non-GCB Subtype Research Articles

Genetic basis of PD-L1 overexpression in diffuse large B-cell lymphomas

AbstractDiffuse large B-cell lymphoma (DLBCL) is one of the most common and aggressive types of B-cell lymphoma. Deregulation of proto-oncogene expression after a translocation, most notably to the immunoglobulin heavy-chain locus (IGH), is one of the hallmarks of DLBCL. Using whole-genome sequencing analysis, we have identified the PD-L1/PD-L2 locus as a recurrent translocation partner for IGH in DLBCL. PIM1 and TP63 were also identified as novel translocation partners for PD-L1/PD-L2. Fluorescence in situ hybridization was furthermore used to rapidly screen an expanded DLBCL cohort. Collectively, a subset of samples was found to be affected by gains (12%), amplifications (3%), and translocations (4%) of the PD-L1/PD-L2 locus. RNA sequencing data coupled with immunohistochemistry revealed that these cytogenetic alterations correlated with increased expression of PD-L1 but not of PD-L2. Moreover, cytogenetic alterations affecting the PD-L1/PD-L2 locus were more frequently observed in the non–germinal center B cell–like (non-GCB) subtype of DLBCL. These findings demonstrate the genetic basis of PD-L1 overexpression in DLBCL and suggest that treatments targeting the PD-1–PD-L1/PD-L2 axis might benefit DLBCL patients, especially those belonging to the more aggressive non-GCB subtype.

Primary Breast Diffuse Large B Cell Lymphoma: Summarization of 12 Cases

To investigate the clinicopathological manifestation, immunophenotypic features and prognostic factors of patients with primary breast DLBCL (PB-DLBCL). Twelve cases of PB-DLBCL, diagnosed according to the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues, were retrospectively studied. Most patients were admitted to hospital because of painless unilateral breast mass. Out of 12 cases, 5 were in Ann Arbor stage I (41.7%), 7 case were in stage II (58.3%). Most (89.9%) were assigned to non-GCB subtypes, 11.1% were classified as GCB subtype. The patients who recepted treatment were sensitive to chemotherapy and they were all alive following 12 to 92 months. Primary breast DLBCL is extremely rare without specific clinical features. They all respond well to chemotherapy and show good prognosis.

The Distinct Clinical Features and Prognosis of the CD10+MUM1+ and CD10-Bcl6-MUM1- Diffuse Large B-Cell Lymphoma

Introduction: Based on combination of the three markers (CD10, Bcl6 and MUM1), Hans algorithm could divide DLBCL into two groups (GCB and non-GCB subtype). Although MUM1 is used as a post GC marker, cases with coexpression of CD10 and MUM1 (CD10+ MUM1+, double positive or DP), which was classified as GCB subtype according to Hans algorithm, do exsit. However, the differences of clinical characteristics and prognosis between DP and other GCB (GCB excluding DP) is unknown. Additionally, DLBCL without any positive staining of these three markers (CD10¨C Bcl6¨C MUM1¨C, triple negative or TN) were also noted. These cases, based on Hans algorithm, are classified as non-GCB subtype. However, little was known about the difference between TN cases with GCB or other non-GCB (non-GCB excluding TN).Patients and methods: Immunohistochemistry was appiled in this study with the antibodies as follows. CD10 (clone 56C6, Dako), CD20 (clone L26, Abcam), Bcl6 (clone LN22, Dako), and MUM1 (clone MUM1p, Dako), FOXP1 (clone JC12, Abcam), GCET1 (clone RAM341, Abcam), LMO2 (clone 1A9-1, Santa Cruz), Myc (clone Y69; Abcam, cut-off: 40%) and Bcl2 (clone 124; Dako, cut-off: 50%). Fluorescence in situ hybridization (FISH) was carried out according to manufacturer¡¯s instructions on with the following probes: MYC dual-color, break apart translocation probe (Vysis LSI) and IGH/BCL2 dual-color, and dual fusion translocation probe (Vysis LSI). For cases with MYC translocation, BCL6 dual-color break apart rearrangement probe (Vysis LSI) wasfurtheranalyzed. A total of 601 cases were included in the study. Among these, 306 cases were treated with rituximab based chemoimmunotherapy and well follow up. The median follow up time was 24.3 months (1 to 115.4 months).We analyzed the clinical features of different groups: GCB vs. non-GCB; DP vs. other GCB (GCB*) or non-GCB; TN vs. other non-GCB (non-GCB*) or GCB. Besides, we further analyzed the survival differences among above groups in patients who were treated with rituximab based chemoimmunotherapy and well followed up.Results: The incidence of the DP patients was 13.3% (80/601, 95%CI: 10.7%-16.3%). The DP phenotype was more likely to occur in elderly patients (P =0.0432) and associated with poor PS status (P =0.0192) than GCB* phenotype. Additionally, although not statistically significant, the DP group showed higher incidence of double hit lymphoma (DHL) than GCB* patients (10.7% vs 2.9%, P =0.139). However, the DP group showed more frequent incidence of double expression lymphoma (DEL, Myc and Bcl2 coexpression) (P =0.044), MYC rearrangement (P =0.016) and DHL (P =0.029) than non-GCB group. The incidence of the TN patients was 8.8% (53/601, 95%CI: 6.7%-11.4%). There was no difference in any of clinical characteristics between the TN and GCB group. However, advanced Ann Arbor stage (III or IV) (P =0.0442), better PS status (P =0.0321), high-risk IPI (P =0.0042) Myc expression as well as BCL2 rearrangement were significantly more common in the non-GCB* group than the TN group. The DP group showed similar overall survival (OS) (median OS: both not reached, P =0.3650) and progression-free survival (PFS) (median PFS: 47.0 vs. 32.7 months, P =0.0878) with the non-GCB group while the TN group showed similar OS (median OS: both not reached, P =0.9278) and PFS (median PFS: both not reached, P =0.9420) with the GCB group (Fig 1-2).Conclusion: Cases in the DP and TE group might hinder the intrinsic power of Hans algorithm in other studies. More detailed classification of DLBCL based on Hans algorithm may help to identify patients with distinct clinical characteristics and prognosis, thereby improving the stratification of patients for risk-adjusted therapies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

