Mutations In Diffuse Large B-cell Lymphoma Research Articles

Abstract B51: FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy

Abstract Lack of remission or early relapse remains a major clinical concern in diffuse large B-cell lymphoma (DLBCL), with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent targets for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild-type and mutated) on the expression of the rituximab-target, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available Chip-Seq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK-2206 and GDC-0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab-mediated complement-dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild-type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1-AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA-binding defective mutant of FOXO1-H215R, suggesting a FOXO1-mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA, and the analysis of ChIP-Seq data using anti-FOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity effectively suppress CD20 expression in comparison to wild-type FOXO1, through a binding to CD20 promoter. In addition, the disruption of FOXO1 with CRISPR/Cas9 genome editing resulted in CD20 upregulation and improved the cytotoxicity induced by rituximab and survival of mice with sgFOXO1 tumors. Consistently, in primary samples pharmacologic inhibition of FOXO1 activity upregulated surface CD20 levels. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity needs further attention while designing rituximab-based regimens in the therapy of selected lymphomas. Supported by National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwoyak, giulia Morlino, Piotr Zapala, Nina Miazek, Jakub Golab, Magdalena Winiarska. FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B51.

EP300 single nucleotide polymorphism rs20551 correlates with prolonged overall survival in diffuse large B cell lymphoma patients treated with R-CHOP

BackgroundRituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) is used as standard frontline regimen for diffuse large B-cell lymphoma (DLBCL). The landscape of somatic mutations in DLBCL revealed that inactivation of EP300 plays an important role in lymphomagenesis. A common EP300 single nucleotide polymorphism (SNP) rs20551 results in the substitution of valine for isoleucine at codon 997 close to the Bromodomain. However, the association between SNP rs20551 and clinical prognosis in DLBCL patients treated with R-CHOP is unknown.MethodsIn this study we analyzed the EP300 SNP rs20551 and prognosis of 226 DLBCL patients who treated with R-CHOP or R-CHOP-like regimes from 2002 to 2013. Determination of the EP300 SNP rs20551 from genomic DNA was obtained by Sanger chain termination sequencing.ResultIn this study, the frequency of the A and G allele of the EP300 SNP rs20551 in 226 patients were 92.5 and 7.5%, respectively. We did not observe obvious correlation between patients’ disease features and the EP300 SNP rs20551. But the patients with genotype AA had a higher 5-year overall survival rate than those with genotype GA (77.0% vs. 64.7%, p = 0.045). Furthermore, multivariate Cox regression analysis showed that the GA genotype of EP300 SNP rs20551 was an independent poor prognostic factor for DLBCL patients treated with Rituximab-chemotherapy (p = 0.009, HR 2.956, 95% CI 1.315–6.645).ConclusionThis study suggests that EP300 SNP rs20551 might be a useful biomarker to predict the long-term outcome of R-CHOP in DLBCL patients.

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice

AbstractCREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

Diffuse large B-cell lymphoma genotyping on the liquid biopsy

AbstractAccessible and real-time genotyping for diagnostic, prognostic, or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. In this study, we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pretreatment plasma cfDNA from DLBCL patients correctly discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with >90% sensitivity and ∼100% specificity. Plasma cfDNA genotyping also allowed for the recovery of mutations that were undetectable in the tissue biopsy, conceivably because, due to spatial tumor heterogeneity, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients who were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations were acquired in cfDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and noninvasive approach to tracking clonal evolution and the emergence of treatment-resistant clones.

B-cell Function Gene Mutations in Diffuse Large B-cell Lymphoma: A Retrospective Cohort Study

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous subtype of non-Hodgkin lymphoma. In addition to clinical and immunophenotypic characteristics, recurrent gene mutations have recently been identified in patients with DLBCL using next-generation sequencing technologies. The aim of this study is to investigate the clinical relevance of B-cell function gene mutations in DLBCL. Clinical analysis was performed on 680 Chinese DLBCL patients (146 non-CR and 534 CR cases) treated with six cycles of 21-day R-CHOP (Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), alone or followed by two additional doses of rituximab consolidation on patients' own intention. Somatic mutations of B-cell function genes were further screened on 275 (71 non-CR and 204 CR) cases with available tumor samples by targeted sequencing, including genes involved in B-cell receptors (BCRs) pathway (CARD11, LYN, CD79A, and CD79B), Toll-like receptors (TLRs) pathway (MYD88), and tumor necrotic factor receptor (TNFR) pathway (TRAF2 and TNFAIP3). B-cell function gene mutations occurred in 44.0% (121/275) of DLBCL patients. The TLRs and TNFR related gene mutations were more frequently observed in non-CR patients (p=0.019 and p=0.032, respectively). BCRs related gene mutations, as well as revised IPI (R-IPI) and double BCL-2/MYC expression, were independently related to short progression-free survival in DLBCL after CR. The adverse prognostic effect of BCRs related gene mutations could be overcome by two additional doses of rituximab consolidation. These results highlight the molecular heterogeneity of DLBCL and identify a significant role of B-cell function gene mutations on lymphoma progression and response to rituximab in DLBCL.

