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Background A recent laboratory study indicated that statins impaired the antitumor effects of rituximab by inducing conformational changes in CD20. Although these findings raised significant concerns about statin use during rituximab treatment, their clinical significance is unclear. Patients and methods We conducted a retrospective study investigating the effects of statins on the prognosis of diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP). Newly diagnosed DLBCL patients were analyzed (n = 256), including 35 patients taking statins. Results The 3-year progression-free survival rates were 84% and 73% (P = 0.38), while the overall survival rates were 89% and 78% (P = 0.28) for those patients treated with and without statins, respectively. After adjusting for the International Prognostic Index and serum cholesterol level, statin use was not associated with prognosis. Conclusions These results indicate that statins do not influence the clinical prognosis of DLBCL treated with RCHOP. Further studies with larger numbers of patients are warranted to confirm the prognostic significance of statins for patients with DLBCL receiving rituximab-containing chemotherapy.

A 32-year-old woman was referred to our hospital because of diffuse bone pain and a high fever lasting 2 weeks. She had a normal physical examination with no sign of hepatosplenomegaly or adenopathy. The hematological examination revealed no abnormalities; the WBC count was 7,410.0/mm (neutrophils, 84.9%; eosinophils, 0.8%; basophils, 0.4%; monocytes, 1.8%; lymphocytes, 12.2%), hemoglobin was 12.1 g/dL, and platelet count was 221,000/mm. Laboratory tests showed an increased lactate dehydrogenase (409 IU/L; reference range, 120 to 240 IU/L) and C-reactive protein (22.10 mg/dL; reference range, 0.00 to 0.30 mg/dL). Computed tomography (CT) and magnetic resonance imaging found no evidence of abnormalities, while positron emission tomography (PET)/CT with [F]fluorodeoxyglucose (FDG) showed diffuse increased bone marrow uptake (Fig 1). Based on the PET/CT result, we examined a bone marrow aspirate, which revealed 98% lymphoblasts (Fig 2). The leukemic cells in the bone marrow expressed CD19, CD10, CD34, and CD13 antigens. A multiplex real-time quantitative polymerase chain reaction assay showed that the copy number of minor BCR/ABL transcripts was elevated at 1.7 10 copies. She was diagnosed with acute lymphoblastic leukemia with the Philadelphia chromosome, was treated immediately with chemotherapy including imatinib, and achieved molecular complete remission. Routinely, PET/CT is used to find a wide variety of tumors with detection sensitivities and specificities usually exceeding 90%, particularly in cancers of the lung, colon, head and neck, and melanoma. In hematological malignancies, its value for diagnosis and response assessment has been confirmed in lymphoma, myeloid sarcoma, and multiple myeloma. Previous report assessed the response of acute myeloblastic leukemia using PET/CT with [F]fluorodeoxythymidine, although the diagnostic value of PET/CT has not been evaluated thoroughly in leukemia. In several published case reports, PET detected focal bone localization of leukemia, while bone marrow aspiration detected no abnormalities. In our case, leukemic cells were localized in the bone marrow only, while hematological examination of the peripheral blood revealed no abnormalities. PET/CT might be useful for detecting leukemia in cases that are difficult to diagnose, allowing treatment initiation without delay.

Genomic studies have revealed surprising new insights into the genetics of human B-cell lymphomas. Of particular note the lysine-specific methyl transferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and in diffuse large B-cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations in lymphoma are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and promotes lymphoma development in vivo. Integrative analyses of KMT2D function indicate specific and focal effects on H3K4 methylation and gene expression. These include B-cell growth and differentiation signals such as the CD40, JAK/STAT, and B-cell receptor pathways. Moreover, KMT2D targets include frequently mutated tumor suppressor genes such as TNFAIP3/A20, SOCS3, and TNFRSF14/HVEM. In this manner, KMT2D deficiency disrupts the physiological process of B-cell differentiation, facilitates malignant outgrowth, and can impede therapies acting on the CD40 and B-cell receptor pathways. Citation Format: Ana Ortega-Molina, Isaac W. Boss, Andres Canela, Heng Pan, Yanwen Jiang, Chunying Zhao, Man Jiang, Deqing Hu, Ji-Eun Lee, Hua-Tang Chen, Daisuke Ennishi, David W. Scott, Christoffer Hother, Shichong Liu, Xing-Jun Cao, Wayne Tam, Rita Shaknovich, Benjamin A. Garcia, Randy D. Gascoyne, Kai Ge, Ali Shilatifard, Olivier Elemento, Andre Nussenzweig, Ari M. Melnick, Hans-Guido Wendel. Functional characterization of the tumor suppressor lysine-specific methyltransferase KMT2D in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA18.