R-CHOP Therapy Cannot Overcome CD5 Positive Non-GCB Subtype of DLBCL

Introduction: There are various poor prognosis factors in diffuse large B cell lymphoma (DLBCL). CD5 positive (CD5+) is estimated as one of the poor prognosis markers in DLBCL. CD5+ DLBCL is completely distinguished from CLL or Mantle cell lymphoma, and de Novo CD5+ DLBCL is related to a high incidence of cytogenetic abnormalities of 8p21 and 11q13. In many cases, CD5+ DLBCL is associated with an aggressive clinical status and advanced stages. Several chromosomal studies have demonstrated the gene expression was similar to non-GCB type of DLBCL. In the clinical phase, it is easy to express chemo-resistance and CNS invasion. However, the clear characterization and mechanisms of chemo-resistance have not been demonstrated yet. In this study, we examined our previous CD5+ DLBCL patients in our institute, then evaluated the prognosis and clinical characteristics regarding GCB or non GCB type.Methods: We studied 372 newly diagnosed DLBCL patients including 42 cases of CD5+ from 2005 to 2015 in our institution retrospectively. The pathological diagnoses were performed with immunohistochemical analysis by two or three hematological pathologists. CD5 expression was evaluated by immunohistostaining and flowcytometry, then cyclin D1 positive cases were excluded. GCB or non-GCB subtype was evaluated with CD10, bcl-6, and MUM-1 of immunohistostaining. The clinical stage of patients and evaluation of the effect of the therapy were performed using PET-CT scan. The statistical analyses were performed by Dr. SPSS II.Results: In all our treated patients, 350 DLBCL patients (female;161) and 41 CD5+ patients (female 22) could be evaluated. 192 patients were GCB type and 158 patients were non-GCB type. The median age was 64.5 yrs (25-86 yrs) and the median follow up time was 45 months (1-133 months). All patients were treated by rituximab-CHOP (R-CHOP) therapy. CR rate of CD5- DLBCL was 94.2% and CD5+ was 73.1% respectively (p =0.0093). 3.5-year event free survival (EFS) was 79.16% and 52.20% (p <0.0001) and overall survival (OS) was 85.82% and 54.35 %(p <0.0001) (CD5- and CD5+ respectively). In CD5+ DLBCL, GCB type was 11 (36.6%) and non-GCB type was 30 (63.3%). In the non-GCB type of CD5+ cases, IPI low (L) and low intermediate (LI) was 86.6% (18 and 8 each) and high intermediate (HI) was 13.3% (4/30). CR of GCB type in CD5+ was 81.8% and relapse rate was 22.2% compared with CR of non-GCB in CD5+ was 70.0% and relapse rate was 52.4% respectively. All HI cases of non-GCB type could not get CR and died within 6 months. 3.5-year EFS of CD5+ DLBCL according to GCB and non-GCB were 81.82% and 40.6% (p =0.1214) and OS was 90.9% and 40.05% (p =0.0154), respectively. 3.5-year EFS of CD5- DLBCL according to GCB and non-GCB were 82.12% and 69.23% (p =0.0173), and OS of CD5- was 88.51% (GCB) and 82.12%(non-GCB) (p =0.0061) respectively.Discussion: CD5+ showed poor prognosis in our treated DLBCL, however, GCB type of CD5+ had similar OS and EFS as CD5- DLBCL. Non-GCB type of CD5+ demonstrated significantly poor prognosis compared to GCB subtype or CD5- DLBCL. The clinical status of CD5+ cases have been generally reported as having aggressive status, however, in our CD5+ of non-GCBcases, IPI L and LI were 86%. In spite of many populations of L and LI of IPI, the prognosis was poor. From these results, it suggested that it is not necessary to estimate CD5+ of GCB subtype as poor prognosis in R-CHOP therapy, however, CD5+ non-GCB type indicated independent poor prognosis factors in DLBCL. Additionally, R-CHOP therapy could induce CR at once in IPI L and LI cases, however it could not contribute to good prognoses. We should investigate other treatment strategies for improving outcomes of CD5+ non-GCB subtype of DLBCL. DisclosuresMishima:Chugai Pharmaceutical CO., LTD.: Consultancy. Yokoyama:Chugai Pharmaceutical CO., LTD.: Consultancy. Nishimura:Chugai Pharmaceutical CO., LTD.: Consultancy. Hatake:Chugai Pharmaceutical CO., LTD.: Other: lecture speaking.

