Evaluating the Clinical Utility of Real-Time Comprehensive Whole Exome Sequencing and RNA-Seq for Patients with Diffuse Large B-Cell Lymphoma

Gene expression profiling has shown that diffuse large B-cell lymphoma (DLBCL) clusters into three major subtypes based on similarity in expression patterns to their cell of origin (COO): germinal center B-cell-like (GCB-DLBCL), activated B-cell-like DLBCL (ABC-DBLCL) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes of DLBCLs are associated with distinctly different overall survival rates after standard immunochemotherapy. However, clinical and prognostic heterogeneity remains within COO subsets and strategies are needed to further stratify patients to identify and target high-risk subsets. A comprehensive genomic analysis of COO on a clinically defined set of DLBCL cases has not been performed and the aim of this study was to use whole-exome sequencing (WES) data from 58 paired tumor-normal DLBCL samples to assess association of known DLBCL genomic alterations with cell COO as well as for identification of novel and relevant genetic biomarkers.To investigate genomic alterations associated with DLBCL subsets, we analyzed WES and genome wide copy number data from 58 paired tumor-normal DLBCL tumors. Gene expression profiling or Hans classification was performed to determine DLBCL COO subtype; 31 patients were classified as GCB and 27 as non-GCB. The WES data were used to 1) assess the association of known DLBCL genomic alterations with COO, and 2) identify novel alternations associated with COO. Statistical analysis was performed and the data were ranked by significance (p≤ 0.05) within each DLBCL subtype. In total, 45 genomic abnormalities were analyzed for their association with either GCB, non-GCB or both. Mutations in CREBBP, EZH2, MEF2B, FOXO1 and REL have also been reported as GCB driver mutations and we observed GCB patients with these mutations, but the mutation clustering was not wholly associated with GCB. MYC double-hits were exclusively found in the GCB-subtype group. For the non-GCB cases we found that mutations in MAP2K3 and MYD88 were significantly associated with this subtype(p< 0.05). In addition to mutational patterns, we identified several copy number alterations (CNA) across both groups. Chromosomal losses in GCB patients were found at chromosomes 10q11.21-10q24.23, 4q12-4q35.2, 3q12.1-3q29, 4p12-4p16.3, 10p11.21-10p15.3, and 14q11.2-14q24.3 whereas gains were localized to 7q11.1-7q36.3, 7p11.2-7p22.3, and 1q21-1q32.1 (p < 0.05). No CNA was observed to directly associate with non-GCB patients, however, a loss at 9p21 and gains at 9q24.1 and 18q21.33 trended with the non-GCB subtype, supporting previous reports. Loss at 10q23.31 or a gain in 2p13-2p12 have been reported as being specific for GCB and our data confirmed the association of 10q23.31 with GCB while a gain at 2p13-2p12 (REL)was found in both subtypes. To further understand genomic differences between DLBCL subtypes, we evaluated the relative percentage of each genomic feature. 18/45 (40%) were only observed in GCB patients whereas 2/45 (5%) were specific to the non-GCB subtype. The majority (25/45, 55%) overlapped between the two subtypes.Throughout our analysis we noted that 7 non-GCB cases lacked any of the driver mutations analyzed in the study. While all cases carried mutations, they consisted of low frequency mutations that were not specifically associated with COO. 2/7 cases had a gain at 9p24.1 that included CD274 and JAK2. Because 9p24.1 gains have not been fully defined in DLBCL, we reviewed all cases and identified 4 (7%) with a 9p24.1 gain, 3 of which were non-GCB and 1 GCB. One non-GCB case was EBV+ and none of the cases showed evidence of PMBCL. Of the 9p24.1 cases, three had RNAseq data available and we found that PDL1 and JAK2 expression was elevated (12 fold p< 0.01 and 7 fold p< 0.01, respectively) when compared to the 9p24.1 normal cases (n=32). While outcome was not the focus of the study, we did note that 6/7 cases that lacked driver mutations achieved event free survival at 24 months (EFS24).Taken together, this analysis has further characterized the genetic profile of each COO subtype and has identified novel GCB CNAs which require independent replication. Additionally, we identify a subgroup of non-GCB DLBCL patients that do not harbor known driver mutations and require further genomic study to better resolve the biology of these tumors. Together, these data provide insight on the genetic heterogeneity of DLBCL and identify genetic variants that may inform subtype specific therapy. DisclosuresNowakowski:Morphosys: Research Funding; Celgene: Research Funding; Bayer: Consultancy, Research Funding. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