Several authors have reported interstitial pneumonia (IP) during rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy, while others have encountered Pneumocystis jirovecii pneumonia during rituximab-combined bi-weekly CHOP. Herein, we report that 13 of 90 (14%) patients developed IP during R-CHOP therapy, compared with none of 105 patients treated with CHOP alone as a historical control. There were no differences in baseline data between patients undergoing the two therapies. Among R-CHOP-treated patients, serum beta-D-glucan was increased in 8 of 12 (75%) IP patients compared with none of 30 non-IP patients examined. In five IP patients who underwent sputum evaluation, two were positive for P. jirovecii by the polymerase chain reaction and another two were positive for Candida albicans. No other organisms were detected as causative pathogens. Treatment with steroids, sulfamethoxazole-trimethoprim (ST), and antifungals was effective. Our results suggest that R-CHOP raises the incidence of IP, possibly through increasing the susceptibility to P. jirovecii and fungal infection. The need for prophylactic antifungals and ST during R-CHOP should be evaluated by randomized controlled trials.

Several studies have shown that the frequency of hepatitis C virus (HCV) infection is high in patients with B-cell non-Hodgkin's lymphoma (NHL). In these studies, liver dysfunction during chemotherapy has been demonstrated, but changes in HCV ribonucleic acid (RNA) levels during chemotherapy have not been well documented. In this study, we monitored serum HCV RNA levels and liver function in five HCV-infected patients with B-cell NHL undergoing treatment with rituximab-combination chemotherapy. Increased HCV RNA levels during or after the chemotherapy were observed in all five patients, and a significant increase in transaminases was seen in one case. In this case, serum HCV RNA level dramatically decreased at the time of the increase of transaminases, and this suggested that the cause of liver damage was an immune reaction against hepatocytes with HCV and not any anticancer drug induced liver toxicity. Monitoring of serum HCV RNA levels and transaminases may be helpful to understand the cause of liver dysfunction in patients receiving chemotherapy. However, increases of HCV viral load were not associated with the occurrence of liver dysfunction in this study. Further studies will be necessary to investigate more fully the relationship between changes in HCV viral load and liver function during chemotherapy for HCV-infected patients.

Background: Recent work in both hematologic malignancies and solid tumors has supported the notion that human cancers exhibit marked intra-tumoral heterogeneity (ITH). Results from next generation sequencing (NGS) studies support that subclonal DNA mutations underlie genotypic ITH within a single tumor since the majority of sequence variants are present in only 5-50% of reads for a given tumor sample, and single cell analyses have shown that individual tumor subclones may be ancestrally related in a complex branching hierarchy, suggesting that therapy failures and progressive disease likely arise by Darwinian selection for more aggressive or therapy resistant clones. It has become increasingly clear that it will be important to understand the multi-clonal structure of tumors in order to treat them more effectively. In this study, we sought to use time-of-flight mass cytometry (CyTOF) to explore clonal phenotypic substructure in diffuse large B-cell lymphoma (DLBCL), a diagnostic entity notorious for clinical heterogeneity. Methods: We examined viably frozen single cell suspensions from diagnostic lymph node biopsy samples received for flow cytometric analysis at the BC Cancer Agency. We have thus far acquired CyTOF data from 25 cases of DLBCL using a two-tube, 40-parameter panel encompassing a total of 58 different markers including both surface and intracellular antigens that were selected to reveal heterogeneity within the malignant B-cell population. For each sample acquisition, we included "spiked-in" control cells from pooled reactive (non-malignant) lymph node samples to control for staining variation between antibody/reagent lots and also run-to-run CyTOF instrument drift, facilitated by a CD45 antibody "barcoding" approach. We analyzed the data using a combination of viSNE, Isomap, and PhenoGraph analysis packages. Results: Analysis of individual tumor samples readily distinguished between malignant and residual normal B-cell populations, and also revealed distinct subpopulations among malignant cells of varying degrees of relatedness to one another. These subpopulations were then sorted from one another by conventional FACS from parallel vials of cryopreserved cells using lower dimensional sorting strategies derived from the 40-parameter CyTOF data. Sorted subpopulations will be analyzed by targeted amplicon sequencing for single nucleotide variants identified from whole exome sequencing data obtained from unsorted material to explore the hypothesis that these may represent genotypic subclones. Analysis of multiple tumor samples at once yielded several observations. First, B-cells from reactive lymph nodes and non-malignant B-cells within patient lymphoma specimens reproducibly cluster atop one another, indicating highly similar if not identical phenotypic profiles. Second, the majority of patient DLBCL tumors form cohesive individual clusters, separate and distinct from one another, suggesting that the 40-dimensional panel defines cell populations with sufficient resolution such that each patient's tumor can be uniquely identified. Third, individual DLBCL tumors do not aggregate in tight proximity with one another to the extent that we observe among patient follicular lymphoma (FL) samples, suggesting DLBCL represents a broader diversity of phenotypic classes. Fourth, there is local, but loose aggregation of ABC versus GCB subtypes, but there are also clear outliers and areas of intermingling between ABC and GCB tumors, as defined by immunohistochemistry. Finally, a subset of DLBCL tumors exhibit minor subpopulations that map apart from their corresponding "parent" tumor populations, but yet overlap one another, raising the possibility of divergent evolution away from (or alternatively convergent evolution towards) a common tumor archetype. Conclusions: Taken together, these observations support that novel information can be derived from CyTOF data with important implications for our understanding of both intra- and inter-tumoral heterogeneity in DLBCL. Disclosures Scott: Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