Randomized Phase 2 Open-Label Study of R-CHOP ± Bortezomib in Patients (Pts) with Untreated Non-Germinal Center B-Cell-like (Non-GCB) Subtype Diffuse Large Cell Lymphoma (DLBCL): Results from the Pyramid Trial (NCT00931918)

Randomized Phase 2 Open-Label Study of R-CHOP ± Bortezomib in Patients (Pts) with Untreated Non-Germinal Center B-Cell-like (Non-GCB) Subtype Diffuse Large Cell Lymphoma (DLBCL): Results from the Pyramid Trial (NCT00931918)

Management Strategies and Outcomes for Very Elderly Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma. As the incidence of DLBCL increases with age, the number of elderly patients with DLBCL in our aging society continues to increase. However, patients aged ≥ 80 years old are often frail and carry multiple comorbidities, and are generally underrepresented in prospective clinical trials.Patients and Methods: 207 patients, age ≥80 years of age at diagnosis of de novo DLBCL were identified between 2002 and 2014 at MD Anderson Cancer Center and included in this retrospective analysis. Failure-free survival (FFS) and overall survival (OS) were examined; baseline characteristics and frontline therapy were evaluated for the association with survival outcomes by hazard ratio (HR).Results: Median age was 83 years (range 80-96). Fifty-five percent of the patients were male, 66% had advanced stage and 37% had poor performance status (ECOG PS≥2). Forty-five percent of the patients were IPI high-intermediate or high risk. Eighty patients (39%) were GCB and 49 patients (24%) were non-GCB subtype, respectively, according to the Hans criteria, with 78 patients lacking data for classification. Thirty-two patients (15%) had ≥4 scores by Charlson comorbidity index (CCI, Charlson 1987). Treatment received included R-CHOP (n=144, 70%), R-EPOCH (n=12, 6%), and non-anthracycline based therapies including R-CEOP and R-CVP (n=20, 10%). Sixteen patients (8%) did not receive any treatment due to deteriorated condition or patient's decision. Fifteen patients received other treatment such as radiation and rituximab monotherapy. With a median follow up of 38.1 months, 88 patients (43%) experienced progression/relapse and 123 patients (59%) died. Sixty-two patients (50%) died of lymphoma, 32 patients (26%) died of other co-morbid conditions while in remission and 17 patients (14%) died as a result of treatment complication during frontline therapy. Thirteen patients died during R-CHOP, 2 patients each died during R-CEOP or R-CVP and R-EPOCH, respectively, most were the result of infection or multi-organ failure. The 3-year FFS and OS were 55% and 54%, respectively. After 3-years, almost all deaths were attributed to other diseases (Figure A). Patients who received R-CHOP or R-EPOCH had a significantly longer FFS than patients who received R-CEOP or R-CVP (Figure B). CCI ≥4 was not associated with inferior FFS but was significantly associated with inferior OS. Multivariate analysis for OS adjusted by IPI and frontline treatment revealed that being female was associated with significantly longer OS (HR: 0.50, 95% CI, 0.33-0.75, p<0.01) and patients with beta-2 microglubulin >2.0mg/l had significantly inferior OS (HR: 7.21, 95% CI, 1.67-31.1, p=0.01).Conclusion: Patients with DLBCL aged ≥80 years who received anthracycline-based regimens such as R-CHOP or R-EPOCH had outcomes similar to younger patients with de novo DLBCL. Although they carry higher risks of therapy-related mortality, anthracycline-based regimens are moderately well tolerated, and their use should not be minimized based solely on age. Inclusion of very elderly patients in prospective clinical trials is warranted to identify the most effective management strategy for this population. [Display omitted] DisclosuresWestin:Spectrum: Research Funding. Fanale:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Research Funding; Infinity: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Genentech: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; ADC Therapeutics: Research Funding; Onyx: Research Funding; Gilead: Research Funding. Rodriguez:Orthobiotech: Research Funding. Wang:Celgene: Research Funding. Nastoupil:AbbVie: Research Funding; TG Therapeutics: Research Funding; Genentech: Honoraria; Celgene: Honoraria; Janssen: Research Funding.