Biological and Clinical Relevance of Associated Genomic Alterations in MYD88 L265P and Non-L265P Mutated Diffuse Large B-Cell Lymphoma: Analysis of 361 Cases

Introduction: MYD88 mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of Activated B-Cell like (ABC) Diffuse Large B Cell Lymphoma (DLBCL), leading to constitutive NFkB pathway activation. The frequency of MYD88 mutations in DLBCL and other hematologic malignancies is well described; however, there has not yet been a large-scale study of a MYD88 mutated patient cohort with additional Next Generation Sequencing (NGS), copy number variation (CNV), and gene expression data, in order to thoroughly characterize the associated genomic profiles of these patients. The aims of our study were to compare the L265P and non-L265P mutations in terms of pathological and genetic features, to better detail the genomic background associated with MYD88 mutations in order to delineate patients potentially sensitive to targeted therapies, and to define the prognostic value of MYD88 mutations according to different genomic contexts.Methods: A cohort of 361 DLBCL patients (94 MYD88 mutant and 267 MYD88 wild-type) was selected among the prospective, multicenter and randomized LNH-03B and LNH09-7B (NCT01195714) LYSA trials, as well as among patients sequenced at our institution as part of routine procedure. Cell of origin (COO) classification was obtained with HGU133+2.0 Affymetrix GeneChip arrays for 214 patients, with RT-MLPA for 77 patients1 and with Hans immunohistochemistry (IHC) method for 49 patients. All cases were submitted to next generation sequencing (NGS) focusing on 34 genes (Lymphopanel2) in order to analyze associated mutations and copy number variations (CNVs), as well as IHC, FISH, and clinical and prognostic analyses.Results: Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis segregated subgroups according to associated genetic alterations among patients with either MYD88 L265P or non-L265P mutations. As such, clustering separated MYD88 L265P mutated DLBCL with associated PIM1 (52%), CD79B (52%), KMT2D (42%), and PRDM1 (32%) mutations, as well as MYD88 L265P mutated DLBCL with CDKN2A/B (67%/50%), PRDM1 (57%) and TNFAIP3 (52%) CNVs. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFkB pathway activation, although the majority of ABC MYD88 L265P mutant cases harbor downstream NFkB alterations, which can potentially predict BTK inhibitor resistance. Of note, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated CD79B mutations significantly improved the survival of MYD88 L265P mutant ABC DLBCL in our cohort both in OS (p=0.02) and PFS (p=0.01), whereas the association of CARD11 or TNFAIP3 alterations did not impact survival. Interestingly, MYD88 mutant DLBCL cases were significantly more likely to experience central nervous system (CNS) relapse than MYD88 WT cases (p=0.02), as were MYD88 L265P mutant cases specifically (p=0.03). This result still tended toward statistical significance when considering only ABC patients (7 of 11 ABC CNS-relapsing cases were MYD88 mutant, p=0.1) but would have to be confirmed in a larger cohort.Conclusions: This study highlights the relative heterogeneity of MYD88 mutant DLBCBL, adding to the field's knowledge of the distinct genetic backgrounds of these subgroups. Our data highlights the theranostic and prognostic relevance of examining MYD88 and associated genomic alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy.1. Mareschal S, Ruminy P, Bagacean C, et al. Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study. The Journal of molecular diagnostics: JMD. 2015;17(3):273-283.2. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study. Clinical cancer research: an official journal of the American Association for Cancer Research. 2016;22(12):2919-2928. DisclosuresSalles:Novartis: Consultancy, Honoraria; Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Somatic Mutations of Epigenetic Regulator Genes in Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive types of B-cell lymphoma with high heterogeneity, accounting for 30-40% of newly diagnosed non-Hodgkin lymphoma (NHL), and dysfunction of epigenetic regulation has been found as a common and important feature of B cell lymphomas. To identify epigenetic associated genes mutations in DLBCL, including KMT2D, CREBBP, EP300, EZH2 and MEF2B, we sequenced tumour DNA from 226 Chinese DLBCL cases by applying next generation sequencing technology (NGS). A total of 679 consecutive Chinese patients with previously untreated DLBCL at our institution from December 2006 and January 2016 were enrolled in this study, and we assessed the predictive value of clinical and mutational pattern of epigenetic associated genes in a large single-institution cohort of these patients.Methods. Genomic DNA was extracted from 226 subjects with DLBCL formalin-fixed paraffin-embedded tumor tissue, using a QIAamp DNA FFPE Tissue Kit (Qiagen). Specific primers, producing amplicons about 200 bp at the coding regions of the genes of interest , were designed at the UCSC website (http://genome.ucsc.edu/cgi-bin/hgGateway ). Microfluidic PCR reactions ran in a 48 ¡Á 48 Access array system (Fluidigm) with FastStart High Fidelity PCR system (Roche) and high-throughput DNA sequencing was performed on Illumina Genome Analyzer IIx (GAIIx) and HiSeq2000 systems, according to the manufacturer’s instructions. SAMtools version 0.1.19 was used to generate chromosomal coordinate-sorted bam files and to remove PCR duplications. Sequences for epigenetic associated genes were obtained from the UCSC Human Genome database, using the corresponding mRNA accession number as a reference, and those containing splice-site, nonsense or coding-region indel mutations, were selected for Gene Ontology analysis. All of the results were also confirmed by Sanger sequencing. Baseline characteristics of patients were analysed using two-sided c2 test. Overall survival (OS) was estimated using the Kaplan-Meier method and compared by log-rank test. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Covariates demonstrating significance with P<0.100 on univariate analysis were included in the multivariate model. Statistically significance was defined as P<0.05. All statistical analyses were carried out using Statistical Package for the Social Sciences (SPSS) 20.0 software (SPSS Inc., Chicago, IL, USA).Results. Overall, 105 of 226 Chinese DLBCL cases were identified to have at least one mutation in epigenetic regulator genes. Somatic mutations in KMT2D were most frequently observed (24.3%), followed by CREBBP, EP300, EZH2 and MEF2B (15.5%,10.6%,4.4% and 2.2%, respectively)(Figure1,A,B). Association of mutated genes according to the conceptual classification. Circos plot of mutated genes according to the function is shown, and overlap mutations between epigenetic regulator genes mutations were frequently observed (Figure1, C). Clinically, mutation-positive DLBCL patients presented shorter OS than patients those without mutations (P=0.0286, Figure 1,D) among 226 DLBCL cases. A total of 679 Chinese DLBCL cases were enrolled in univariate analysis, and R-IPI, Complete Remission (CR), epigenetic related mutations were significant prognostic factors for OS. In further multivariate analysis, R-IPI (RR=2.72,95%CI=1.619-4.567,P<0.000), CR (RR=0.129,95%CI=0.076-0.219,P<0.000), epigenetic related mutations (RR=1.605,95%CI=1.007-2.557,P=0.046) are independent prognostic factor for OS.Conclusion. Our study provided the mutational spectrum of epigenetic regulator genes in DLBCL, and the relationships between mutations and clinic suggested some therapeutic efficiency. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transformation of Chronic Lymphocytic Leukemia Towards Richter´s Syndrome Is Induced By AKT Activation