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.

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.

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.

Diffuse Large B-cell Lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin Lymphoma (NHL). Despite its improved outcome with R-CHOP chemotherapy, 40% of patients still suffer with relapsed or refractory disease. Further investigation is needed to understand the complexity of DLBCL. Time-of-flight mass cytometry (CyTOF) is a recently developed technology that combines traditional flow cytometry with mass spectrometry and supports analysis of up to 30-40 parameters simultaneously at the single cell level with minimal spectral overlap (Bendall et al, Science 2011). In this study, we sought to develop a CyTOF method for phenotypic analysis of DLBCL to obtain high resolution profiles of the malignant clone(s) represented in individual tumor samples and resolve any underlying population substructure that might be informative in understanding the clinical and biologic heterogeneity of this disease. We designed a two-tube assay for this study. Tube #1 contained 35 cell surface markers including CD45, CD19, CD20, CD22, CD79B, IgM, IgD, Ig Kappa, Ig Lambda, CD5, CD10, CD23, CD43, CD38, CD138, CD44, CD21, CD24, CD40, CD72, CD80, CD45RA, CD49D, CD49F, CD62L, CD25, CD27, CD30, CD127, CD184, CD194, CD200, CD34, HLA-DR, and CD3. Tube #2 contained 37 markers in total including 17 cell surface markers overlapping with Tube #1 plus an additional 20 intracellular markers (BCL2, BCL6, IRF4/MUM1, LMO2, MYC, MCL1, MEF2B, KAT3B/P300, CBP, FOXP1, RUNX1, Ikaros, EZH2, BMI1, NOTCH1, CARD11, IkBa, phospho-Rb, Ki67, and CyclinD2). Metal-conjugated antibodies were purchased from DVS Sciences or unconjugated antibodies labeled in-house using DVS MaxPar metal labeling kits. Data were acquired using a DVS CyTOF2 instrument. We examined both fresh and viably frozen single cell suspensions from diagnostic lymph node biopsy samples received for flow cytometric analysis at the BC Cancer Agency. We used SPADE (spanning-tree progression analysis of density-normalized events) for initial data analysis. We first performed preliminary validation studies including cross-comparison of mass cytometry (CyTOF2) vs. flow cytometry (BD Canto2) datasets and fresh vs. previously frozen cell suspension material. Although individual marker intensities using matched antibody clones were in general lower by CyTOF, eight-parameter surface staining results were qualitatively comparable between the two platforms. Also there were essentially no differences observed in CyTOF staining profiles between fresh and previously frozen samples. Preliminary clustering analysis of cell populations using SPADE revealed clear separation between normal and malignant B cell populations as well as apparent substructure to the malignant population in a subset of DLBCL samples. These findings suggest intratumoral heterogeneity can be resolved by high dimensional CyTOF analysis. Ongoing efforts will focus on determining if phenotypically defined subsets show enrichment for subclonal mutations. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent studies have established that concurrent MYC and BCL2 protein expression by immunohistochemistry (IHC) identifies a subgroup of patients with diffuse large B-cell lymphoma (DLBCL) with a poor outcome. Classic dual translocation MYC/ BCL2, so called ‘double hit' disease, is associated with a high risk of central nervous system (CNS) relapse; however the impact of concurrent MYC and BCL2 protein expression on the risk of CNS relapse remains unknown. Further, robust biological markers that accurately predict the risk of CNS relapse in DLBCL would also be of value in clinical practice. Methods: Cases of pre-treatment formalin fixed paraffin embedded DLBCL in two tissue microarrays were independently scored by two expert hematopathologist (GWS and