A 220-Gene Targeted Next-Generation Sequencing Panel for the Detection of Variants in Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, and Mantle Cell Lymphoma: Application to a Cohort of 85 Formalin-Fixed Paraffin-Embedded Diffuse Large B-Cell Lymphoma Biopsies

Background: Recent advancements in comprehensive sequencing technologies has enabled researchers to uncover numerous somatic variants within many disease types, but their respective contributions to disease pathogenesis and potential roles as outcome biomarkers are less well-defined. Even fewer studies have integrated somatic mutation events with genomic gain and loss events of respective genes, mostly due to the need to utilize a second platform to robustly assess genomic copy number. To further understand the roles of these genomic aberrations in the disease biology of mature B-cell neoplasms, we developed a next-generation sequencing (NGS) panel of 220 genes for profiling of Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), and secondarily for Chronic Lymphocytic Leukemia (CLL).Methods: The 220 genes represented in the panel included genes based on reported frequency of mutations, targets of therapeutic agents, involvement in disease-enriched pathways, prognostic value, and associated with targeted therapy resistance. Also included were genes that mapped to sites of genomic gain and loss commonly observed in these neoplasms. Of all the genes, 163 were enriched for DLBCL, 144 for FL, 44 for MCL, and 107 for CLL. Probes were designed to cover the coding exons of the 220 genes using a hybrid-capture approach (Nimbledesign, Roche, Inc.), with a total capture size of 0.92 Mb comprising 4086 target regions. Optimization of DNA fragmentation and library preparation was performed for DNA extracted from formalin-fixed paraffin-embedded (FFPE) biopsy material, being the most common intended tissue type and also for which tumor burden could be assessed. Sequencing was performed using a Miseq sequencer (Illumina, Inc.) with the goal to enable detection of somatic variants down to an allele variant frequency (AVF) of 5%. CLC Genomics Workbench (Qiagen, Inc.) was used for alignment and variant calling. Filtering was performed to exclude synonymous, intronic, and untranslated region variants, variants within homopolymer regions (>6), and variants detected below 5% AVF.Results: The average depth of coverage achieved across an initial sample set of over 50 FFPE DLBCL specimens was approximately 350X, with only one sample having more than 10% of the target regions not achieving a coverage of 60X or greater (within 95% of the target region). As part of a proof-of-concept study, we sequenced a cohort of 85 DLBCL FFPE samples from a single institution from patients undergoing biopsy as part of their routine clinical care. Clinical outcome, cell-of-origin subtype (by the Hans method) and genomic gain/loss data by array-CGH were available. All studies were performed with IRB approval. Of the 85 cases, 80 had also been submitted to bi-directional Sanger's sequencing for Exons 5-8 of TP53 to establish accuracy of variant detection. Of cases completed to date, additional TP53 mutations have been detected by NGS due to an AVF below that detectable by Sangers or only detected in one direction. In addition, of the 14 cases completely sequenced and filtered to date, EZH2 variants in four samples have been detected in the GCB subtype while four CD79B variants were in samples of the non-GCB subtype, consistent with previously reported associations. Furthermore, six of the eight samples which harbored an EZH2 or CD79B variants had mutations within the catalytic domain at Y646 (EZH2) or the ITAM residue Y196 (CD79B), both of which have clinical implications. Initial assessment of the ability of the panel to infer copy number showed concordance with aCGH in detecting large chromosomal aberrations. The sensitivity and specificity of this approach is currently being optimized.Conclusion: Overall then, a hybrid-capture NGS panel has been developed, targeting genes enriched for involvement in the most frequent subtypes of mature B-cell neoplasms and optimized for application to FFPE biopsy specimens. While emerging pathogenomic patterns observed in the cases sequenced to date have confirmed previously observed patterns, completion of the NGS analysis of the 85 DLBCL cohort will permit more complex integrated analysis of somatic variants and genomic gain/loss with pathologic and clinical outcome data. DisclosuresFriedman:Cancer Genetics Inc.,: Employment, Equity Ownership. Guttapalli:Cancer Genetics, Inc.: Employment, Equity Ownership. Ma:Cancer Genetics, Inc.: Employment, Equity Ownership. Thodima:Cancer Genetics, Inc.: Employment, Equity Ownership. Kamalakaran:Cancer Genetics, Inc.: Employment, Equity Ownership. Houldsworth:Cancer Genetics, Inc.: Employment, Equity Ownership.

Differences in Cell of Origin (COO) Affect Outcomes in Caucasian(C) but Not in African American(AA) Patients with Diffuse Large B-Cell Lymphoma(DLBCL)