Richter's syndrome (RS) is an aggressive transformation of Chronic Lymphocytic Leukemia (CLL) to Diffuse Large B Cell Lymphoma (DLBCL) refractory to current therapies with dismal prognosis. Richter Syndrome arises from CLL cells independent of common DLBCL mutations. Frequently, mutations in p53, CDKN2 or cMyc genes are involved, but a significant proportion displays no specifically acquired driver mutation.We could observe activation of AKT in 6 out 48 Richter syndrome biopsies by positive staining for active phosphorylated AKT while in CLL lymph nodes, DLBCL and Burkitt´s Lymphoma no phospho-AKT by IHC could be observed.However in primary patient CLL cases we could detect varying levels of pAKT by Western blot, elevated levels were identified predominantly in patients harboring high-risk mutations such p53, ATM, NOTCH1 and XPO1. Furthermore, B-cell receptor mediated stimulation of the PI3K/AKT axis provided protection towards genotoxic stress induced apoptosis via post-translation stabilization of MCL1. This provides subsequently a synergistic induction of apoptosis by combining idelalisib and bendamustin.Thus we analyzed the functional impact of AKT signaling using a conditional constitutive allele for AKT (AKT-C) specifically activated using CD19-Cre and Cγ1-Cre fro post-GC-activation. AKT activation alone could not induce a malignant phenotype, however we could demonstrate that Eµ-Tcl-1 mice with AKT-C develop Richter Syndrome. Both in EµTCL1:CD19-CreAKT-C (TCA) and EµTCL1:Cγ1- CreAKT-C (TCγ1A) mice developed a high-grade lymphoma phenotype leading to decreased survival. Transformed cells displayed blastoid characteristics with significantly increased cellular size and the histomorphological features of DLBCL. Large transformed cells show high percentage of KI67-positive staining (>90%) and frequent mitotic figures.Here, AKT-mediated GSK-3b inhibition and subsequent cMyc and Mcl-1 stabilization might transform CLL to RS cells and combinatory treatments with DNA-damaging and PI3K-inhibiting compounds revealed promising therapeutic results. Collectively, we have identified AKT signaling as an oncogenic signaling pathway in progression of CLL towards Richter´s syndrome and generated the first murine Richter Syndrome model (TCA and TCγ1A) providing novel mechanistic insights into the molecular understanding of Richter’s transformation that is amenable to model therapeutic strategies and to address the efficacy of synergistic treatment combinations. DisclosuresKlapper:Roche, Novartis, Amgen, Takeda: Research Funding. Hallek:Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements

Several classes of signaling proteins contain autoinhibitory domains that prevent unwarranted signaling and coordinate the induction of activity in response to external cues. CARD11, a scaffold protein critical for antigen receptor signaling to NF-κB, undergoes autoregulation by a poorly understood inhibitory domain (ID), which keeps CARD11 inactive in the absence of receptor triggering through inhibitory intramolecular interactions. This autoinhibitory strategy makes CARD11 highly susceptible to gain-of-function mutations that are frequently observed in diffuse large B cell lymphoma (DLBCL) and that disrupt ID-mediated autoinhibition, leading to constitutive NF-κB activity, which can promote lymphoma proliferation. Although DLBCL-associated CARD11 mutations in the caspase recruitment domain (CARD), LATCH domain, and coiled coil have been shown to disrupt intramolecular ID binding, surprisingly, no gain-of-function mutations in the ID itself have been reported and validated. In this study, we solve this paradox and report that the CARD11 ID contains an unusual array of four repressive elements that function cooperatively with redundancy to prevent spontaneous NF-κB activation. Our quantitative analysis suggests that potent oncogenic CARD11 mutations must perturb autoinhibition by at least three repressive elements. Our results explain the lack of ID mutations in DLBCL and reveal an unusual autoinhibitory domain structure and strategy for preventing unwarranted scaffold signaling to NF-κB.

