hallmark of antigen-presenting cells, including B cells, and is indispensable for the initiation of antigen specific immune responses. It has been shown that certain B cell lymphoma entities are able to evade immune recognition by downregulation of MHC molecules on the tumor cell surface. We have previously identified recurrent chromosomal rearrangements of CIITA, the master regulator of MHC class II transcription, as one possible mechanism to reduce MHC class II expression in primary mediastinal large B-cell lymphoma (PMBCL) and classical Hodgkin lymphoma (cHL) (Steidl et al., Nature 2011). Furthermore, we have recently described a 1.6kb breakpoint cluster region within intron 1 of CIITA and have shown in a small sample set of PMBCL cases that deletions, insertions and single nucleotide variants (SNV) are commonly found within this genomic region (Steidl, ASH abstract # 437, 2011). Therefore, we aimed to explore the frequency of these alterations and the correlation with CIITA and MHC class II protein expression in a larger cohort of PMBCL cases and to further characterize their functional significance. Methods: We have comprehensively analyzed 45 diagnostic PMBCL samples for the presence of coding sequence mutations as well as alterations within the promoter III region and the first 3kb of intron 1 using deep amplicon sequencing (Illumina TruSeq) and/or Sanger sequencing. In addition, we characterized the PMBCL-derived cell lines U2940 and Med-B1 by whole transcriptome paired-end sequencing (RNA-seq). To elucidate the functional consequences of the coding sequence mutations identified in these two cell lines we performed retroviral transductions of wild type CIITA and CIITA mutants in a CIITA and HLA-DR expression-negative cell line (DEV, nodular lymphocyte predominant Hodgkin lymphoma-derived). We subsequently analyzed CIITA mRNA expression using qRT-PCR and HLA-DR surface expression using flow cytometry. Furthermore, we applied immunohistochemistry (IHC) to determine expression levels of CIITA and HLA-DR in a large cohort of PMBCL samples represented on two tissue microarrays (TMA, n=149). The TMAs were also used for fluorescence in-situ hybridization (FISH) to evaluate the presence of copy number alterations or translocations of the CIITA locus. Results: FISH was interpretable in 115 samples with a CIITA break-apart (CIITA-ba) frequency of 33.9% (39/115). Correlative analyses revealed that decreased CIITA protein expression by IHC was significantly correlated with the presence of CIITA-ba (P=0.019), whereas HLA-DR expression was not correlated with CIITA-ba status alone (P=0.219). However, we could demonstrate a positive correlation between protein expression of CIITA and HLA-DR (Pearson r=0.45, P Using RNA-seq, we have detected two missense mutations in the Med-B1 cell line affecting both alleles in functionally relevant protein domains. Furthermore, we identified a novel NUBP1-CIITA fusion transcript in U2940 also harboring an SNV on the other allele resulting in the transcription of an elongated protein due to the loss of the original stop codon. Ectopic expression of these CIITA mutants in DEV, which has been shown to have undetectable levels of CIITA and HLA-DR due to a biallelic CIITA inactivation, revealed that these individual SNVs showed a diminished capability to restore HLA-DR surface expression in comparison to wild type CIITA as measured by flow cytometry. Conclusions: Here we show that the presence of CIITA rearrangements is significantly associated with low CIITA protein levels, and we could demonstrate that protein expression of CIITA and HLA-DR are positively correlated in PMBCL. Furthermore, CIITA is frequently targeted by coding sequence mutations and intronic deletions in PMBCL cell lines and clinical samples. Functional studies demonstrate that genomic alterations in CIITA contribute to downregulation of MHC class II expression in malignant lymphomas and therefore represent a potent mechanism of acquired immune privilege and escape from immune surveillance. Disclosures No relevant conflicts of interest to declare.