KLT or PF and AM) for expression of MYC (Epitomics Y69), BCL2 (Dako 124), CD10, BCL6 and MUM1 by IHC. MYC and BCL2 positivity were defined as ≥ 40% and ≥ 50% cells with staining, respectively, in accordance with previously established cutoffs (Johnson, JCO 2012; 30). Cases with discordant scores were reviewed by a third hematopathologist (RDG) to reach a consensus. Cell of origin (COO) was assigned according to the Hans IHC algorithm (Hans, Blood 103: 2004) as well as by the recently described gene expression profiling Lymph2Cx 20 gene assay based on NanoString technology (Scott, Blood 2014; 123) in the subset of patients with ≥ 40% tumor content. Patients treated with at least one cycle of R-CHOP chemotherapy with curative intent were included and those with established CNS disease at diagnosis were excluded. Results: 447 patients were identified with the following baseline clinical characteristics: Median age 65 y (16-92y); males n=280, 63%; performance status ≥ 2, n= 147, 33%; stage 3 or 4 disease n=242, 54%; elevated LDH n=219, 47%; EN > 1 n= 80, 17%. With a median follow-up of 6.75 years for living patients, the 3 year time to progression, progression-free and overall survival for all patients were 68%, 66%, and 73%, respectively. In total, 131 (29%) were MYC+BCL2+ and 316 (71%) were non-MYC+BCL2+. By COO assignment using the Hans algorithm (n=444), 192 were non-GCB (43%) and 252 were GCB (57%) and by the Lymph2Cx (n=308); 103 were ABC (33%), 172 were GCB (56%) and 33 (11%) were unclassifiable. The 2 year cumulative risk of CNS relapse for the whole cohort was 4.3%. The cumulative risk of CNS relapse was higher in cases that were MYC+BCL2+ (2 year risk 9.4% vs 2.4%, P=0.001) with similar results obtained if classic MYC+BCL2+ double hit cases are excluded. There were no cases of CNS relapse in cases MYC+ alone by IHC. By COO, patients with a non-GCB phenotype by the Hans algorithm had an increased risk of CNS relapse (2 year risk 6.9% vs 2.6%, P=0.03) and similarly, cases assigned as ABC DLBCL by the Lymph2Cx assay also identified a group with a higher risk of CNS relapse compared to GCB cases (9.5% vs 2.5%, P=0.03) (Figure 1). In Cox regression multivariate analysis including the COO (Hans), IPI group (0/1 vs 2/3 vs 4/5) and MYC/BCL2 IHC, only the IPI (HR 2.18, P=0.02) and MYC+BCL2+ IHC (HR=3.76, P=0.007) were associated with an increased risk of CNS relapse. Similar results were obtained using the Lymph2Cx COO designation. Within the IPI risk groups, MYC+BCL2+ status further stratified patients in the intermediate risk group (IPI 2 or 3, n=206) into a higher risk group (2 year CNS relapse 12.6%) and a low risk group (2 year CNS relapse 2.9%) (P=0.01). A similar trend was observed in the high IPI risk group (IPI 4 or 5, n=86, 2 year CNS relapse MYC+BCL2+ 17.2% vs 4.7%, P=.0.18) but it was not useful in the low IPI risk group (IP1 0 or 1 (n=155), 2 year CNS relapse 4% vs 1%, P=0.39) where the overall risk was low. Within the COO subgroups, MYC+BCL2+ status also defined a group at high cumulative risk of CNS relapse within the non-GCB subtype (12.9% vs 3%, P=0.001) and by the Lymph2Cx defined ABC subtype (16.9% vs 2.2%, P= 0.03) and a trend was observed for GCB defined by Lymph2Cx (6.6% vs 1.5%. P=.08) but not by Hans criteria (P=0.40). Conclusion: Concurrent expression of MYC and BCL2 protein in DLBCL defines a group of patients at high risk of CNS relapse, independent of the IPI and COO. MYC+BCL2+ status may help to further risk stratify patients in the intermediate and high IPI risk groups and within the ABC subtype to identify patients who should undergo additional diagnostic testing and in whom to explore the effectiveness of prophylactic CNS strategies. Figure 1 Figure 1. Disclosures Savage: F Hoffmann-La Roche: Other. Sehn:Roche: Research Funding. Connors:Seattle Genetics, Inc.: Research Funding; Roche: Research Funding. Gascoyne:Hoffman La-Roche: Research Funding.

