Aberrant Somatic Hypermutation Research Articles

Abstract 786: Genomic distinctions of HIV- and EBV-associated DLBCL in a diverse African cohort

Abstract Lymphoma, a leading cancer cause of death among people living with HIV (PLHIV) globally, poses a pressing public health challenge in Sub-Saharan Africa (SSA), the epicentre of the HIV epidemic. Despite its prevalence, the genetic underpinnings of lymphoma in Black Africans and PLHIV remain poorly defined, contributing to regional disparities. This study aims to elucidate the impact of HIV status and EBV on the genetic landscape, molecular subtypes and survival outcomes of diffuse large B-cell lymphoma (DLBCL) in ethnically diverse SSA patients. We established a cohort of 117 newly diagnosed DBLCL cases (55 HIV+, 21 EBV+, 62 Black Africans), with histopathologic and clinical annotation from a public teaching hospital in Cape Town, South Africa (2010-22). Tumor specimens underwent histologic review, whole exome sequencing (WES) and bulk whole transcriptome sequencing. Germline variants were filtered by paired normal WES (when available) and normal references augmented by supervised machine learning in tumor-only specimens. Aberrant somatic hypermutation (ASHM) was assessed in curated genes within paired samples. Microsatellite instability (MSI) was assessed via MSI-sensor. Canonical genetic aberrations reproduced in this cohort included recurrent SNVs in TP53, CARD11, TET2, SOCS1, MYD88, and KMT2D with differences in relative proportion compared to the NCI consortium cohort being more attributable to HIV or EBV status than ethnicity. HIV+ cases harbored frequent mutations in TET2, BRAF, and KRAS, with fewer in NFKB pathway genes including CARD11, IKBK, and BTK. ABC-type HIV+ cases manifested higher expression of genes in the JAK-STAT pathway. EBV+ cases exhibited distinct genomic features including amplifications in NOTCH1, CARD11, NFKBIE, MYC, CD274, and JAK2, as well as frequent gain-of-function (GOF) mutations in the NOTCH1 PEST domain (40% vs 3.8% q=0.04). Tumor mutation burden was consistent across ethnic and virological strata (median mutations 139-167, p>0.05). Notably, ASHM was not identified as a predominant mutational signature in either HIV-positive or negative cases, and 4 HIV+ DLBCL cases exhibited MSI, corroborated by protein-affecting mutations in mismatch repair genes. The 2-year overall survival (OS) was less than 40%, markedly inferior to published Western cohorts, even when controlled for clinical prognostic factors and treatment regimen. This study highlights genetic and biological distinctions in DLBCL due to HIV and EBV. Key findings include the absence of ASHM correlates of pathogenic driver mutations, challenging a prevailing hypothesis of HIV-associated lymphomagenesis, and the identification of MSI in HIV+DLBCL. The dismal survival of this cohort and distinct genomic features, especially in EBV+DLBCL, highlight the urgency of further biological interrogation and inclusive clinical trials for these demonstrably underserved populations. Citation Format: Katherine Antel, Aleksander Nesmelov, Meng-Xuan Wu, Charlotte Slome, Dharshnee Chetty, Raymond Kriel, Ekaterina Postovalova, Robert Redd, Martin Aryee, Delphine Armand, Lauren McGrath, Nina Xiong, Jenna Oosthuizen, Karryn Brown, Andrey Tyshevich, Svetlana Podsvirova, Felix Frenkel, Natalia Kuzkina, Jessica Scher, Cagdas Tazearslan, Ksenia Zornikova, Nikita Kotlov, Aleksander Bagaev, Nathan Fowler, Arnold Freedman, Scott Rodig, Estelle Verburgh, Mark A. Murakami. Genomic distinctions of HIV- and EBV-associated DLBCL in a diverse African cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 786.

Abstract P04: Role of glucocorticoid receptor in normal and malignant germinal center B cells