Introduction: DLBCL is an aggressive mature B-cell neoplasm with a low incidence in African Americans and other minority groups. The differences in outcomes based on DLBCL Cell of Origin(COO) immunophenotypic subtypes as defined by the Hans algorithm using CD10, Bcl-6, and MUM1 namely germinal center B-cell (GCB) and non-GCB have been based on predominantly Caucasian patients (pts) and their impact on outcomes in AA patients have not been well studied.Material and Methods: A retrospective review of clinicopathologic data from patients diagnosed and treated for DLBCL at UAB between 2002-2011 was conducted following IRB approval .The data collected included patient demographics (age, sex, self identified race), DLBCL subtype (GCB vs. non-GCB based on the Hans algorithm), disease stage, first line therapy regimens and patient outcomes. We analyzed group differences in both C and AA pts with GCB and non-GCB subtypes of DLBCL in all the parameters mentioned above using the chi square test for categorical variables,. In addition, OS was examined using Kaplan Meier curves and the log rank test. We performed univariate and multivariate analyses to examine the effects of variables of interest on OS. All results were considered statistically significant at α=0.05 level.Results: We included a total of 259 pts in our analysis after excluding patients with missing demographics, disease related data, primary mediastianal DLBCL, missing first line therapy data and outcomes data as well as race identified as other than C or AA. 45 patients (17.4%) of patients self identified as AA and 214 (82.6%) self identified as Caucasian. The mean age of presentation for AA pts was 52 yrs compared to 58 years for C patients (p=0.045) and 28 (62.2%) AA patients presented with advanced Stage III and IV disease compared to 89 (44.6%) of C patients (P=0.04) both being statistically significant. Based on COO characterization using immunophenotype by Hans algorithm, 27 AA pts (60%) and 145(67%) of C pts were GCB by immunophenotype(p=0.317). 95% of all patients received R-CHOP as first line therapy , 3.5% received R-CVP and the remaining received a combination of Rituximab alone, FCR or radiation therapy .On analyzing OS based on COO, AA patients did not demonstrate a significant difference in OS based on COO with both GCB and non-GCB groups having a median OS of 84 months ( p=0.74)( 95% CI 72-121 months). However, the median OS for C pts with GCB phenotype was 104 months compared to 25 months for the non-GCB phenotype and the median OS was 55 months +/-12.6 months for the C pt group as a whole( p<0.001 by log rank test )which was highly statistically significant.Multivariate analyses of different factors affecting OS showed only COO (p<0.001), age(p=0.07) and stage of DLBCL at presentation(p=0.027) to affect OS significantly.Race,Gender and IPI score did not appear to impact OS significantly.Conclusion: Our study aimed to study the impact of COO and Race on OS in DLBCL especially in AA pts where this has not been well characterized. Our results show that as expected the incidence of DLBCL is lower in AA patients compared to C pts. However, DLBCL presents at younger age and with more advanced stage in AA than in C patients. Although distribution of COO is similar in AA and C patients, our findings suggest that COO may not have the same effect on prognosis among AA pts as it does among the C pt population where we see non-GCB patients do significantly worse than their GCB counterparts. This may reflect a different disease biology in AA patients that has yet to be understood. Our study limitations include not taking into account disease relapse, second line therapy and effect of autologous bone marrow transplantation on overall survival. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase 1 Study of Tazemetostat (EPZ-6438), an Inhibitor of Enhancer of Zeste-Homolog 2 (EZH2): Preliminary Safety and Activity in Relapsed or Refractory Non-Hodgkin Lymphoma (NHL) Patients

Introduction: The histone methyl transferase EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and responsible for methylation of lysine 27 of histone H3 (H3K27), a modification of DNA associated with repressed transcription when trimethylated (H3K27me3). Aberrant EZH2 activity has been implicated as an oncogenic driver in non-Hodgkin's lymphoma (NHL). Here we report the phase 1 first-in-human experience to date with tazemetostat in patients (pts) with NHL.Methods: Tazemetostat was administered orally twice daily (BID) to subjects in five dose cohorts (100 mg [n=6], 200 mg [n= 3], 400 mg [n=3], 800 mg [n=14], 1600 mg [n=12], and one food effect cohort (400 mg [n=7]). Tumor response assessments were performed every 8 weeks. Archival tumor tissue from NHL pts was analysed for EZH2 hot spot mutations Y646X, A682G and A692V (by either amplicon-based next generation sequencing [NGS] or cobas ® EZH2 Mutation Test [in development]), and for additional somatic mutations by NGS focusing on a panel of 39 genes commonly mutated in NHL. In addition, cell-of-origin in Diffuse Large B-cell Lymphoma (DLBCL) patients was determined by immunohistochemistry on archival tumor tissue using the Hans algorithm (Blood, 2004).Results: As of 9-July 2015, 45 pts were enrolled to this trial (CT.gov: NCT01897571). To date 19 NHL pts: 13 DLBCL, 5 follicular lymphoma (FL) and 1 marginal zone lymphoma (MZL) were enrolled. Results to date on the 26 solid tumor pts have been reported separately (ECC, 2015). Adverse events (AE) occurring in >10% of the 45 pts regardless of attribution were: asthenia, anorexia, constipation, nausea, dysgeusia, vomiting and muscle spasms with 5 grade 3 or greater related AE's: thrombocytopenia, neutropenia, hypertension, anorexia and transaminase elevation. The median age of the NHL patients enrolled was 62 yrs (range 23-82) and 74% of pts were male. Of the fifteen evaluable NHL pts, objective responses were seen in: 5/9 DLBCL, 3/5 FL and 1/1 MZL. The majority of objective responses occurred at the Recommended Phase 2 Dose of 800 mg BID. EZH2 status in patient tumors was determined for 14/19 NHL patients (n=3 data pending, n=2 tissue unavailable) with 13/14 found to be wild-type (WT) and one patient, who experienced an ongoing PR at week 16, expressing an Y646H mutation. Updated data including duration of response will be presented. In addition, 10/13 patients had evidence of somatic mutations in >1 non-EZH2 genes among the 39 genes tested (allelic frequency >10% with coverage >1000X) known to be commonly mutated in NHL, e.g. MYD88 & CARD11, or involved in epigenetic signalling, e.g. EP300 & CREBBP. TableRelapsed or refractory NHLTotal(n=19)Evaluable(n=15)Best Response CR+PRaBest Response SDaDLBCLGCB4220Non-GCB6520undetermined3210FL5531MZL1110aper IWG (Cheson, 2007), complete response (CR), partial response (PR), stable disease (SD)Conclusions: Tazemetostat demonstrates a safety profile favorable for chronic dosing and objective responses in pts with either EZH2 WT or mutant B-cell lymphoma, including both GCB and non-GCB sub-types of DLBCL. EZH2 pathway genes and genes commonly somatically mutated in lymphoma are under investigation to elucidate the mechanisms and biomarkers of clinical response to tazemetostat. Based upon the observed safety and efficacy profile of tazemetostat, a phase 2 trial in relapsed or refractory DLBCL and FL pts, stratified by cell-of-origin and EZH2 mutation status, is enrolling. DisclosuresRibrag:Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thomson:Epizyme, Inc: Employment. Keilhack:Epizyme: Employment, Equity Ownership. Blakemore:Epizyme: Employment. Reyderman:Eisai: Employment. Kumar:Eisai: Employment. Fine:Epizyme: Employment. McDonald:Epizyme: Employment. Ho:Epizyme, Inc: Employment.