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.

Abstract 3101 Background: Natural killer (NK/) T-cell lymphoma, nasal type, is an aggressive form of extranodal lymphoma that is common in Asia, but rare in Europe and North America. Although NK/T-cell lymphoma involves upper aerodigestive sites such as the nasal cavity and nasopharynx, it frequently involves other extranodal sites, including the gastrointestinal tract, bone marrow, adrenal gland, and skin. Because of the frequency of extranodal site involvement, pretreatment evaluation of disease extension is important for staging and treatment. Recently, 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) was evaluated for its usefulness in the prognosis and treatment response of aggressive B-cell lymphoma and Hodgkin's lymphoma, and relevant results were obtained. Many studies have evaluated the value of PET/CT for various types of B-cell lymphomas, but other similar studies involving T-cell and NK-cell lymphomas are rare. In the present study, we compared the utility of PET/CT and conventional modalities, particularly CT, in extranodal NK/T-cell lymphoma. Patients and Method: From January 2006 to April 2010, 19 untreated patients with extranodal NK/T-cell lymphoma (11 males and 8 females; median age 61 years; range 13–90 years) were included in the study. PET/CT and conventional procedures (e.g., CTs and biopsies) were compared and evaluated for their abilities to detect tumor lesions and their influence in staging and treatment strategies. PET/CT was performed as an initial staging procedure. In addition to PET/CT, all patients underwent initial staging workups, including whole-body CT with contrast media, biopsies from the bone marrow and other sites, and panendoscopies of the upper aerodigestive tract. Patients were evaluated for clinical stage by both evaluation modalities (PET/CT and conventional modalities) according to the Ann Arbor Staging System, and treatment strategies were first planned based on staging results. Following PET/CT, the clinical stage was reevaluated in each patient, and treatment strategies were decided based on the re-staging results. Result: Seven patients (37%) had bone marrow involvement, eight (42%) were in Ann Arbor stage I–II, and 11 (58%) had a systemic dissemination. Most patients with systemic dissemination (9/11, 82%) had cutaneous lesions. The median number of disease sites was 4 (range, 1–15). The median number of positive lesions was 3 (range, 1–15) by PET/CT compared to 2 (range, 0–14) by CT (p = 0.12). Using PET/CT, 108 lesions (93%) were detected, and at least one FDG-avid lesion was observed at initial staging workups in all patients. In contrast, 70 lesions (60%) were detected by CT, and three patients (16%) did not show any positive lesion. Two lesions (2%) at the nasal cavity that were detected by biopsy were undetectable by either PET/CT or CT. The nodal and extranodal regions were separately evaluated by PET/CT and conventional modalities. In total, 28 nodal lesions were detected: all (100%) were positive by PET/CT and 26 (93%) by CT. Conversely, 89 extranodal lesions were detected, and 83 (93%) and 44 (49%) were positive by PET/CT and CT (p = 0.003), respectively. For the detection of upper aerodigestive lesions, PET/CT and CT demonstrated similar results: 23 lesions (92%) vs. 17 (68%), respectively. Notably, PET/CT was superior to CT in detecting cutaneous lesions [31 lesions (100%) vs. 19 lesions (61%), respectively; p = 0.026]. Bone marrow involvement was confirmed pathologically in only seven patients; four cases (57%) were positive by PET/CT and none by CT. Using conventional modalities, 11 patients (58%) were in the localized stage and eight (42%) were in the advanced stage. Using PET/CT, eight (42%) were in stage I–II and 11 (58%) were in stage III–IV. Most patients received chemotherapy plus local irradiation in stage I–II, and intensive chemotherapy with or without hematopoietic stem cell transplantation in stage III–IV. One patient did not receive treatment because of unwillingness for treatment and older age. PET/CT findings altered the stage and treatment strategy in four (21%) and two cases (11%). Conclusion: In extranodal NK/T-cell lymphoma, PET/CT demonstrated a high detection rate for nodal and extranodal lesions, except in the bone marrow. PET/CT may have an impact on treatment strategy and is essential for risk-adapted treatment. Disclosures: No relevant conflicts of interest to declare.