Abstract We have reported that super-enhancers (SEs) are specifically hypermutated by Activation-Induced Deaminase (AID)-induced Aberrant Somatic Hypermutation in >90% of diffuse large B cell lymphoma (DLBCL) cases (Bal et al. Nature 2022). Analysis of 122 primary DLBCL biopsies and cell lines identified more than 80 SEs that are recurrently hypermutated, with an average of 12 hypermutated SE/case. Hypermutated SEs are predominantly linked to genes encoding regulators of B cell development and well-known lymphoma oncogenes, including BCL6, BCL2 and CXCR4. We showed that specific hotspot mutations prevent binding and transcriptional downregulation by transcriptional repressors leading to target gene dysregulation. Among these, hypermutation of the SEs linked to BCL2 and CXCR4 abrogates the binding of the glucocorticoid receptor (GR)/transcription factor encoded by NR3C1. As a result, BCL2 and CXCR4 escape GR-mediated transcriptional repression leading to their de-regulated expression in germinal center (GC) B cells. Together with the observation that the NR3C1 gene is genetically inactivated in a small fraction of DLBCL, and the relevance of glucocorticoid-based therapy in DLBCL, these results prompted a comprehensive analysis of the role of GR in normal and malignant B cells. We report that GR is detectable in the nucleus of the great majority of GC B cells, suggesting that it is active as a transcription factor. Similarly, DLBCL primary cases (including GCB-, ABC- and unclassified DLBCL) display GR nuclear expression. Analysis of a conditional GC-specific Nr3c1 knock-out (KO) mouse model showed that GC B cells form normally in the absence of Nr3c1. However, the transcriptional profile of Nr3c1-KO GC B cells was significantly altered compared to wild-type cells. By integrating the GR binding profile obtained in normal human GC B cells with the transcripts differentially expressed in Nr3c1-KO GC B cells, we identified a set of about 2,000 genes that are directly modulated by GR during the GC reaction. Pathway enrichment analysis showed that GR controls several signaling pathways, suggesting a modulatory role on the BCR and CXCR4 pathways. In addition, GR targets display a significant overlap with the BCL6 transcriptional network, suggesting a cooperative role in GC B cell development. Notably, GR was shown to regulate pathways involved in plasma cell differentiation, while repressing targets associated with memory B cell development. In DLBCL cases, several NR3C1 binding sites are recurrently targeted by mutations, suggesting that genetic impairment of GR activity is heterogeneous and extends beyond CXCR4 and BCL2. Together, these data support a role for the glucocorticoid pathway in GC physiology. Recurrent inactivation of the NR3C1 gene or part of its transcriptional regulatory program may contribute to DLBCL pathogenesis, with implications for presently unknown specific roles of glucocorticoids in the therapeutic regimens for DLBCL. Citation Format: Clarissa Corinaldesi, Antony B Holmes, Elodie Bal, Laura Pasqualucci, Katia Basso, Riccardo Dalla-Favera. Role of glucocorticoid receptor in normal and malignant germinal center B cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P04.

Aberrant Somatic Hypermutation at Super-enhancer Drives B Cell Lymphoma Transformation.

Aberrant Somatic Hypermutation at Super-enhancer Drives B Cell Lymphoma Transformation.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

RCHOP Plus High-Dose Methotrexate As First-Line Therapy in Large B-Cell Lymphoma with Testis Involvement

Primary testicular lymphoma (PTL) and diffuse large B-cell lymphoma with secondary testicular involvement (DLBCL-T) are rare subtypes of lymphomas associated with poor prognosis and a high risk of central nervous system (CNS) relapse. The optimal CNS prophylaxis strategy for these subtypes remains uncertain. High-dose methotrexate (HD-MTX) is debated for CNS prophylaxis, but its efficacy in this setting is unknown. This study aimed to retrospectively analyze the use of HD-MTX in combination with chemotherapy (R HD-MTX CHOP) as frontline treatment for PTL and DLBCL-T and to compare the biological characteristics of these two forms of testicular involvement. Data from patients who received R HD-MTX CHOP as first-line treatment for DLBCL-T or PTL between 2010 and 2020 were collected retrospectively. The study included 15 patients, 5 with PTL and 10 with DLBCL-T. The overall response rate was 86%, with 73% of patients achieving complete remission. Treatment-related adverse events were generally manageable, with febrile neutropenia being the most common. Two patients experienced CNS relapse, both from the DLBCL-T subgroup, with a median time of 10.7 months from diagnosis to CNS relapse. None of the PTL patients receiving HD-MTX experienced relapse during the median follow-up of 6.08 years (Figure 1A), compared to 8/10 DLBCL-T (p=0.007). All five PTL cases had a non-germinal center phenotype, while 7 out of 10 DLBCL-T cases were non-germinal center. One DLBCL-T case exhibited high-grade lymphoma. Myc expression was positive in 3 out of 10 patients assessed, of whom all were double expressors. Bcl-2 expression was positive in all 13 assessable patients, while Bcl-6 expression was positive in 6 patients. Myc, Bcl-2, and Bcl-6 rearrangements were assessed in 10 patients and were present in 1 patient who was found to have triple-hit lymphoma. No other patient had double-hit lymphoma or any significant rearrangement. Polysomy of Myc, Bcl-2, and/or Bcl-6 was found in 2 patients. PDL1 expression was observed in one out of six patients analyzed, while no PD1 expression was detected. In 8 out of 15 patients with available tissue material, next-generation sequencing using a panel targeting 43 lymphoid genes was performed. Among these 8 patients, 87% exhibited gain-of-function mutations in MYD88 (MYD88 L265P), and 62% had missense mutations in CD79B at position Y196. Multiple mutations in the PIM1 gene, a known target of aberrant somatic hypermutation, were also observed in 7 patients. Non-recurrent mutations were detected in various genes, including PRDM1, CD58, B2M, CREBBP, CIITA, CARD11, CDKN2A, EP300, CCDND3, FOXO1, IRF4, NFKBIE, and TCF3 (Figure 1B). Ig rearrangement was analyzed in 4/9 patients with available DNA. All 4 were clonal, with distinct but mutated VH segments (sequence identity between 81 and 89%). This study suggests that HD-MTX in combination with RCHOP chemotherapy is highly effective in preventing CNS relapse in PTL but less so in DLBCL-T. DLBCL-T had a higher risk of CNS relapse and poorer outcomes compared to PTL. Further studies are needed to determine the optimal timing and sequence of HD-MTX in DLBCL-T, as well as alternative treatment approaches. PTL and DLBCL-T have distinct clinical presentations and responses to frontline chemotherapy with HD-MTX prophylaxis. PTL may benefit from HD-MTX in preventing relapse, while alternative treatments should be explored for DLBCL-T. Future studies should consider PTL and DLBCL-T as separate entities to better understand their characteristics and optimize treatment strategies.