Background Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype accounting for ∼40% of all non-Hodgkin lymphomas. Although R-CHOP has significantly improved outcome in DLBCL, 35% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Immune cell signatures have been shown to impact survival in DLBCL (Lenz et al, NEJM 2008). Recurrent mutations and/or copy number alterations (CNAs) in genes that impact the tumor's immune-cell crosstalk have recently been described, but their clinical impact is unknown. Moreover, mutations and copy-number loss involving genes that foster immune escape has been implicated as a dominant oncogenic mechanism in DLBCL (Challa-Malladi et al, Can Cell 2011). We sought to determine the relationship between genetic alterations involving immune-cell crosstalk pathways and survival in a cohort of DLBCL patients treated with R-CHOP. Methods Following our initial analysis of 91 whole transcriptome sequencing (RNA-seq) including 40 with whole genome sequencing, we validated SNVs in the genes of interest by Sanger sequencing in the tumor and matched germline DNAs if available. RNA-seq-derived gene expression data were also used to determine the cell of origin (COO) distinction. Affymetrix SNP 6.0 microarrays were used to ascertain the CNAs in all 91 DLBCL samples. Clinical outcome correlations were analyzed by Cox regression and the log-rank test in the 82 patients treated with R-CHOP (median follow-up; 82 months). Results Using next-generation sequencing, we identified 97 SNVs in 16 genes (B2M, CD274, CD276, CD58, CD83, CIITA, NLRC4, NLRC5, RFXAP, RFXANK, TNFRSF14, TNFRSF25, TNFSF9, TAP1, TAP2 and TAPBP) that were selected based on an extensive literature review implicating these genes in immune-cell crosstalk pathways in DLBCL. Among them, eight genes were mutated in more than one case including B2M (7%), CD58 (7%), TNFRSF14 (7%), CIITA (4%), TNFSF9 (2%), NLRC5 (3%), CD83 (11%), and RFXAP (2%). These eight loci were further analyzed for CNAs, gene expression and outcome correlations. SNP 6.0 microarrays revealed either focal heterozygous or homozygous deletions in B2M (22%), CD58 (14%), TNFSF9 (10%), and heterozygous deletions in TNFRSF14 (12%), CIITA (3%), NLRC5 (7%), CD83 (2%), and RFXAP (7%). Amongst the 91 cases, 35 cases (38%) had no genetic aberration. 56 cases (62%) had at least one aberration, while 29 (32%) had multiple genetic aberrations indicating that genetic aberrations associated with immune-cell crosstalk are not mutually exclusive in DLBCL. Mutations and CNAs were not COO specific, with the exception of TNFRSF14, which was significantly restricted to the GCB-subtype (p=0.001). We evaluated outcome correlations for each genetic aberrations in the 82 cases with uniform therapy, including two recognized major histocompatibility class (MHC) groups; MHC class I (B2M and NLRC5) and MHC class II (CIITA, RFXAP and CD83). In univariate analysis, the presence of genetic aberrations in the MHCI group and NLRC5 individually were prognostic (5yr PFS, 59% vs 78%, p=0.025, 44% vs 77%, p=0.005, respectively), but not the MHCII group or other individual loci. In subgroup analyses according to COO, both MHCI and MHCII genetic aberrations were associated with inferior 5yr PFS in the ABC subtype (MHCI, 27% vs 79%, p Conclusions Recurrent genetic alterations involving genes related to immune-cell crosstalk in DLBCL are frequent (56%), co-occur and independently contribute to outcome in patients treated with R-CHOP. MHCI and MHCII perturbations are only prognostic in the ABC subtype. Disclosures: No relevant conflicts of interest to declare.

Introduction Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels. Methods To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency’s Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries. Results FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript. Conclusion Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise. Disclosures: No relevant conflicts of interest to declare.

Abstract 295 Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin lymphoma (NHL), accounting for approximately 30–40% of all new lymphoma cases. While standard therapy using rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) has significantly increased the survival of DLBCL patients, approximately one third of DLBCL patients still remain unresponsive to or relapse after standard treatment. Further investigation into the genomic architecture of DLBCL will contribute to elucidating the causes of the poor outcomes in this subgroup of patients. While the copy number and the gene expression profiles of DLBCL specimens have been well described as separate analyses, a large-scale high resolution integration of both orthologous measurements has yet to be reported. The integration of these two data types in a clinically well-annotated cohort of DLBCL is crucial as it can potentially distinguish driver from passenger genomic aberrations and reveal associations with clinical outcome. Methods and Patients: Affymetrix SNP 6.0 microarrays were used to ascertain the copy number profiles in 151 pretreatment biopsies of DLBCL that were representative of the population of DLBCL patients treated at the British Columbia Cancer Agency. Clinical outcome data were available for all 151 patients with 142 patients receiving R-CHOP or R-CHOP-like treatment. Matching RNA-seq libraries were used to quantitate the gene expression levels in 91 samples. The SNP 6.0 pre-processing method cRMAv2 was used to generate raw probe intensities that were then normalized to 1258 HapMap3 SNP 6.0 arrays. Copy number state calls were predicted using HMM-Dosage. RNA-seq data were aligned using the split-read aware aligner GSNAP and gene expression values were generated using the metric reads per kilobase of transcript per million mapped reads. DriverNet