Abstract 2700 Poster Board II-676 Background: Hepatitis B virus reactivation after systemic chemotherapy including rituximab is a well-documented complication. However, no studies have investigated the influence of hepatitis C virus (HCV) infection for hepatic toxicity of diffuse large B-cell lymphoma (DLBCL) patients treated by rituximab containing chemotherapy. The prognostic value of HCV infection in DLBCL in the era of rituximab was also unclear. Herein we conducted a multicenter retrospective analysis to compare the outcome and hepatic toxicity of DLBCL patients with and without HCV infection treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) as an initial therapy. Methods: We analyzed 548 patients: HCV-positive (n=126) or -negative (n=422) patients with CD20-positive DLBCL receiving RCHOP between January 2004 and March 2008. HCV-negative patients treated during same period with HCV-positive patients in each institute were enrolled. Hepatitis B surface antigen positive patients were excluded in this study. For survival analysis, event-free survival (EFS) and overall survival (OS) were compared according to HCV infection. The definition of severe hepatic toxicity was more than Grade 3 transaminases increase according to National Cancer Institute of Canada criteria. The change of serum HCV-RNA levels was examined in 33 HCV-positive patients. Results: Before the treatment, HCV-positive patients had higher age (P Conclusion: Our data show that HCV infection is not prognostic factor, but was the significantly influential for hepatic toxicity in the patients with DLBCL treated by RCHOP therapy. Further prospective studies are warranted to clear whether antiviral therapy should be added to rituximab and chemotherapy. Disclosures: Kinoshita: Chugai Pharmaceutical Co Ltd, Zenyaku Kogyo Co. Ltd: Honoraria; Zenyaku Kogyo Co. Ltd: Research Funding.

8551 Background: CD5+ DLBCL comprises 5–10% of DLBCL, and shows a high incidence of central nervous system (CNS) relapse. It has been included in the 4th WHO classification as an immunohistochemical subgroup. To clarify the prognosis and incidence of CNS relapse of CD5+ DLBCL in the rituximab-era, we conducted a multicenter retrospective study. Methods: We analyzed 313 patients (pts) with CD5+ DLBCL who received chemotherapy with (n=164) or without rituximab (n=149). The current series includes 107 out of 120 pts described in our previous study (Haematologica, 2008). Intravascular large B-cell lymphoma, primary CNS DLBCL, and secondary CD5+ DLBCL were excluded from the study population. Results: 313 pts showed the following clinical features: median age, 67 (range: 15–93); M:F=163:150; elevated serum LDH level, 71%; stage III/IV, 64%; IPI HI/H, 53%. No significant difference in clinical background such as the IPI and its five components, B symptom, male sex, and bone marrow involvement was found between pts who were treated with and without rituximab. Pts treated without rituximab received more dose-intensive chemotherapies (CHOP14, third-generation regimen, and high dose cytarabine-based regimen) than those treated with rituximab (24% vs. 7%, P No significant financial relationships to disclose.