Discovery of Recurrent Mutations Associated with Chemo-Immunotherapy Relapse in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common form of adult lymphoma. Although comprehensively investigated, it remains a prominently heterogeneous disease. The standard of care for DLBCL is a combination chemo-immunotherapy RCHOP regimen. The 3-year progression free survival (PFS) rate is 70%. Relapses normally occur within 2-3 years and treatment options for relapsed and refractory DLBCL are limited with only 10% of patients achieving a 3-year PFS. Global mRNA expression analysis has been used to further classify DLBCL into molecularly distinct subtypes (GCB and ABC) which display remarkably different outcomes to current therapeutics, with GCB having a better prognostic outcome. Multiple attempts at further restratification of DLBCL cases based on next generation sequencing have only confirmed the striking heterogeneity of the disease; providing little direction for clinical practices. Therefore, further analysis of DLBCL with the intent to stratify patients for clinical response is needed.We utilized whole exome sequencing to reveal alterations in the genome that are associated with relapse and chemotherapeutic resistance in DLBCL. Current literature has focused on DLBCL as an entity without stratification into relapsed/refractory and de novo DLBCL. In addition, published studies have had a clear focus on diagnostic sampling. By discovering and prioritizing somatic mutations associated with relapse and chemotherapeutic resistance, we aim to provide a novel analysis of the mutational heterogeneity of DLBCL to identify new biomarkers for improved stratification of patients for clinical treatment.Method: DLBCL samples from 37 cases (45 biopsies) were collected with normal matched blood (germline). Of these, 17 cases never relapsed (median observation time 3.9 yrs). Whereas, 20 cases relapsed from RCHOP or related therapy (median observation time 4.8 yrs). Of these, 15 biopsies were taken at diagnosis and 13 were taken at relapse including 8 cases with serial biopsies. We performed whole exome capture and paired-end sequencing using the Illumina HiSeq2000 platform. Reads of each sample were mapped with BWA-mem to the human reference genome (build b37 with an added decoy contig). Marking of duplicates was performed with Picard tools; GATK tools were used for two‐step local realignment and base‐quality recalibration. Somatic single nucleotide variant (SNV) detection was performed with MuTect and Strelka. Strelka alone was used for insertion/deletion (InDel) detection.Results: We achieved 288X mean coverage of targeted exonic regions in tumor samples and 125X in normal blood. After application of our bioinformatics pipeline, we identified 44,297 variants in the cohort. The mean mutational SNV burden was 944 and the mean InDel burden was 40 variants per case. In serial biopsies there was a higher mean mutational burden at relapse than diagnosis (1001 to 772, respectively). After filtering synonymous, UTR, non-coding, and low confidence variant calls, we identified 11,743 coding variants corresponding to 6,399 genes (Figure 1). In order to define potential recurrent driver mutations in DLBCL associated with relapse and survival, we applied the Oncodrive algorithm (IntOgen) to the total cohort and subgroups classified by relapse status, survival and subtype. We also applied the Oncocluster algorithm (IntOgen) to the total cohort to identify genes with significant hotspots and clustering which may suggest potential cancer driver mutations. Using a corrected q-value of <.1 we identified 102 potential driver mutations. Within our driver mutations we identified many of the previously discovered genes associated with DLBCL including; MLL2, B2M, CD58, MEF2B, FOXO1, TP53, PIM1, SOCS1, MYC, GNA13, SGK1, TNFAIP3, MYD88, PRDM1, CDKN2A, EZH2 and CIITA. However, as previously reported, our gene list does not extensively overlap with earlier DLBCL exome sequencing reports (Figure 2). This may, in part, be due to differing library preparations, sequencing methods, bioinformatic pipelines and criteria for gene selection, yet it also represents the heterogeneity of DLBCL as a neoplastic entity. Given the underlying heterogeneity of DLBCL, by focusing on relapse and survival status of our cohort the gene variants generated and analyzed in our analysis will provide a novel view of the DLBCL mutational landscape from the perspective of relapse and chemoresistance. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Liquid Biopsy As a Tool for Monitoring the Genotype of Diffuse Large B-Cell Lymphoma