Longitudinal Noninvasive Surveillance & Fragmentomic Characterization of Follicular Lymphoma

Introduction While cell-free DNA (cfDNA) plays an increasingly defined role in aggressive lymphomas, its characteristics in indolent lymphomas are less established. Many follicular lymphoma (FL) patients experience durable remissions and late relapses ( Fig A). We therefore explored circulating tumor DNA (ctDNA) kinetics during long-term blood-based surveillance in patients with FL. In addition to noninvasive detection using somatic mutations, we also evaluated gene expression inference from cfDNA utilizing a novel fragmentomic method to detect histological transformation (tFL). Methods We studied 121 serial samples in a cohort of 17 FL patients (median 7 per patient) with long term clinical and blood-based MRD monitoring (median 5.6 years, max 10.8). We considered serial ctDNA status at 4 milestones: baseline/pre-treatment, post-treatment, in radiographic remission (MRD), and at relapse. We also profiled pre-treatment cfDNA for 9 tFL cases. Cases were genotyped to identify somatic mutations from diagnostic tumor or baseline plasma, with matched PBMCs used to censor germline variants and clonal hematopoiesis. ctDNA status in subsequent samples was evaluated via CAPP-Seq (SNVs) and PhasED-Seq (PVs). The cfDNA fragmentation profiles of pre-treatment FL and control samples were evaluated for inferred gene expression via EPIC-seq (Esfahani Nat Biotech 2022) using a novel 1676-gene panel focused on lymphoid neoplasms, including classic and transformed FL. Results Treatment & Relapse Kinetics Most patients had advanced disease (88% stage III-IV) and Low to Intermediate risk FLIPI (71% 0-2). At first cfDNA evaluation, 9/17 (53%) were previously untreated. Median pre-treatment ctDNA levels were 73.4 hGE/mL in FL, as compared to 236 hGE/mL in DLBCL (Kurtz JCO 2018). Post-treatment, median ctDNA levels dropped to 1.2 hGE/mL. Depth of response was similar between rituximab monotherapy and cytotoxic regimens, with median -2.2 log10 fold change from baseline. MRD Detection PVs were observed in all tumors, consistent with expected aberrant somatic hypermutation in FL. To evaluate MRD detection via PhasED-Seq, we considered plasma timepoints with undetectable ctDNA by CAPP-Seq (n=26) from patients (n=9) with subsequent clinical relapse. MRD was detected in 11 additional specimens (42%) by PhasED-Seq. Among FL patients tested for MRD while in radiographic remission, low-level ctDNA was detectable in 62% of plasma specimens (8/13) obtained within 1 month of negative PET/CT, suggesting potential additional utility for surveillance. Long-term Genomic Stability Sequential biopsies of FL tumors have shown genomic heterogeneity between diagnosis and relapse. We therefore evaluated ctDNA concordance across variant classes & regions. Relative AF was calculated pre-treatment and at relapse (median 2 years between samples, range 1-8 years), normalized by total ctDNA levels. The most stable lesions included missense mutations in CREBBP and TP53, nonsense KMT2D mutations, and BCL2 5'-UTR variants. BCL6 intronic mutations and chr2/chr14 SHM varied between diagnosis/relapse. Plasma versus Cellular Concentrations In contrast to DLBCL, circulating FL cells are frequently detectable at low levels in blood. To compare relative FL concentrations in the cellular versus plasma compartments, we profiled 10 paired specimens from patients with radiographic disease. While tumor-confirmed variants were detected in 7 of 10 cellular specimens, mean allelic frequency (AF) was 5.3x higher in plasma. Fragmentomic Assessment of Transformation We compared gene expression profiles inferred from cfDNA from pre-treatment lymphoma (FL=15, tFL=9) versus control (n=20) samples ( Fig B). Considering genes associated with FL (Huet Lancet Onc 2018), FL patients had significantly higher inferred expression than healthy adults (p=0.036). Likewise, patients with tFL showed higher inferred expression of genes associated with histologic transformation (Gentles Blood 2009, p=0.025). Conclusions Sensitive methods are required for effective MRD assessment in FL given lower ctDNA shedding as compared with DLBCL. Frequent detectable MRD in radiographic remission suggests that integrating molecular surveillance may augment serial imaging in the long-term management of FL. Simultaneous inferred gene expression from FL cfDNA appears to hold promise, including for noninvasive detection of transformation.

Integrated genetic analyses of immunodeficiency-associated Epstein-Barr virus- (EBV) positive primary CNS lymphomas.