Background: Several biomarkers indicating poor prognosis have been reassessed in patients with diffuse large B-cell lymphoma (DLBCL) treated by rituximab combination chemotherapy. However, no studies have investigated the outcome by combining these biomarkers for rituximab treatment. In addition, no large studies have reassessed the outcome of patients with CD5-positive DLBCL treated by rituximab. To determine the most influential biomarkers, we reassessed and investigated the predictive value of three biomarkers, namely Bcl-2 expression, GC phenotype and CD5 expression, in DLBCL patients treated with rituximab in combination with CHOP as an initial therapy. Methods: A total of 121 patients with CD20-positive DLBCL receiving RCHOP at our institute between April 2002 and October 2005 were enrolled in the study. To classify the samples into immunohistochemically defined GC or non-GC phenotypes, we used a previously described algorithm using expression of CD10, Bcl-6, and MUM1 (Hans et al. Blood, 2004). For examination of the CD5 expression, we defined the cases as CD5-positive if CD5 expression was detected by immunohistochemical and flow cytometric analyses. We also assessed the outcome of patients according to International Prognostic Index (IPI). Results: Expression of Bcl-2 was detected in 79 of the 121 (65%) patients, while CD5 expression was positive in 11 patients (9%). Overall, 48 of the 121 (40%) patients expressed a non-GC phenotype. CD5-positive patients displayed a significantly poorer progression-free survival (PFS) and overall survival (OS) than CD5-negative patients (2-year PFS, 18% vs. 73%, P Conclusion: These results demonstrated that CD5 expression was the most influential prognostic factor among the biomarkers examined and associated with a very poor outcome, even after rituximab treatment. To confirm this conclusion, further large studies of CD5-positive DLBCL patients are required. Cox’s hazard regression analysis of PFS and OS Variable RR 95% CI P RR indicates relative risk; CI, confidence interval; GC phenotype, non-GC type worse; and IPI, higher IPI worse PFS CD5 13.57 4.260–42.947

Background: In recent years, it has been reported that addition of rituximab to CHOP chemotherapy significantly improves event-free survival (EFS) and overall survival (OS) compared with standard CHOP alone in patients with diffuse large B-cell lymphoma (DLBCL). On the other hand, gastric DLBCL is a good clinical outcome without surgical excision in the group of primary extranodal DLBCL. Localized gastric DLBCL shows very good EFS and OS treated with standard CHOP plus radiation therapy without rituximab. Methods: We analyzed retrospectively the prognosis between R-CHOP and CHOP alone in patients with localized gastric DLBCL. All 24 patients diagnosed between July 1999 and July 2006 in our hospital, were included in this study. The pathology was reviewed by a hematopathologist and confirmed to be de novo DLBCL according to the WHO classification. The staging system was using by Lugano meeting classification. Patients with stage IIE and IV were excluded. All patients were initially treated with six cycles of CHOP or R-CHOP regimen consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. If we evaluated partial response after six cycles of chemothrapy, the patients have been added radiation therapy. Results: Initial characteristics of the 24 patients are summarized in Table 1. The median age of patients at time of inclusion was 67.5 yr (range 30–83 yr). The median follow up of surviving patients was 30 months. CR rates between CHOP and R-CHOP were 91.7% vs. 83.3%, p=1.000, and overall response rates were 100% vs. 83.3%, p=.478, respectively. 2 years progression-free survival and overall survival were no statistical significance difference between two groups (Log rank test: P=.138, P=.739). Conclusion: These results suggested that addition of rituximab to CHOP chemotherapy does not improve clinical outcome of localized gastric DLBCL. The patients with localized gastric DLBCL did not get the clinical benefits of rituximab. As you know, rituximab needs high costs, so we recommend standard CHOP regimen plus radiation therapy in patients with localized gastric DLBCL, without rituximab combination regimen. | Characteristic | CHOP (n=12) | R-CHOP (n=12) | P | |:-------------- | ----------- | ------------- | ----- | | Age | | | 1.000 | | =60 | 9 | 8 | | | Male | 7 | 6 | 1.000 | | Stage (Lugano) | | | .890 | | I | 4 | 3 | | | II 1 | 7 | 9 | | | Unknown | 1 | | | | IPI score | | | .867 | | 0,1 | 7 | 7 | | | 2 | 4 | 5 | | | Unknown | 1 | | | Characteristics of the 24 patients with localized gastric DLBCL