Introduction. Accessible and real-time genotyping for diagnostic, prognostic or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Since DLBCL lacks a leukemic phase, tumor genotyping has so far relied on the analysis of the diagnostic tissue biopsy. Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of cancer-gene somatic mutations. Accessing the blood has obvious sampling advantages in the monitoring of mutations in real-time. Also, cfDNA is representative of the entire tumor heterogeneity, thus allowing to identify mutations from tumor cells residing in non-biopsied sites. Here we aimed at tracking the DLBCL genetic profile using plasma cfDNA.Methods. The study was based on 26 consecutive DLBCL patients (age >65=15, male:female ratio=11:15) presenting in different Ann Arbor stages (III-IV=13) and age-adjusted IPI risk scores (2-3=13), and provided with cfDNA from plasma collected at diagnosis, during R-CHOP course, at the end of treatment and at progression. Paired normal genomic DNA from granulocytes was also collected for comparative purposes to filter out polymorphisms. A targeted resequencing panel including the coding exons and splice sites of 59 genes (207 kb) that are recurrently mutated in mature B-cell tumors was specifically designed to allow the recovery of at least one mutation in >90% of DLBCL. Ultra-deep next-generation-sequencing (NGS) of the gene panel was performed on MiSeq (Illumina) (coverage >2000x in >80% of the target) using a SeqCap library preparation strategy (NimbleGen). The somatic function of VarScan2 was used to call non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors. The study cohort was divided in a training set of 17 patients provided with paired tumor DNA from the diagnostic tissue biopsy that was used for the set up of the ultra-deep NGS strategy, and an extension set of 9 patients lacking the tissue biopsy.Results. A total of 76 cfDNA samples (26 pretreatment, 28 during treatment, 18 at the end of treatment, and 4 at time of progression) were evaluated. Pretreatment cfDNA genotyping disclosed somatic mutations of heterogeneous abundance (median mutated molecules/ml of plasma: 3168, range 1.73-6.5x104) in known DLBCL-associated genes, including MLL2 (33%), TP53 (25%), CREBBP and TNFAIP3 (21%), EZH2, TBL1XR1, PIM1 (17%), B2M, BCL2, CARD11, CCND3, FBXW7 and STAT6 (13%) (Fig. 1A). The results of genotyping on cfDNA from plasma and of genomic DNA from tumor cells of the diagnostic biopsy (gold standard) were compared to derive the diagnostic accuracy of cfDNA genotyping (Fig. 1B). Genotyping of the paired plasma cfDNA correctly identified 79% of the tumor biopsy mutations. Most of the tumor variants not discovered in the cfDNA had a low representation in the tumor biopsy (median allelic abundance=5.7%; range 0.8-54%). Consistently, ROC analysis showed that cfDNA genotyping had the highest sensitivity (92%) if mutations were represented in >15% of the alleles of the tumor biopsy. Plasma cfDNA genotyping also disclosed a number of additional somatic mutations (~2 per case, range 1-6) that were not detectable in the tissue biopsy, including mutations of clinically relevant genes. Longitudinal analysis of plasma samples under R-CHOP chemotherapy showed a rapid clearance of the DLBCL mutations in the cfDNA already after the first cycle among responding patients (Fig. 1C). Among patients that were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations appeared in cfDNA that conceivably marked resistant clones selected during the clonal evolution process taking place under the pressure of treatment (Fig 1D).Conclusions. Overall, these results provide the proof of principle that cfDNA genotyping of DLBCL: i) is as accurate as genotyping of the diagnostic biopsy to detect somatic mutations of allelic abundance >15% in DLBCL; ii) allows the identification of mutations that are otherwise absent in the tissue biopsy conceivably because restricted to clones that are anatomically distant from the biopsy site; and iii) is a real-time and non-invasive way to track clonal evolution and emergence of treatment resistant clones. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Genetic Aberrations in Diffuse Large B Cell Lymphoma

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<0.0001). In univariate analyses we identified several gene mutations and copy number aberrations significantly associated with survival in all patients. Of these genes, MYD88 and TP53 mutations were associated with significantly inferior TTP in the ABC subtype (p=0.04) and GCB subtype (p=0.002), respectively, while TMEM30A, CREBBP, PIM1 and BTG1 mutations were associated with prognosis in DLBCL. Our analyses confirm the poor prognosis conferred by TP53 mutations in DLBCL and, importantly, identified several novel genetic alterations associated with survival stratified by COO distinctions.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. DisclosuresNo relevant conflicts of interest to declare.

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

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both biologically and clinically. Although a number of genetic alterations have been associated with the pathogenesis of DLBCL, survival association of most of the identified mutations remains to be shown. Here we have used exome and RNA sequencing (RNAseq) data to identify novel genetic events associated with treatment outcome after chemoimmunotherapy.Initial screen was performed with eight primary tumor samples and matched normal DNA using exome sequencing. The patients had clinically high risk DLBCL, and were treated in a Nordic phase II protocol with dose dense chemoimmunotherapy and CNS prophylaxis. After stringent filtering (VarScan2 p < 0.05), and removal of known germline variants, 967 somatic point mutations and indels in 561 different genes were identified. Of these, 436 were non-synonymous, and 27 were detected in more than one patient. These included both novel and several previously described DLBCL associated mutations, such as MYD88, CD79B, B2M and PTEN.To correlate the identified mutations with survival, we used RNAseq and survival data of 92 DLBCL samples from the Cancer Genome Characterization Initiative (CGCI) repository. The mutational status of the 436 genes identified in our screen was assessed with Bambino, which confirmed 405 of these genes also to be mutated in the CGCI set. Kaplan-Meier survival estimates per gene with at least one single nucleotide variant (SNV; with criteria of minimum 3 reads and 10% coverage and known polymorphisms filtered out) revealed 17 genes that were associated with overall survival (OS, p<0.05, minimum 10 samples mutated).We highlight mutations affecting Deltex1 (DTX1), which functions as an E3 ubiquitin ligase and targets Notch1 intracellular domain for degradation. In the study cohort, 14% of the patients had a non-synonymous DTX1 mutation. Mutations were equally distributed between GCB and ABC DLBCLs and low (0-2) and high (3-5) International Prognostic Index (IPI) risk groups. According to Kaplan-Meier analysis, the 5-year PFS rate for the patients with DTX1 mutations was significantly worse as compared to ones without mutations (46% vs 78%, p=0.010). The corresponding OS values were 54% and 85% (p=0.006). In multivariate analysis with IPI, DTX1 mutation status remained as an independent prognostic factor for both PFS (RR, 2.83; 95% CI, 1.17-6.85, p=0.021) and OS (RR, 3.51; 95% CI, 1.41-8.71, p=0.007). Mutational status was also found to correlate with the expression levels; the patients with mutations had lower DTX1 mRNA levels (p=0.006). When DTX1 mutations were analyzed according to different functional domains, 11 out of 16 (65%) were located in the first exon encoding the WWE 1 domain and a binding site for intracellular domain of Notch1. Interestingly, the patients with mutations in exon 1 had a significantly worse survival in comparison to other patients (Figure 1).Taken together, the results demonstrate that DTX1 mutations in DLBCL are an independent predictor of survival after chemoimmunotherapy, and mutations enriched in exon 1 identify cases with dismal prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PP-082 DELAYED ENGRAFTMENT DUE TO CANDIDA NORVEGENSIS IN HDC AND SCT PATIENT