analyses were utilized to predict functionally relevant driver genes and outcome correlations in R-CHOP treated patients were performed using Cox regression and the log-rank test. Results: The copy number landscape derived from the SNP 6.0 microarrays revealed previously reported large scale chromosomal deletions in chromosome 6p and amplifications in chromosomes 3, 7 and 18. By integrating the gene expression with copy number data, we found that gene copy number was correlated with its own gene expression (classified as being cis-correlated) in 23.5% of genes. In addition, we investigated copy number aberrations which were highly correlated with gene expression across the genome (classified as trans-correlated). This analysis revealed aberration hotspots in genomic locations 3q26-q28 (TBL1XR1, BCL6, TP63), 17p12 (NCOR1, MAP2K4), 18q11.1-q11.2 (RBBP8) and 22q11.21 (BID, IL17RA) suggesting that these hotspots regulate important pathways that may contribute to the pathogenesis of DLBCL. We identified previously reported focal amplifications (e.g. REL) and deletions (e.g. B2M, CDKN2A). Moreover, we identified novel focal deletions, including homozygous deletions, in chromatin modifying genes: LCOR (7.9%), RCOR1 (9.9%), and NCOR1 (17.9%), all of which were cis-correlated and were validated using fluorescence in situ hybridization. DriverNet analyses identified RCOR1 deletions as one of the main driver alterations. RCOR1 deletions were also found to be associated with progression-free survival (5-year progression-free survival: deleted 40% vs. non-deleted 75%, p=0.0188). Discussion: Our systematic integration of SNP 6.0 and RNA-seq data confirmed findings of previous studies and also revealed novel genomic aberration hotspots and highly focal and frequent deletions in chromatin modifying genes. Results derived from our large-scale high resolution data set indicate the feasibility and efficacy of integrative genomic analyses in revealing novel and pathogenetically relevant genomic aberrations in lymphoid cancers. The discovery of the association of RCOR1 deletions with progression-free survival suggests that RCOR1 deletions could be used as a prognostic marker and might indicate a molecular phenotype that can be targeted by novel therapeutic agents in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 430 Introduction: 25–30% of patients with advanced stage classical Hodgkin lymphoma (cHL) develop progressive disease despite treatment with ABVD. While more intensive upfront treatments, such as BEACOPP, result in a higher failure free survival, this comes at the expense of increased toxicity and morbidity. 10–15% of patients with advanced stage cHL will ultimately succumb to the disease, with similar outcomes using different upfront regimens likely reflecting the planned use of second-line high dose therapy. The International Prognostic Factors Project Score, a prognostic tool developed using freedom from progression of disease as the outcome, has been used in clinical practice and has guided clinical trials. We hypothesized that biological factors, as measured by gene expression profiling in pretreatment formalin fixed paraffin embedded tissue (FFPET) samples, might provide a robust predictor of overall survival (OS). Method and Patients: NanoString technology was used to quantitate 261 mRNA species in diagnostic FFPET samples from patients in the Intergroup E2496 trial, which compares ABVD with Stanford V chemotherapy in locally advanced and advanced stage cHL. The 261 genes included individual genes and genes that represent cell and cellular process signatures that have been associated with outcome in cHL. Total RNA was extracted from whole section scrolls from the 309 FFPET blocks available. A penalized binary logistic regression model with leave-one-out cross validation was used to produce a parsimonious predictive model for OS. A threshold was selected that maximized the Chi square of the Log Rank (Mantel-Cox) test between the identified low and high risk groups. An independent cohort of patients with advanced stage cHL enriched for treatment failure, consisting of 59 patients from British Columbia treated with ABVD and 71 patients from Portugal treated with Stanford V, was used to test the generated model and threshold. Results: Gene expression of adequate quality was produced in 293 of the 309 samples (95%). At a median follow up of 5.3 years, 36 (12%) of the 293 patients had died. 51 genes were differentially expressed (t-test p < 0.05) with respect to OS, including genes that are part of macrophage, cytotoxic/NK cell and apoptosis signatures. A predictive model for OS was produced with an area under the curve (AUC) of the receiver operating characteristic (ROC) curve of 0.73. The predictor identified a high-risk group, comprising 39% of the cohort, with a 77% estimated 5-year OS compared with 96% in the low risk group (Figure 1A, log-rank p < 0.0001). The predictor was validated in the independent cohort giving an AUC of the ROC curve of 0.73 and an estimated 5-year OS of 71% in the high-risk group compared with 91% in the low risk group (Figure 1B, log-rank p = 0.006). Discussion: We have produced a parsimonious gene expression-based predictor of OS in cHL, developed in, and applicable to, widely available FFPET using NanoString technology. The predictor identifies, at diagnosis, a clinically meaningful proportion of patients at significantly higher risk of death when treated with ABVD or Stanford V. Further evaluation of the gene expression signature with alternative treatments, ideally in the context of clinical trials, is needed to understand the impact of more intensive cytotoxic therapies versus novel targeted approaches. Disclosures: Horning: Genentech: Employment, Equity Ownership. Connors:Seattle Genetics: Consultancy, Research Funding.