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV+PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV-PCNSL. Instead, EBV+PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+PCNSL. In addition to novel insights into the pathobiology of EBV+PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

BTG2 SUPER‐ENHANCER MUTATIONS DISRUPT TFAP4 BINDING AND DYSREGULATE BTG2 EXPRESSION IN DIFFUSE LARGE B‐CELL LYMPHOMA

Introduction: Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy, remains incurable in ∼40% of patients. Coding-genome sequencing efforts identified several genes/pathways altered in this disease, as well as genetic subgroups of potential clinical relevance. However, the large non-coding portion of the genome remained largely unexplored. We recently identified a pervasive hypermutation mechanism targeting active super-enhancers (SEs) in >90% of DLBCL and leading to dysregulation of multiple genes, including well-known lymphoma oncogenes (Bal et al., Nature 2022). As evidence of oncogenic relevance, we demonstrated that mutational hotspots in the BCL6, BCL2 and CXCR4 SEs impair the binding of specific transcriptional repressors, preventing the gene negative regulation and creating oncogenic dependencies in DLBCL cells. Here we aimed to define the pathogenic role of mutations targeting the intragenic SE (iSE) of the BTG2 gene, the second most commonly mutated in DLBCL. BTG2 encodes a member of the B-cell translocation gene (BTG)/TOB family involved in transcriptional co-activation and modulation of mRNA abundance. BTG2 is also a recurrent target of somatic missense mutations (6%–11% of DLBCL), suggesting a major role in the pathogenesis of this disease. Methods: We screened 243 DLBCL cases for the presence of mutational hotspots within the BTG2-iSE, and combined in silico prediction, DNA-binding assays (Reverse-ChIP, electromobility-shift assay, ChIP-qPCR), RNA-seq and CRISPR/Cas9 editing approaches in isogenic BTG2 mutant versus WT DLBCL cell lines to identify transcription factors bound to the SE and disrupted by the mutation. Results: We identified a recurrent mutational cluster affecting the BTG2-iSE in 52/243 (21%) DLBCLs, with preferential enrichment in ST2 subgroup. CRISPR-Cas9 mediated correction of the mutation in 3 DLBCL cell lines led to counter selection and reduced BTG2 expression, consistent with oncogenic addiction. In silico motif prediction and in vitro DNA-binding assays, followed by validation in multiple isogenic DLBCL cell lines, identified TFAP4 as a major transcription factor that binds to the WT, but not to the mutated site. TFAP4 is an important regulator of B-cell proliferation and cell fate decisions, which can function as a transcriptional activator or repressor in germinal center B-cells, and acts downstream of/in parallel with c-MYC. Of note, introduction of SE hotspot mutations in WT DLBCL cells was associated with increased BTG2 expression, confirming a direct link between SE mutations and deregulated gene expression through escape from TFAP4-mediated suppression. Conclusions: These findings suggest a major role for BTG2 deregulation by SE aberrant somatic hypermutation in the pathogenesis and heterogeneity of DLBCL, with implications for precision classification and potential therapeutic targeting of DLBCL. Keywords: aggressive B-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Genetic subdivisions of follicular lymphoma defined by distinct coding and noncoding mutation patterns

AbstractFollicular lymphoma (FL) accounts for ∼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning–derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.

Large B-cell Lymphomas of Immune-Privileged Sites Relapse via Parallel Clonal Evolution from a Common Progenitor B Cell.

Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP.

GEN-13 PAIRED MUTATIONAL ANALYSIS IN SECONDARY NERVOUS SYSTEM LYMPHOMA AND PCNSL SYSTEMIC RELAPSE REVEALS DRIVER MUTATION CANDIDATES IN THE CENTRAL NERVOUS SYSTEM

Abstract Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma confined to the brain, eyes, and the spinal cord. The mechanism of central nervous system (CNS) tropism in PCNSL has not been fully elucidated. Diffuse large B cell lymphomas (DLBCLs) occasionally present with distant recurrence, which can involve inside and outside the CNS. Secondary central nervous system lymphomas (SCNSLs) are CNS relapse of systemic lymphoma. PCNSLs also rarely present with systemic relapse. We have previously reported in our study of whole exome sequencing that PCNSLs harbor frequent mutations in genes of B cell receptor pathway members and aberrant somatic hypermutation (aSHM) target genes. Although several genetic alterations were identified as more frequent in PCNSLs compared with systemic lymphomas, specific genetic alterations which serve as the driver for CNS tropism in PCNSLs has not been identified. In order to search for mutations which might serve as driver mutations in the CNS, we have performed targeted sequencing in paired samples from patients with recurrent lymphomas, either SCNSLs or PCNSL systemic relapses, using Ion Torrent multiplex PCR. Mutational profiles were compared between the primary and recurrent tumor. Six cases (four SCNSL cases and two PCNSL systemic relapse cases) were analyzed. Of note, in the SCNSL cases, several de novo mutations were enriched only among the recurrent CNS tumors. Among these mutations, BTG2 mutations were observed in 3/4 (75%), and B2M and KLHL14 mutations were observed in 2/4 (50%) cases. In the two PCNSL systemic relapse cases, KMT2D mutations were enriched only in the recurrent systemic tumors. It is suggested that these de novo mutations in the recurrent CNS tumors might serve as driver mutations in the CNS. Further analysis in larger cohorts, and functional studies are required in order to validate these findings.