Prognostic value of immunophenotype in patients with diffuse large B-cell lymphoma in the immune chemotherapy era

Objective To study the prognostic value of immunophenotypes in the patients with diffuse large B-cell lymphoma (DLBCL) and its prognostic significance of immunohistochemical markers for rituximab. Methods The data of 186 initial DLBCL patients were retrospectively analyzed. According to the international prognostic index, all patients were divided into relatively high-risk group and relatively low-risk groups, and the patients in both groups were divided into chemotherapy (CHOP) group and immune chemotherapy (R-CHOP) group, respectively. Immunohistochemical method and Hans typing method were applied to immune classification in all of groups. The prognostic significances of the immunological subtypes in different prognosis group were compared by Log-rank test. Results The 5-year OS rate of the GCB subtype patients in the CHOP group was significantly higher than that of the non-GCB subtype (58.82 % vs 32.00 %, χ2 = 8.482, P= 0.004), while that was no significant difference between GCB and non-GCB subtypes patients in the R-CHOP group (72.97 % vs 61.54 %, χ2 = 2.694, P= 0.101). Further analysis results showed that non-GCB subtype patients treated with R-CHOP had significantly higher 5-year OS rate than those treated with CHOP (61.54 % vs 32.00 %, χ2 = 7.385, P= 0.007). In the 107 cases of low risk group, there was no significant difference in 5-year OS rate between GCB and non-GCB subtype patients in the CHOP group or in the R-CHOP group (all P> 0.05), and there was no significant difference in 5-year OS rates between the CHOP and R-CHOP groups in the GCB group or in the non-GCB group (all P> 0.05). In the 79 cases of high risk group, the 5-year OS rate in the GCB subtype group was significantly higher than that in the non-GCB subtype in the CHOP group (40.00 % vs 22.72 %, χ2 = 3.978, P= 0.045), while there was no significant difference in 5-year OS rates between GCB and non-GCB subtype patients in the R-CHOP group (62.50 % vs 42.31 %, χ2 = 2.072, P= 0.150). Conclusions By immunohistochemistry, DLBCL can be divided into two immunophenotype subgroups, which have guiding significance for early prognosis and therapeutic strategies, but which are no longer or weak in prognostic significance in the era of post-rituximab. R-CHOP improve significantly survival of non-GCB subgroup patients. However, its function needs to be further explored in the GCB subgroup. Key words: Lymphoma, large B-cell, diffuse; Immunophenotyping; Prognosis; Rituximab

High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma.

Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20% of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5%) and 17p13.1 (2.5%) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development.

FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco–Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients.

CD5 positivity is an independent adverse prognostic factor in elderly patients with diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. Age over 60 years is one of the five parameters of the International Prognostic Index (IPI), which is the most important clinical prognostic predictor in DLBCL. A previous study on German DLBCL patients over 60 years of age showed that immunoblastic morphology, but not germinal center B cell-like (GCB)/non-GCB subtype, correlated with short survival. We collected 174 DLBCL cases over 60 years of age in Taiwan and performed immunophenotyping and detection of Epstein-Barr virus (EBV)-encoded RNA (EBER) by in situ hybridization. Of the cases, 5.2 % were positive for CD5 and 5.7 % positive for EBER. Neither immunoblastic morphology nor GCB/non-GCB subtype correlated with survival. In univariate analysis, adverse prognostic factors included IPI ≥ 3 (P < 0.000001), B symptoms (P = 0.000075), bone marrow/peripheral blood involvement (P = 0.017), EBER positivity (P = 0.0013), and CD5 positivity (P = 0.016). In multivariate analysis, CD5 positivity was the only independent adverse prognostic factor (HR = 3.16; 95 % CI = 1.34-7.47; P = 0.0087) in addition to IPI ≥ 3 (HR = 3.07; 95 % CI = 1.84-5.11; P = 0.000018). Surprisingly, despite an overall 5.2 % incidence of central nervous system (CNS) relapse in our patients, none of the CD5+ cases experienced CNS relapse (P = 1.00). This is in stark contrast to the more frequent CNS relapse in Japanese CD5+ DLBCL patients. EBER positivity was associated with IPI ≥ 3 (P = 0.010), stage III-IV (P = 0.0082), and B symptoms (P = 0.011). In multivariate analysis, EBER positivity was not an independent adverse prognostic factor (P = 0.81), its effect being due likely to accompanying adverse clinical parameters.

Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas.

Background:Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines.Methods:We analysed expression of NGF, BDNF, and their receptors (Trk, p75NTR) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75NTR axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies.Results:A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75NTR, TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75NTR was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model.Conclusions:This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups.

High prevalence of the MYD88 mutation in testicular lymphoma: Immunohistochemical and genetic analyses.

The activating mutation of MYD88 has been identified in diffuse large B-cell lymphoma (DLBCL). We investigated the mutational status and both the gene amplification and protein expression of MYD88 in 23 cases of testicular DLBCL. To detect the MYD88 mutations, we employed the allele-specific PCR and Sanger sequencing. MYD88 gene amplification and protein expression were analyzed by quantitative PCR and by immunohistochemistry, respectively. There were 17 cases of primary testicular DLBCL: 94% (16/17) exhibited a non-Germinal center B-cell (non-GCB) subtype, 82% (14/17) showed the MYD88 L265P, and 65% (11/17) had intense expression of MYD88. When compared with normal lymph nodes, the MYD88 is significantly amplified in primary testicular DLBCL. However, the amplification status showed no correlation with its mutational status or protein expression. Moreover, neither the MYD88 mutational status nor the expression pattern affected overall survival. Six cases were secondary testicular DLBCL with an 83% (5/6) and an 80% (4/5) incidence of the non-GCB subtype and of the MYD88 L265P, respectively. In conclusion, we demonstrated a high prevalence of the non-GCB subtype and the common MYD88 L265P in both primary and secondary testicular DLBCL. Our data suggest that the MYD88 mutation is a fairly consistent genetic feature in testicular DLBCL.

Abstract 3893: Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations

Abstract Primary central nervous system lymphoma (PCNSL) is defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system. Although PCNSL cannot be histologically distinguished from extracerebral DLBCL, its prognosis is quite poor. To gain insights into the transforming mechanism of PCNSL, we conducted whole-exome sequencing for 44 PCNSL specimens. Genomic DNA was extracted from tumors and their matched normal samples. Exome fragments were captured by using SureSelectXT Human All ExonV5+lncRNA, and subjected to deep sequencing with the HiSeq 2000 system. There were 8 cases with germinal center B-cell-like (GCB) subtype and 34 cases with non-GCB subtype. Whole-exome sequencing of tumor and paired normal yielded sequencing data with a mean coverage of 156X and 80X, with 98% and 99% of the targeted bases covered by ≥20 independent reads, respectively. Of 17,832 somatic mutations identified in the PCNSL specimens, 11,611 (65%) were nonsynonymous single nucleotide variations. Predominant nucleotide substitution was a C&amp;gt;T transition (57%) especially in the context of the sequence GCG. We also identified 798 insertions/deletions. Interestingly, MYD88 mutations were detected in 35 of 44 patients (79.5%), and PIM1 mutations were detected in 42 patients (95.5%). Also, a number of genetic aberrations were shown to activate the NF-κB pathway. These results suggest that MYD88 and PIM1 mutations have important roles in the development of PCNSL and could be the therapeutic targets of this intractable disorder. Citation Format: Motoo Nagane, Kazutaka Fukumura, Toshihide Ueno, Jeunghun Lee, Yukiko Shishido-Hara, Mitsuaki Shirahata, Kazuhiko Mishima, Koichi Ichimura, Akitake Mukasa, Yoshitaka Narita, Ryo Nishikawa, Hiroyuki Mano. Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations. [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 3893. doi:10.1158/1538-7445.AM2015-3893

Expression of CD68, cyclin D1 and rearrangement of bcl-6 gene are adverse prognostic factors in diffuse large B-cell lymphoma