Whole-Genome Sequencing of Primary Central Nervous System Lymphoma and Diffuse Large B-Cell Lymphoma

Somatic Mutations in E3 Ubiquitin Ligase Deltex 1 Are Associated with Survival in Diffuse Large B-Cell Lymphoma

Relapsed diffuse large B-cell lymphoma is shaped by molecular subtypes and genomic alterations

Background: Lymphoma arises in lymph nodes. Infiltration of malignant lymphomatous cells in the organs other than lymph node is termed as extranodal lymphoma (ENL). Almost any organ in the body can get affected. The most frequently involved system is gastrointestinal tract, followed by lung, liver, spleen, bone, and skin. Primary central nervous system (CNS) lymphoma is also well documented. Origin of tumor from non-lymph-nodal tissue is termed as primary ENL, whereas hematogenous spread of disease from lymph nodes to extranodal tissue is secondary ENL. Extranodal involvement is more common in non-Hodgkin’s lymphoma (NHL) than Hodgkin’s disease. Aims and Objectives: The aim of the study was to study the prevalence of primary ENLs at unusual sites. Materials and Methods: A prospective study was conducted for a period of 3 years to study the prevalence of primary ENLs at rare sites. Results: In our study, the participants were having a mean age of 50 years and showed a bimodal distribution. A total 16 cases were male (59%) and rest were female (11 cases, 41%). We found that B-symptoms were present in 19 cases (70%) and absent in the remaining. Most of the cases were of B- cell type (20 cases, 74%) and rest were T-cell type (seven cases, 26%). The most common extranodal site being gastrointestinal tract (10 cases, 37%) followed by CNS (four cases, 15%), spleen (two cases, 7%), tonsil (two cases, 7%), and cutaneous site (two cases, 7%). Other less common sites included breast, paranasal sinuses, mediastinum, floor of mouth, nasopharynx, testis, and thyroid. NHL was the only variant present in all the cases. Diffuse large B-cell lymphoma (DLBCL) was the most common variant present in 11 cases (37%). The DLBCL was found to be aggressive in most of the cases with most of them being activated B-cell type, ABC (70%) and rest being Germinal center B-cell type, GCB (30%). Conclusion: A broad spectrum of extranodal organs is involved in various subtype of lymphoma. This study concluded that ENLs can occur at any location. The fact that a lymphoma is a primary ENL may be a factor in the treatment plan and prognosis. The lymphoma subtype B cell or T cell type, and the primary organ or tissue of origin can all be important prognostic factors.

Identification of Genes Frequently Mutated In FL and DLBCL with Transcriptome, Genome and Exome Sequencing

Targeting EGR1-Medited Metabolic Reprogramming to Overcome Ibrutinib Resistance in Aggressive B Cell Lymphomas

Cell of Origin and Genomic Profile of Primary Central Nervous System Lymphoma Determined Using the Nanostring Lst Assay and Ultra Deep Targeted Next Generation Sequencing

Akt activation confers an inferior survival in patients with activated B-cell subtype of diffuse large B-cell lymphoma: a report from The International DLBCL Rituximab-CHOP Consortium Program

Treatment with Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019) Results in Durable Remissions in Patients with Relapsed or Refractory Diffuse Large B Cell Lymphomas of Germinal Center and Non-Germinal Center Origin, "Double Hit" Diffuse Large B Cell Lymphomas, and Transformed Follicular to Diffuse Large B Cell Lymphomas

