THE MUTATIONAL LANDSCAPE OF DOUBLE/TRIPLE‐HIT HIGH‐GRADE B‐CELL LYMPHOMA WITH BCL2 REARRANGEMENT (DH/TH‐BCL2) – AN LLMPP PROJECT

Introduction: The poor outcome of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement (DH/TH) highlights the need for identifying specific targetable biology. Although a gene expression signature (DHITsig) shared by DH/TH-BCL2 tumours has confirmed a unified biology, the genetic features of these tumours beyond MYC/BCL2 rearrangement are not well established. Here, we explore the mutational landscape of DH/TH-BCL2 to identify key molecular features that may inform on targetable tumour cell vulnerabilities. Methods: Diagnostic biopsies of 102 DH/TH-BCL2 tumours (morphology: 60 diffuse large B-cell lymphoma (DLBCL); 42 high-grade – intermediate or blastoid) were identified with break-apart fluorescent in situ hybridization (FISH). Tumours with DLBCL morphology had no history of indolent lymphoma and were sourced from BC Cancer where unbiased FISH testing was applied. Among tumours of high-grade morphology, 29% had a history of indolent lymphoma. High confidence somatic mutations (SNVs/Indels) were identified on whole genome or exome sequencing using an ensemble of variant callers (Strelka2, LoFreq, Mutect2, SAGE). Mutation frequencies among recurrently mutated genes were compared to Burkitt lymphoma (BL; Grande et al. Blood 2019) and GCB DHITsig-neg DLBCL (Schmitz et al. NEJM 2018) to identify genes with significant differences in the recurrence of non-silent mutations (FDR < 0.05). Genetic subtypes were assigned using the LymphGen data portal. Results: The most frequently mutated genes in DH/TH-BCL2 were in chromatin modifiers KMT2D, CREBBP, and EZH2, along with BCL2 and TNFRSF14 – genes commonly mutated in follicular lymphoma (FL) and EZB DLBCL. Consistently, 92% of tumours were classified as EZB by LymphGen. Comparisons of DH/TH-BCL2 to GCB DHITsig-neg DLBCL revealed less frequent alterations of genes involved in NF-κB signaling and immune surveillance. Mutations in germinal centre dark zone (DZ) regulatory genes FOXO1 and CCND3 were more frequent in DH/TH-BCL2 – genes that are also recurrently mutated in BL. While mutations in NF-κB signaling pathways and immune surveillance were also uncommon in BL, these tumours were readily distinguishable from DH/TH-BCL2 by the absence of “FL-like” mutations and more frequent alterations to ID3, DDX3X, FBXO11, ARID1A, and SMARCA4. Conclusion: The similarity to FL is suggestive of an evolutionary trajectory through an FL/FL-like common progenitor, where DH/TH-BCL2 diverges from this trajectory by adopting a DZ-like identity. This is supported by near universal DHITsig positivity of both DH/TH-BCL2 and BL, suggesting this signature more accurately identifies a DZ biology that may be at least partly promoted by MYC rearrangement and mutations in FOXO1 and CCND3. Infrequent mutations promoting immune escape in DH/TH-BCL2 and BL is consistent with a decreased selective pressure due to the “immune cold” nature of the DZ. Keywords: Genomics, Epigenomics, and Other -Omics, Aggressive B-cell non-Hodgkin lymphoma, Pathology and Classification of Lymphomas No conflicts of interests pertinent to the abstract.