Identification of Genetic Subtypes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma subtype and is characterized by heterogeneous clinical trajectories. While certain genetic abnormalities are highly prevalent, heterogeneity exists also at the molecular level. Yet, unlike diffuse large B-cell lymphoma, where genetic subtypes have been described in multiple studies, the taxonomy of FL has not been clearly established to date. While clusters defined by STAT6 mutations or aberrant somatic hypermutation have been described in FL by Crouch et al. (Blood Advances, 2022), its landscape of molecular alterations has been insufficiently dissected into biologically distinct subgroups. The recognition of such subtypes may pave the way for treatment individualization. Here, we sought to define novel genetic subtypes in FL by means of targeted DNA sequencing and clustering. Methods: Samples of FL grade 1 to 3A (i.e. classic FL) were accrued from biorepositories of participating centres in Canada, Northern Europe and Australia, as well as from two prospective clinical trials (E4402 and E2408). We performed hybridization-based targeted DNA-sequencing of 57 genes that are recurrently mutated in FL. Moreover, we included targeted DNA sequencing data from a previously published dataset (PMID 27959929). All cases were analyzed using a uniform computational pipeline. Samples were removed from the analysis if the mean on-target coverage was <50X. Single nucleotide variants were identified using Mutect2 and we applied a variant allele fraction cut-off of 0.1. Bernoulli mixture-model clustering was applied using variants as features for the identification of genetic subtypes. Cluster stability was assessed by bootstrap consensus. Clusters were tested for their association with outcome measures. As a further means to annotate genetic clusters, we generated genome-wide methylation data using the Illumina EPIC platform. Results: Following quality control, the total number of pre-treatment samples available for clustering was 715 (543 from formalin-fixed and paraffin-embedded tissue samples and 172 from fresh-frozen). The average number of mutations and mutated genes per sample were 5.9 (range 1-20) and 4.8 (range 0-14), respectively. An analysis of interactions between somatic mutations revealed both expected (e.g. mutual exclusivity of CREBBP and EP300 mutations) and unexpected patterns (e.g. co-occurring mutations of GNA13 and MEF2B), overall justifying the pursuit of unsupervised clustering. After consensus clustering, the optimal clustering solution resolved 5 genetically distinct groups, henceforth referred to as C1 to C5 (Figure). The C1 cluster was characterized by variants in genes involved in the mTOR signaling pathway (ATP6AP1, RRAGC, ATP6V1B2); the C2 cluster by low mutation burden; the C3 cluster by mutations involving TNFAIP3, TP53 and MYD88; the C4 cluster by GNA13 and MEF2B mutations; and the C5 cluster by STAT6, CREBBP, EZH2 and TNFRSF14 mutations. The number of mutations located in RGYW or WRCY motifs, suggestive of somatic hypermutation, was highest in the C4 and the C3 clusters (P < 0.001). Applying a previously reported methylation-based clock (epiCMIT) to the subset of samples with methylation data (n=345), we found that samples in the C2 cluster had the lowest proliferative history. Concordantly, the C2 cluster was characterized by a lower degree of global hypomethylation and lower average methylation of CpG islands, whereas samples in clusters C1, C3, C4 and C5 were characterized by not just longer proliferative histories, but also by higher mean methylation in CpG islands. In terms of clinical associations, the C2 cluster was significantly associated with limited-stage disease presentation. In an exploratory outcome analysis of 165 patients treated with immunochemotherapy, the C3 and C5 clusters were associated with inferior time to progression. Conclusion: Here, we identified novel genetic subtypes that are distinguishable not just by unique sets of mutations, but also by their clinical associations and methylation profiles. The novel genetic profiles shed light on core pathways distinctly tied to each subtype. Targeted DNA sequencing of a limited gene panel represents a potentially accessible method of subtyping FL in the clinical setting, with implications for the understanding of responses to targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNA Methylation-Based Burkitt Lymphoma Epitypes Have Distinct Molecular and Clinical Features