To study expression of CD68, cyclin D1 protein and rearrangement of bcl-6 gene impact on the prognosis of diffuse large B-cell lymphoma (DLBCL). Gets paraffin samples of the 105 cases DLBCL with the detailed follow-up information, and were studied by using immunohistochemical EnVision method for CD3, CD10, CD20, CD68, cyclin D1, bcl-6, MUM 1, SOX-11 immunolabeling. The DLBCL were classified into germinal center B cell-like (GCB) subtypes and non-germinal center B cell-like (non-GCB) subtypes according to Hans'algorithm. Application of fluorescence in situ hybridization (FISH) technique to detect the bcl-6 gene rearrangement. The relationship between CD68, cyclin D1 protein, the bcl-6 gene and the curative effect of chemotherapy and survival was analyzed using statistical software. Respectively by GCB type, non-GCB type immune phenotype and CHOP, R-CHOP chemotherapy group, compare the curative effects. 105 patients had GCB 19 cases (18.1%), non-GCB 86 cases (81.9%), CD68 expression was 18 cases (17.1%), cyclin D1 high expression 36 cases (34.3%), bcl-6 gene rearrangement in 21 cases (21.9%), there is no correlation among the three (P > 0.05). One-way analysis of variance showed that age ≤ 60 years, clinical stage I-II, IPI score 0 to 2 points, LDH (U/L) < 245 IU/L,GCB subtypes, R-CHOP therapy, the prognosis of patients with better (P < 0.05), But gender, primary site no correlation with prognosis (P > 0.05). CD68, cyclin D1 high expression, bcl-6 rearrangement had poor prognosis (P < 0.05). Stratification analysis results show GCB-type or non-GCB type with high expression of CD68 contrast alloimmune phenotype groups had a poor prognosis, non-GCB type with high expression of cyclin D1 and rearrangement of bcl-6 gene had a poor prognosis (P < 0.001, P = 0.02). Treatment scheme of layered display, the CHOP treatment, significantly correlated with overall survival with high expression of CD68, cyclin D1 (P < 0.05), the R-CHOP treatment, there was no statistically significant difference between CD68, cyclin D1 high expression and overall survival (P = 0.428 and 0.168). Multivariate COX model analysis showed that high expression of CD68 (P = 0.026), high expression of cyclin D1 (P = 0.003) and high levels of LDH (P = 0.005) were adverse prognostic factors independent. high expression of CD68, cyclin D1 and rearrangement of bcl-6 gene suggests poor prognosis, CD68, cyclin D1 protein and bcl-6 gene can be used as a prognostic indicator in patients with DLBCL.

Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma in the Elderly: A Matched Case-Control Analysis.

BackgroundEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) in the elderly has rarely been reported. This study aimed to explore the clinical characteristics and prognosis of this entity.MethodsIn situ hybridization (ISH) analysis of Epstein-Barr virus (EBV) and immunohistochemistry was performed in 230 tumor specimens from consecutive de novo DLBCL patients over 50 years old. A matched-case control analysis (1:3) was utilized to compare EBV-positive and EBV-negative DLBCL in the elderly.ResultsA total of 16 patients (7.0%) were diagnosed with EBV-positive DLBCL. Of these 16 cases, the median age was 62 years, with a male to female ratio of 11:5. Elderly EBV-positive DLBCL patients had a higher incidence of non-germinal center B-cell (non-GCB) subtypes (87.5%) and high Ki67 (75%) and CD30 expression (93.8%). For EBV-positive patients undergoing initial chemotherapy, 7 of 16 (43.8%) had complete remission, 2 (12.5%) had partial remission, 2 (12.5%) had stable disease, and 5 (31.3%) had progressive disease. The median overall survival was 9 months for the EBV-positive patients. A matched-case control analysis suggested that EBV-positive patients had inferior survival outcomes compared with EBV-negative patients (3-year progression-free survival [PFS]: 25% vs. 76.7%, respectively; 3-year overall survival [OS]: 25% vs. 77.4%, respectively; P<0.001).ConclusionEBV-positive DLBCL of the elderly is associated with an inferior clinical course and inferior survival outcomes. The role of EBV in this disease and the optimal management of this subgroup warrants further investigation.

IX. Is it only about MYC? How to approach the diagnosis of diffuse large B-cell lymphomas.

Keywords: diffuse large B-cell lymphoma; cell of origin; genetic alterations; double hit; double expresser; MYC; BCL2; BCLU

Loss of IRF8 Inhibits the Growth of Diffuse Large B-cell Lymphoma.

IRF8 is a transcription factor with a critical role in B lymphocyte development and functions. Its role in human diffuse large B-cell lymphoma (DLBCL), however, remained elusive. In this study, using shRNA-mediated knockdown of IRF8 expression, we found that the loss of IRF8 significantly reduced the proliferation of DLBCL cells (P<0.05). Mechanistically, decreasing the levels of IRF8 led to a suppression of the phosphorylation of p38 and ERK, molecules critical for B cell proliferation. Furthermore, using a xenograft lymphoma mouse model, we found that the loss of IRF8 significantly inhibited the growth of lymphomas in vivo (P<0.05). Immunohistochemical analysis of human DLBCL tissues revealed that the levels of IRF8 were significantly greater in non-germinal center B-cell-like (non-GCB) subtype than that in GCB subtype (P<0.05). Analysis of public available data also suggested that the expression levels of IRF8 mRNA in human DLBCL tissues were inversely correlated with patients' overall survival time. Taken together, this study suggested that IRF8 may play an oncogenic role in human DLBCL by promoting cell proliferation.