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous subtype of non-Hodgkin lymphoma. In addition to clinical and immunophenotypic characteristics, recurrent gene mutations have recently been identified in patients with DLBCL using next-generation sequencing technologies. The aim of this study is to investigate the clinical relevance of B-cell function gene mutations in DLBCL. Clinical analysis was performed on 680 Chinese DLBCL patients (146 non-CR and 534 CR cases) treated with six cycles of 21-day R-CHOP (Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), alone or followed by two additional doses of rituximab consolidation on patients' own intention. Somatic mutations of B-cell function genes were further screened on 275 (71 non-CR and 204 CR) cases with available tumor samples by targeted sequencing, including genes involved in B-cell receptors (BCRs) pathway (CARD11, LYN, CD79A, and CD79B), Toll-like receptors (TLRs) pathway (MYD88), and tumor necrotic factor receptor (TNFR) pathway (TRAF2 and TNFAIP3). B-cell function gene mutations occurred in 44.0% (121/275) of DLBCL patients. The TLRs and TNFR related gene mutations were more frequently observed in non-CR patients (p = 0.019 and p = 0.032, respectively). BCRs related gene mutations, as well as revised IPI (R-IPI) and double BCL-2/MYC expression, were independently related to short progression-free survival in DLBCL after CR. The adverse prognostic effect of BCRs related gene mutations could be overcome by two additional doses of rituximab consolidation. These results highlight the molecular heterogeneity of DLBCL and identify a significant role of B-cell function gene mutations on lymphoma progression and response to rituximab in DLBCL.

PP-083 SECONDARY ALL IN MULTIPLE MYELOMA PATIENT WHO UNDERWENT HDC AND SCT

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous subtype of non-Hodgkin lymphoma. In addition to clinical and immunophenotypic characteristics, recurrent gene mutations have recently been identified in patients with DLBCL using next-generation sequencing technologies. The aim of this study is to investigate the clinical relevance of B-cell function gene mutations in DLBCL. Clinical analysis was performed on 680 Chinese DLBCL patients (146 non-CR and 534 CR cases) treated with six cycles of 21-day R-CHOP (Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), alone or followed by two additional doses of rituximab consolidation on patients' own intention. Somatic mutations of B-cell function genes were further screened on 275 (71 non-CR and 204 CR) cases with available tumor samples by targeted sequencing, including genes involved in B-cell receptors (BCRs) pathway (CARD11, LYN, CD79A, and CD79B), Toll-like receptors (TLRs) pathway (MYD88), and tumor necrotic factor receptor (TNFR) pathway (TRAF2 and TNFAIP3). B-cell function gene mutations occurred in 44.0% (121/275) of DLBCL patients. The TLRs and TNFR related gene mutations were more frequently observed in non-CR patients (p = 0.019 and p = 0.032, respectively). BCRs related gene mutations, as well as revised IPI (R-IPI) and double BCL-2/MYC expression, were independently related to short progression-free survival in DLBCL after CR. The adverse prognostic effect of BCRs related gene mutations could be overcome by two additional doses of rituximab consolidation. These results highlight the molecular heterogeneity of DLBCL and identify a significant role of B-cell function gene mutations on lymphoma progression and response to rituximab in DLBCL.

