INTRODUCTION: Multiple myeloma (MM) evolution is complex and heterogeneous. Hyperdiploidy (HRD) and translocations affecting the immunoglobulin heavy chain (IGH) locus are historically considered initiating genomic events of MM. The potential impact of genomic events acquired before known MM initiating events has never been addressed.

METHODS: To investigate whether genomic deletions are acquired before and after known MM initiating events we interrogated whole-genome sequencing (WGS) data from patients with newly diagnosed MM (NDMM, n=319) and relapsed MM (RRMM, n=59). Our newly developed analytical and chronological workflow integrating single nucleotide variants (SNV), structural variants (SV), and copy number variants (CNV) comprises two main steps. First, we estimated the molecular time (i.e. corrected ratio between duplicated and non-duplicated clonal SNV) of large clonal chromosomal duplications and identified the earliest set of chromosomal gains in each patient. Second, within each early large chromosomal gain we identified clonal SV mediating CNV loss. A deletion on a gain can generate three possible scenarios: 1) one of the duplicated alleles is lost after the gain (i.e. post-gain) causing a CNV jump from 3:1 to 2:1 (total alleles : minor alleles); 2) there is a deletion before the duplication (i.e., pre-gain) causing a CNV jump from 3:1 to 1:0. 3) the deletion occurs on the minor, non-duplicated allele, causing a CNV jump from 3:1 to 2:0. Timing the deletion in relation with the chromosomal duplication is impossible in this scenario.

RESULTS: Molecular time data were successfully generated for 249/319 (78%) NDMM and 51/59 (86%) RRMM patients. Restricting our analysis to NDMM with HRD without canonical IGH translocations, 16/170 (9%) of patients acquired deletions before the earliest multi-chromosomal gains, suggesting MM precursors can acquire deletions before HRD. Investigating the entire series, post-gain deletions were observed in 126/417 (30%) samples considering both early and late time windows. Leveraging a background model, we demonstrated that pre-gain events involved more tumor-suppressor genes (TSG) than expected by chance, including TCF3, ATM and TRAF3. In contrast, oncogenes were involved less than expected. In post-gain deletions, both oncogenes and TSG were involved more than expected by chance .

To validate and assess the impact of deletions on driver gene expression we investigated WGS and RNAsequencing data from the MMRF CoMMpass study (n=752 NDMM). Because of the low coverage not allowing for molecular time estimation, we limited our analysis to HRD without IGH translocations. Pre-gain and post-gain deletions were observed in 47/431 (11%) and 225/431 (52%) of patients, respectively. We defined loss and gain of function events based on whether the expression level of a gene affected by the deletion was in the first or fourth quartile, respectively. Pre-gain deletions were mostly associated with downregulation of TSG expression [n=31 events in 13/431 (3%) patients]. In contrast,post-gain deletions had a more heterogenous impact with loss- and gain-of-function events. Gain-of-function events were driven by two main mechanisms: 1) the deletion joined an oncogene with a distal regulatory region inducing its overexpression [n=44 events in 6/431 (1.3%) patients]; 2) the deletion caused a new and expressed fusion [n=231 events in 109/431 (25%) patients], resulting in either loss- or gain-of-function. Surprisingly, post-gain deletions had also a major impact on TSG expression. In 60 patients (14%), we observed 146 post-gain deletions where the affected tumor suppressor gene (TSG) expression was downregulated to the level of cases with monoallelic and biallelic deletions, even though two alleles were retained. Finally, to validate these findings, we investigated 16 RRMM patients with available WGS, scATAC-seq and scRNA-seq and observed additional evidence of TSG downregulation after both pre- and post-gain deletions.

CONCLUSION: Leveraging a large cohort of NDMM we show that somatic deletions can be acquired before HRD trisomies that are assumed to be initiating events. Furthermore, post-gain deletions emerged as a new mechanism inducing TSG down-regulation, despite an apparently diploid gene status.

Dogan:Seattle Genetics: Consultancy; Physicians' Education Resource: Consultancy, Honoraria; EUSA Pharma: Consultancy; Loxo: Consultancy; Peer View: Honoraria; Incyte: Consultancy; Takeda: Other: Research Funding; Roche: Other: Research Funding. Lesokhin:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; ArcellX: Consultancy; Janssen: Honoraria, Research Funding. Davies:sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; BMS / Celgene: Membership on an entity's Board of Directors or advisory committees; pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Millenium Pharmaceuticals: Research Funding; Incyte: Research Funding; KaryoPharm: Research Funding; Mundipharma: Research Funding; Johns Hopkins University: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Research Funding; Dietmar-Hopp-Foundation: Research Funding; Chugai: Honoraria, Patents & Royalties, Research Funding; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Research Funding; Array Biopharma: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Research Funding; Glycomimetics: Research Funding; GSK: Honoraria, Other: Travel Support, Research Funding; Heidelberg Pharma: Research Funding; Hoffman- La Roche: Research Funding; Molecular Partners: Research Funding; MSD: Research Funding; Morphosys AG: Research Funding; Pfizer: Honoraria, Patents & Royalties: Travel Support, Research Funding; Takeda: Research Funding; Novartis: Honoraria, Other: Travel Support, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees. Weisel:Oncopeptides: Consultancy, Honoraria; Roche Pharma: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Other: Research grant to institution; Stemline: Honoraria; Takeda: Consultancy, Honoraria, Other: Research grant; BeiGene: Consultancy, Honoraria; Menarini: Consultancy, Honoraria; Novartis: Honoraria; Bristol Myers Squibb/Celgene: Consultancy, Honoraria, Other: Research grant to institution; AstraZeneca: Honoraria; Karyopharm: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Research grant to institution; GlaxoSmithKline: Consultancy, Honoraria, Other: Research grant to institution; Pfizer: Consultancy, Honoraria; Adaptive Biotech: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Research grant to institution; AbbVie: Consultancy, Honoraria, Other: Research grant to institution. Fenk:Janssen, Amgen, GSK, Takeda, BMS: Honoraria. Mai:BMS, GSK, Janssen, Stemline, Takeda: Other: Travel expenses; BMS, Takeda, Sanofi, Janssen, GSK: Research Funding; BMS, GSK, Jansen, Stemline, Takeda: Honoraria; BMS, GSK, Janssen, Stemline, Takeda: Consultancy. Korde:Amgen, Janssen, Epizyme, AbbVie: Research Funding; CCO, OncLive, Intellisphere, Remedy Health: Consultancy; Janssen: Other: Advisory Board. Usmani:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Research Funding; SecuraBio: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; TeneoBio: Membership on an entity's Board of Directors or advisory committees; EdoPharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Array Biopharma: Research Funding; Merck: Research Funding; Moderna: Membership on an entity's Board of Directors or advisory committees; K36 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; SkylineDX: Membership on an entity's Board of Directors or advisory committees, Research Funding. Landgren:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Adaptive: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees.

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2023
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