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

Our current understanding of the mutation spectrum of relapse/refractory patients is limited. Several published studies describing the mutational landscape of Diffuse Large B cell Lymphoma (DLBCL) have focused, by design, on diagnostic (pre-treatment) biopsies alone, while re-biopsy of patients who are refractory to first line therapy or who relapse on treatment is not standard of care in DLBCL. We have performed whole exome sequencing on 47 post treatment DLBCL core needle biopsies, 8 with matched diagnostic biopsies. Samples were obtained during a phase II trial prior to the start of treatment with the BCR targeted agent fostamatinib (1,2). Patients had progressed following therapy with an anthracycline-based regimen such as R-CHOP and had a median of 3 prior therapies (range 1-8). We compared the whole exome somatic variant and copy number calls from the post treatment and matched pairs to 2 cohorts of primary DLBCL (n = 112) analyzed with the same mutation caller (3,4). The average mutation rate between the pre and post treatment samples (n = 250, 282 respectively) and the paired diagnostic and post treatment biopsies were similar. However, we found that the mutation spectrum between the paired biopsies differed. Known DLBCL hotspot mutations such as MYD88 L265P remained consistent between pre and post treatment biopsies, however novel mutations in known DLBCL targets were found to emerge in the post treatment biopsies. 1. Veldman-Jones, M. et al. Reproducible, quantitative and flexible molecular sub-typing of clinical DLBCL samples using the NanoString nCounter system. Clin Cancer Res (2014) 2. Flinn I, B.N., Blum KA, et al. A Phase II Trial to Evaluate the Efficacy of Fostamatinib in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL). American Society of Hematology (San Francisco, 2014). 3. Zhang, J. et al. Genetic heterogeneity of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A 110, 1398-403 (2013). 4. Pasqualucci, L. et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 43, 830-7 (2011). Citation Format: Danielle Greenawalt, Kate Byth, Zhongwu Lai, Justin Johnson, Ambar Ahmed, Brian Dougherty, Kenneth Thress, Michael Zinda, Winnie S. Liang, John Carpten, Stephen Fawell, J. Carl Barrett. Whole exome sequencing of pre and post treatment diffuse large B cell lymphoma reveals the mutation spectrum of the relapse/refractory patient population. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1706. doi:10.1158/1538-7445.AM2015-1706

MicroRNA-Mediated Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B-Cell Lymphomas.

Expression of the cancer testis antigen PRAME is associated with response to immune checkpoint inhibitor and rituximab priming in treatment-naïve diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid malignancy in the world which is mainly divided to ABC type and GCB type. Ibrutinib benefits patients with ABC subtype DLBCL by inhibiting Bruton's tyrosine kinase (BTK), but there are still a lot of patients who are resistant to Ibrutinib. In this study, we found that post-translational modification played a regulatory role in mediating Ibrutinib resistance in DLBCL. The expression of USP13 was significantly higher in Ibrutinib-resistant U2932 cells compared with Ibrutinib-sensitive WSU-DLCL2 cells, and its expression was further increased both in WSU-DLCL2 and U2932 after exposure to Ibrutinib. USP13 overexpression significantly promoted cell growth and decreased Ibrutinib sensitivity in DLBCL while knockdown of USP13 significantly increased Ibrutinib-induced apoptosis and improved the sensitivity to Ibrutinib. Further studies showed that USP13 interacted with FHL1 and stabilized the expression of FHL1 via deubiquitination, thereby mediating the phosphorylation of ERK1/2, resulting in the decrease expression of Bax and the increase expression of Bcl-xL, thus mediating Ibrutinib resistance in DLBCL. The results of immunohistochemical staining of tumor tissue also indicated that the expression level of USP13 was negatively related to the prognosis of DLBCL patients. These new findings provided an experimental basis for overcoming Ibrutinib resistance in DLBCL and showed that USP13 is a potential therapeutic target and prognostic marker in DLBCL.