Histone acetyltransferase-deficient p300 mutants in diffuse large B cell lymphoma have altered transcriptional regulatory activities and are required for optimal cell growth

BackgroundRecent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP. The majority of these mutations reduce or eliminate the catalytic HAT activity. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300ΔC-1087), whose expression is essential for cell proliferation.MethodsUsing results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300ΔC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300ΔC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300ΔC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300.ResultsWe show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300ΔC-820), but no wild-type p300. The p300ΔC-820 protein has a wild-type ability to localize to subnuclear “speckles,” but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300ΔC-820 in SUDHL2 cells reduced their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300ΔC-1087 resulted in increased expression of mRNA and protein for REL target genes A20 and IκBα, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18.ConclusionsThe high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

BCL2 mutations in diffuse large B-cell lymphoma

BCL2 is deregulated in diffuse large B-cell lymphoma (DLBCL) by the t(14;18) translocation, gene amplification and/or nuclear factor-κB signaling. RNA-seq data have recently shown that BCL2 is the most highly mutated gene in germinal center B-cell (GCB) DLBCL. We have sequenced BCL2 in 298 primary DLBCL biopsies, 131 additional non-Hodgkin lymphoma biopsies, 24 DLBCL cell lines and 51 germline DNAs. We found frequent BCL2 mutations in follicular lymphoma (FL) and GCB DLBCL, but low levels of BCL2 mutations in activated B-cell DLBCL, mantle cell lymphoma, small lymphocytic leukemia and peripheral T-cell lymphoma. We found no BCL2 mutations in GC centroblasts. Many mutations were non-synonymous; they were preferentially located in the flexible loop domain, with few in BCL2-homology domains. An elevated transition/transversions ratio supports that the mutations result from somatic hypermutation. BCL2 translocations correlate with, and are likely important in acquisition of, additional BCL2 mutations in GCB DLBCL and FL. DLBCL mutations were not independently associated with survival. Although previous studies of BCL2 mutations in FL have reported mutations to result in pseudo-negative BCL2 protein expression, we find this rare in de-novo DLBCL.

TET2 mutations in Diffuse Large B-Cell Lymphoma: The Role of TET2 in the Regulation of Methylation Patterns at TET2 Target Genes

Abstract 1364 Introduction:Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011). Aims:In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis. Methods:DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR. Results:We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells. Conclusion and further analyses:Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation. Disclosures:Christensen:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.

Abstract LB-186: Somatic Y641 mutations in EZH2 alter the balance of di- and tri-methylation of H3K27 in diffuse large B-cell lymphomas

Abstract EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. PRC2 activity is essential for maintaining the self-renewal capacity of embryonic and adult stem cells and the dynamic regulation of this activity is critical for proper development and differentiation. Dysregulation of H3K27 methylation is implicated in tumorigenesis and occurs through multiple mechanisms. Elevated levels of EZH2 are known to correlate with poor prognosis in a number of solid tumors including prostate and breast. Inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2, have been described in several tumor types including multiple myeloma, esophageal squamous cell carcinoma, and renal cell carcinoma. More recently, somatic mutations in EZH2 were identified in myelodysplastic syndrome (MDS), follicular lymphoma (FL), and GCB diffuse large B cell lymphoma (DLBCL). While the mutations in MDS are often homozygous and encompass missense, nonsense, and frame shift mutations at multiple regions of the protein, the mutations in DLBCL and FL are heterozygous and occur at a single residue (Y641) suggesting that the effect of these mutations on PRC2 activity could be quite different between MDS and lymphoma. Utilizing a biochemical approach with recombinant PRC2 containing either wild-type or mutant EZH2, we demonstrate that Y641 mutants exhibit an altered substrate preference. In contrast to wild-type EZH2 which prefers an unmodified or mono-methylated K27 residue, Y641 mutants act primarily on a di-methylated H3K27 with little activity for unmodified or mono-methylated K27. Consistent with these biochemical data, we find that when compared to wild-type lymphoma cell lines those harboring Y641 mutations have elevated levels of H3K27me3 and reduced H3K27me2. To further characterize the substrate specificity of PRC2 complexes containing either WT or mutant EZH2, we utilized an epigenetic peptide library which contained unmodified and modified peptides from histone H2A, H2B, H3 and H4. In addition, we have conducted cell-based studies to understand the effect of these mutations on H3 methylation and the regulation of EZH2 target genes. These data and the implications of these findings for the treatment of FL and DLBCL will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-186. doi:10.1158/1538-7445.AM2011-LB-186