Introduction: Genome-wide hypomethylation coupled with hypermethylation of tumor suppressor genes is a hallmark of many cancers. Consistent changes in DNA methylation patterns are also a feature of immune cells as they undergo terminal differentiation and maturation. In previous studies, it has been shown that malignant B cells harbor a combination of de novo methylation while retaining some epigenetic features characteristic of their cell of origin. Although information on the genetic features of Burkitt Lymphoma (BL) is expanding, limited information exists on the methylation landscape and its role in pathogenesis. We sought to identify consistent DNA methylation changes and their interplay between BL pathogenesis and other features such as EBV status and somatic mutations. We performed an integrative analysis using comprehensive genome-wide CpG methylation profiling, whole genome sequencing, and gene expression analysis. Methods: Samples were collected as part of the Burkitt Lymphoma Genome Sequencing Project (BLGSP). DNA from 218 primary BL tumors (154 pediatric (111 EBV+), 64 adult (19 EBV+)), 13 BL cell lines (6 EBV+), and 6 normal centroblast (CB) samples were profiled using the Illumina EPIC array. DNA and RNA from biopsies and cell lines underwent whole genome and RNA sequencing to enable the correlation of DNA methylation status with gene expression and genomic alterations. Nine of the BL samples were separately subjected to whole genome sequencing using the PromethION (Oxford Nanopore) to infer genome-wide methylation landscape. Publicly available data from 1595 B cell samples representing both normal subpopulations and tumor subtypes were used for further comparison. To search for methylation patterns within BL, beta values from the 10000 most variable CpGs were used as features for non-negative matrix factorization (NMF) clustering. Results: Clustering revealed two distinct epigenetic subgroups (epitypes) within BL, which we have named hyperBL (cluster1) and hypoBL (cluster2). We found pediatric and adult tumors to be proportionally split across the two epitypes; however, we noted an over-representation of EBV+ tumors in hyperBL. PCA analysis comparing BL with the additional B-cell samples revealed a separation between the epitypes, with hyperBL projecting farther along the trajectory of B cell differentiation, suggestive of a more terminally differentiated B-cell. Investigating the differential methylation between epitypes revealed hyperBL to be associated with greater hypermethylation compared to hypoBL and CBs. The hypermethylated CpGs in hyperBL mainly affected promoter regions within CpG islands while hypomethylated CpGs were mostly intergenic or in open seas. Using PromethION data to allow for genome-wide analyses, we found hypermethylated regions of hyperBL were enriched for binding motifs for several transcription factors involved in B cell development including: IRF4, PAX5, YY1, BCL6, SP1, STAT3, and KLF9. We used mitotic clock estimates based on DNA methylation (epiCMIT) and found hyperBL had significantly elevated values, suggesting a greater proliferative history of these tumors. To further establish a molecular basis for these epitypes, we searched for gene expression differences and distinct driver mutation patterns. The genomes of hyperBL tumors harbored an overall greater mutation burden and significantly higher mutation burden within regions affected by aberrant somatic hypermutation (aSHM), which was associated with a higher frequency of mutations attributed to the SBS9 mutational signature. Gene expression differences indicate hypoBL tumors have upregulation of IRF4 induced pathways. To determine the clinical relevance of the unique epigenetic clusters we conducted survival analyses and found hyperBL samples to have significantly inferior progression free survival. Conclusion: This work identifies novel epigenetic subgroups within BL with characteristic genetic differences between them. HyperBL is associated with hypermethylation, a higher mutation burden, elevated rates of aSHM, and inferior PFS. These results establish a subset of patients with distinct epigenetic, molecular and clinical features and further elucidates key mechanisms underlying BL pathogenesis. Together these data highlight the need and potential for strategies that exploit these epigenetic changes as a therapeutic target. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-Wide Methylation and Haplotype-Resolved Aberrant Somatic Hypermutation Patterns in B-Cell Lymphomas