Background: In recent years, diffuse large B-cell lymphoma (DLBCL) has been classified into the germinal center B-cell (GC) type, the activated B-cell (ABC) type, and the type 3 using global gene expression profiling or immunohistochemical staining. It has been reported that the GC type DLBCL showed significantly longer survival than the non-GC (ABC and the type 3) type DLBCL treated with CHOP or CHOP like regimen not using rituximab. Methods: We analyzed retrospectively the prognosis between the GC and non-GC types of DLBCL treated with R-CHOP regimen. All 50 patients with DLBCL, diagnosed between July 2003 and July 2005 were included in this study. The pathology was reviewed by hematopathologist and confirmed to be de novo DLBCL according to the WHO classification. Patients with primary CNS- and post-transplant lymphomas were excluded. GC type or non-GC type DLBCL was determined by immunohistochemistry such as the expression patterns of CD10, BCL-6, and IRF-4 (MUM1). All patients were initially treated with six cycles of R-CHOP regimen consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. If we evaluated partial response after six cycles of R-CHOP, the patients have added radiation therapy. Results: The patients consisted of 30 GC type and 20 non-GC type DLBCL with a median age of 61.0 yr (range 31–83 yr). The median follow up of surviving patients was 24 months. CR rate between GC and non-GC types were 57.0% vs. 75.0%, p=0.186, and overall response rate were 87.0% vs. 90.0%, p=0.929, respectively. The median of progression free survival was 17.3 months vs. 19.6 months, p=0.80. There is no statistical significance difference between two groups. Conclusion: These results suggest that addition of rituximab to CHOP regimen improves clinical outcome of non-GC type DLBCL as well as GC type DLBCL.

Introduction: MALT lymphoma, even if advanced stage, is known to have indolent outcome. However, previous reports showed that chemotherapy only could not improve the disease progression. Recently a few reports demonstrated rituximab monotherapy could not acquired enough response for the patients with advanced MALT lymphoma. We retrospectively analyzed outcomes of patients with advanced MALT lymphoma treated with rituximab monotherapy or rituximab-CHOP like regimen (R-CHOP), and discussed about the treatment of this entity. We also performed the immunohistochemcal study of bcl-2 and reverse-transcription polymerase-chain-reaction (RT-PCR) analysis of API2-MALT1, and evaluate whether the existence of bcl-2 or API2-MALT1 makes any influence on the response to rituximab treatments. Patients and methods: Between October 2001 and October 2005, 106 patients (pts) were treated in our institute and 21 pts with advance-staged MALT lymphomas were included in this study. Advanced stage with GI tract MALT lymphoma was defined as a stage II1+2 or IV according to Lugano staging system. On the other hand, advanced stage with non-GI tract lymphoma was defined as a stage III or IV. In this study, immunohistochemical staining with bcl-2 and RT-PCR analysis of API2-MALT1 were performed. Results: Twenty-one pts (12 men and 9 women) were treated with rituximab treatment and a median age was 65 years (27–83 years). Stage II+III pts and IV pts were 9 pts (22.9%) and 12 pts (57.1%), respectively. Three of five HP-positive pts did not respond antibiotic therapy. The remaining HP-negative 16 pts received rituximab therapy as first line treatment. Of the patients who required systemic chemotherapy, 16 pts were treated with R-CHOP; 5 pts with rituximab monotherapy. The overall response rates (ORR) were 100% (CR rate = 62.5%) in the R-CHOP group, 60% (CR rate = 60%) in rituximab monotherapy, respectively. The median follow up was 2.2 years. Relapse rate were 6.2% (1/17) in the R-CHOP group, 40% (2/5) in the rituximab monotherapy, respectively. Bcl-2 was expressed in 11 of 17 tissues tested, and CR rates were 63.3/37% in bcl-2 +/− patients. Grade 3 to 4 cytopenia was observed in 87.5% of patients in R-CHOP group, but not in rituximab monotherapy. Grade3 to 4 non-hematological toxicity was not seen in both groups. Conclusion: R-CHOP exhibited impressive efficacy with one failure among 17 patients. ORR was also significantly better in R-CHOP group than in rituximab monotherapy. The efficacy of rituximab monotherapy was similar to previous reports. These results strongly suggest that R-CHOP improves the response of advance-staged MALT lymphoma. In this study, bcl-2 and API2-MALT1 did not contribute to response to rituximab treatment. R-CHOP is expected to prolong relapse-free survival compared to rituximab monotherapy or chemotherapy only. Scince hematologic toxicity in R-CHOP group is severe, R-CVP regimen which is less toxicity may be necessary to be evaluated.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.