Introduction Diffuse large B-cell lymphoma (DLBCL) is a genetically, phenotypically, and clinically heterogenous disease. Multiple molecular classification systems using either genomic aberrations or gene expression profiles are used to delineate subgroups with shared biology. The cell of origin (COO) gene expression-based classification system stratifies DLBCLs into germinal center B-cell-like (GCB) and activated B-cell-like subgroups of DLBCL. More recently, the double-hit signature (DHITsig) has been used to further stratify GCB DLBCLs into DHITsig+ and DHITsig-, where the former is characterized by a gene expression profile reminiscent of the GC dark zone and inferior outcomes. Currently, patterns of somatic driver mutations can only partially explain the expression of DHITsig in some tumors. Genetic features such as DNA methylation have been shown to be associated with transcriptional repression and altered chromatin structure. We hypothesized that resolving DNA methylation patterns genome-wide using long read whole genome sequencing (LR-WGS) may reveal epigenetic differences associated with differential gene expression profiles within the DHITsig+ and DHITsig- subgroups and in the context of histologic transformation from follicular lymphoma. Methods Whole genome sequencing data of 10 patient samples and 4 cell line GCB DLBCL samples were generated using both short read (SR-WGS; Illumina) and LR-WGS (Oxford Nanopore Technologies, PromethION). The complementary approach combines somatic variant calling from highly accurate SR data with DNA methylation calling from LR data. GCB samples were selected based on the NanoString DLBCL90 assay and further subdivided into DHITsig+ (N=4) and DHITsig- (N=3). RNA sequencing data of 9 patient samples and 3 GCB DLBCL cell lines were generated and analyzed using Salmon and DESeq2. Simple somatic mutations from SR-WGS data were called using an ensemble of somatic variant callers, which confirmed all cases to belong to the EZB genetic subgroup. Methylated bases were called with Nanopolish followed by a differential methylation analysis using the DSS package in R to identify differences in methylation between both subgroups. Differentially methylated regions (DMRs) were annotated for GC B-cell specific superenhancers, CpG islands, and genes. Using WhatsHap and Nanomethphase, heterozygous single nucleotide polymorphisms (SNPs) from SR and LR data were used to phase LRs into haplotypes. Haplotype analyses were performed to determine the phase of aberrant somatic hypermutation (aSHM) and methylation patterns in the context of proximal translocation breakpoints. Results Most DMRs were found to be associated with regulatory regions such as promoters, superenhancers, and CpG islands. We found methylation differences between DHITsig+ and DHITsig- subgroups in 1430 superenhancers known to be active in GC B-cells. Within these superenhancer regions, DHITsig+ tumors were typically hypomethylated relative to DHITsig-. Several DHITsig classifier genes, including WNK2, TNFSF8, and IL21R, had methylation patterns inverted relative to the typical expression pattern (i.e. low methylation with high expression). Similarly, among 633 differentially methylated promoters, several including TCF3 and WNK2 demonstrated the same inverted methylation pattern. CpG islands showed distinct patterns of methylation in both subgroups that were inversely associated with expression, this also included genes differentially expressed between DHITsig+ and DHITsig- tumors. The DHITsig- group had increased levels of methylation of DHITsig genes in both promoters and gene bodies suggesting a functional role for gene body methylation. Finally, we observed that a high density of mutations near the BCL2 transcription start site consistently phased into the same haplotype as the BCL2-IGH translocation in five tumours, demonstrating the allele specificity of aSHM associated with these events (Panel B). Conclusions These results demonstrate differences in methylation between the subgroups. The association of hypomethylated superenhancers, promoters, and CpG islands with genes over-expressed in DHITsig+ suggests these methylation patterns may contribute to the DHITsig phenotype. These results may aid in improving GCB DLBCL patient classification and reveal factors contributing to its heterogeneity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Towards a Unified Genetic Classification System for Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell neoplasm and the most common lymphoid malignancy worldwide. DLBCL, NOS is a genetically and biologically complex entity that remains uniformly treated at diagnosis despite the growing appreciation for subgroups with molecular distinctions. There are various approaches for assigning DLBCL into one or more molecular subgroups using either gene expression and/or mutation patterns. Several genetic classification systems have been proposed, each sharing commonalities. Distinctions between the definitions of individual classes and the ability to reproducibly assign individual patient samples to subgroups using each system remains a significant barrier to their implementation, further emphasized by up to 50% of patients that remain unclassified. The disparities among the genetic features considered by each classification scheme further limits the ability to consolidate, let alone adopt, these for research and clinical studies. We aimed to develop a classification system that unifies and extends the robustness of existing classification algorithms. Methods: We assembled whole genome sequencing (WGS) data from 900 tumors from DLBCL, Burkitt (BL), follicular (FL) and other mature B-cell neoplasms to comprehensively determine the set of significantly mutated genes (SMG) and common targets of aberrant somatic hypermutation (aSHM). These were intersected with the features used by the current DLBCL classification systems and a curated set of lymphoma genes to tabulate the mutation matrix. Common structural rearrangements involving oncogenes were determined from WGS data and the copy number state of focal high-level recurrent alterations was united with simple somatic mutations. For genes containing mutation hot-spots each was considered separately. Results: Clustering all cases using non-negative matrix factorization (NMF) analysis resulted in a solution with 7 distinct genetic subgroups (Figure 1). Many of these directly resemble clusters in the existing systems with either expanded coverage or refinement of a subgroup into two. First, the BNZ cluster was highly enriched for any aberration in BCL6, 3'UTR mutations in NFKBIZ, and truncating mutations in NOTCH2. Unlike the similar BN2/C1/NOTCH2 groups defined previously, the NFKBIZ mutations were weighted more highly than those of NOTCH2 in defining the BNZ subgroup. The cluster we named MCaP resembled the MCD/C5/MYD88 group. Tumors classified in this subgroup had evidence of aSHM with frequent non-coding mutations in PIM1,CD44, GRHPR, PIM2, and BCL11A among others. Another cluster, MP3, was enriched for coding mutations or aSHM in the MYC TSS (MYC_aSHM), which were generally correspondent with IG::MYC rearrangements. MP3 tumors were characterized by TP53 mutations, including hotspots, and significantly depleted for aberrations in BCL2, BCL6 and lower mutation rates at aSHM targets SGK1, PAX5, CIITA, RHOH and others. Notably, tumors in this subgroup were enriched for IRF4 translocations, suggesting potential overlap with large B-cell lymphoma with IRF4 rearrangement. Our approach further refined both the the ST2/C4 and EZB/C3/BCL2 classes into two smaller subgroups, aSEL/aSCI and EGB/ETB, respectively. The EGB tumors were enriched for GNAI2 mutations, and MYC translocations involving the BCL6 enhancer, and were more often associated with MEF2B hotspot mutations. Notably, ETB was significantly enriched for mutations in the lysine acetyltransferase (KAT) domain in CREBBP, while EGB was significantly enriched for non-KAT domain mutations. ETB tumors were further characterized by mutations in STAT6 and FOXO1 hotspot regions, MYC rearrangements with other non-IG partners, and TNFRSF14 mutations, largely resembling the mutational profile of follicular lymphomas. Conclusions: We present here a refined classification of DLBCL genetic subgroups that combines previously described classification algorithms and extends it to allow classification of more tumors into additional subgroups that preserve the major divisions of existing systems. This represents an important advancement that will facilitate further understanding of genomic complexity in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma

AbstractBurkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

HL-370 Fragmentóme Profiling Reveals Distinct Molecular Subgroups of Classic Hodgkin Lymphoma

Classic Hodgkin lymphoma (cHL) is a phenotypically heterogeneous disease with enigmatic biology and pathogenesis. By leveraging ctDNA analysis, here we identify molecular groups of cHL with phenotype- and outcome-associated signatures. Identification and biological characterization of novel cHL subgroups. We sequenced 202 cHL patients targeting ~344 kb of genomic space. The length of ctDNA mapped reads was extracted, and unsupervised clustering was performed. PET/CT scan and 71 plasma cytokines were obtained for clinical correlation. Single-cell RNA sequencing was performed for 8 patients. To identify molecular subgroups within cHL, we focused on the fragmentation profile of cfDNA, a non-genetic way of detecting tumor-derived DNA in the cfDNA samples. We identified two distinct clusters, one with a fragmentation profile close to healthy (N=135), named mono-nucleosomal, and a submono-nucleosomal cluster characterized by a global shift toward shorter fragments, whose length was less than the typical wrap around the nucleosome (N=67). We explored whether cHL subtypes defined by the cfDNA fragmentation profile are clinically and biologically validated. Patients with the submono-nucleosomal fragmentome showed more frequent advanced-stage B symptoms and elevated ESR at the clinical level, higher tumor volume (TMTV and TLG) at the radiomic level, and lower PFS in terms of outcome. Tumor mutations and immune microenvironment are pathophysiologic features of cHL. The submono-nucleosomal cluster carried a higher tumor mutation burden because of heavier aberrant somatic hypermutation (ASHM). The submono-nucleosomal cluster had an immune-suppressive microenvironment enriched of T-regs and higher plasmatic levels of T-regs chemoattractants and inducers of PD1 expression (IL6, CCL2, CCL4). Conversely, mono-nucleosomal cHL had a microenvironment enriched of TFH cells and cytotoxic CD8 TEM cells and associated with cytokines and chemokines that are known for being attractants and activators of effector T cells (IL-1a, CCL21, CXCL12, IL-17A). Our observations indicate that cHL subgroups belonging to the submono-nucleosomal cluster have a more aggressive disease, a higher mutation load and more neoantigens likely due to denser ASHM, and an immune-suppressive microenvironment. Instead, cHL subgroups belonging to the mono-nucleosomal cluster have a less aggressive disease, a lower mutation load and lower neoantigens likely due to lighter ASHM, and a tumor-suppressive microenvironment.

Molecular subclusters of follicular lymphoma: a report from the United Kingdom’s Haematological Malignancy Research Network

AbstractFollicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer’s heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future.

Aberrant somatic hypermutation of CCND1 generates non-coding drivers of mantle cell lymphomagenesis

Aberrant somatic hypermutation (aSHM) can target proto-oncogenes and drive oncogenesis. In mantle cell lymphoma (MCL), CCND1 is targeted by aSHM in the non-nodal subtype (nnMCL), giving rise to exon1 encoded mutant proteins like E36K, Y44D, and C47S that contribute to lymphomagenesis by virtue of their increased protein stability and nuclear localization. However, the vast majority of somatic variants generated by aSHM are found in the first intron of CCND1 but their significance for mantle cell lymphomagenesis is unknown. We performed whole-genome and whole-transcriptome sequencing in 84 MCL patients to explore the contribution of non-coding somatic variants created by aSHM to lymphomagenesis. We show that non-coding variants are enriched in a MCL specific manner in transcription factor-binding sites, that non-coding variants are associated with increased CCND1 mRNA expression, and that coding variants in the first exon of CCND1 are more often synonymous or cause benign amino acid changes than in other types of lymphomas carrying a t(11;14) translocation. Therefore, the increased frequency of somatic variants due to aSHM might be a consequence of selection pressure manifested at the transcriptional level rather than being a mere mechanistic consequence of misguided activation-induced cytidine deaminase (AID) activity.

Deletion of murine Rhoh leads to de-repression of Bcl-6 via decreased KAISO levels and accelerates a malignancy phenotype in a murine model of lymphoma

ABSTRACT RHOH/TFF, a member of the RAS GTPase super family, has important functions in lymphopoiesis and proximal T cell receptor signalling and has been implicated in a variety of leukaemias and lymphomas. RHOH was initially identified as a translocation partner with BCL-6 in non-Hodgkin lymphoma (NHL), and aberrant somatic hypermutation (SHM) in the 5ʹ untranslated region of the RHOH gene has also been detected in Diffuse Large B-Cell Lymphoma (DLBCL). Recent data suggest a correlation between RhoH expression and disease progression in Acute Myeloid Leukaemia (AML). However, the effects of RHOH mutations and translocations on RhoH expression and malignant transformation remain unknown. We found that aged Rhoh−/− (KO) mice had shortened lifespans and developed B cell derived splenomegaly with an increased Bcl-6 expression profile in splenocytes. We utilized a murine model of Bcl-6 driven DLBCL to further explore the role of RhoH in malignant behaviour by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice. The loss of Rhoh in Bcl-6Tg mice led to a more rapid disease progression. Mechanistically, we demonstrated that deletion of Rhoh in these murine lymphoma cells was associated with decreased levels of the RhoH binding partner KAISO, a dual-specific Zinc finger transcription factor, de-repression of KAISO target Bcl-6, and downregulation of the BCL-6 target Blimp-1. Re-expression of RhoH in RhohKOBcl-6Tg lymphoma cell lines reversed these changes in expression profile and reduced proliferation of lymphoma cells in vitro. These findings suggest a previously unidentified regulatory role of RhoH in the proliferation of tumour cells via altered BCL-6 expression. (250)