Abstract: Emergence of drug resistance to all available therapies is the major challenge to improving survival in myeloma. Cereblon (CRBN) is the essential binding protein of the widely used immunomodulatory drugs (IMiDs) and novel CRBN E3 ligase modulator drugs (CELMoDs) in myeloma, as well as certain proteolysis targeting chimeras (PROTACs), in development for a range of diseases. Using whole-genome sequencing (WGS) data from 455 patients and RNA sequencing (RNASeq) data from 655 patients, including newly diagnosed (WGS, n = 198; RNASeq, n = 437), lenalidomide (LEN)-refractory (WGS, n = 203; RNASeq, n = 176), and pomalidomide (POM)-refractory cohorts (WGS, n = 54; RNASeq, n = 42), we found incremental increases in the frequency of 3 CRBN aberrations, namely point mutations, copy losses/structural variations, and a specific variant transcript (exon 10 spliced), with progressive IMiD exposure, until almost one-third of patients had CBRN alterations by the time they were POM refractory. We found all 3 CRBN aberrations were associated with inferior outcomes to POM in those already refractory to LEN, including those with gene copy losses and structural variations, a finding not previously described. This represents the first comprehensive analysis and largest data set of CBRN alterations in myeloma patients as they progress through therapy. It will help inform patient selection for sequential therapies with CRBN-targeting drugs.

Abstract: The acquisition of a multidrug refractory state is a major cause of mortality in myeloma. Myeloma drugs that target the cereblon (CRBN) protein include widely used immunomodulatory drugs (IMiDs), and newer CRBN E3 ligase modulator drugs (CELMoDs), in clinical trials. CRBN genetic disruption causes resistance and poor outcomes with IMiDs. Here, we investigate alternative genomic associations of IMiD resistance, using large whole-genome sequencing patient datasets (n = 522 cases) at newly diagnosed, lenalidomide (LEN)-refractory and lenalidomide-then-pomalidomide (LEN-then-POM)-refractory timepoints. Selecting gene targets reproducibly identified by published CRISPR/shRNA IMiD resistance screens, we found little evidence of genetic disruption by mutation associated with IMiD resistance. However, we identified a chromosome region, 2q37, containing COP9 signalosome members COPS7B and COPS8, copy loss of which significantly enriches between newly diagnosed (incidence 5.5%), LEN-refractory (10.0%), and LEN-then-POM-refractory states (16.4%), and may adversely affect outcomes when clonal fraction is high. In a separate dataset (50 patients) with sequential samples taken throughout treatment, we identified acquisition of 2q37 loss in 16% cases with IMiD exposure, but none in cases without IMiD exposure. The COP9 signalosome is essential for maintenance of the CUL4-DDB1-CRBN E3 ubiquitin ligase. This region may represent a novel marker of IMiD resistance with clinical utility.

Abstract: Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)–based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.

Abstract: Introduction: Multiple myeloma (MM) is a genetically heterogeneous disease where risk stratification and outcomes are associated with translocations involving the immunoglobulin (Ig) loci and MYC, copy number abnormalities including gain(1q), del(1p), and del(17p), as well as mutations. Additionally, MM tumors may harbor rare mutations in genes that are targetable in other tumors, such as in IDH1 and IDH2. Therefore, we designed a comprehensive MM targeted sequencing panel to interrogate the common genomic abnormalities in MM and validated it against known standards. Methods: The targeted panel was designed to include the exons of 228 genes which are either frequently mutated, associated with prognosis or risk stratification, clinically actionable, or sites of important copy number abnormalities. Additional targets were added across the genome to identify hyperdiploidy. These targeted regions encompass the mutation detection part of the panel and involve approximately 990 kb. The Ig loci and region surrounding MYC were tiled to capture translocations and copy number changes. In total, this translocation part of the panel involves approximately 4.7 Mb. The mutation and translocation panels are manufactured separately and combined during the assay resulting in a 5:1 sequencing ratio, respectively, which prevents over-sequencing of the large translocation panel. 100 ng DNA extracted from CD138+ bone marrow cells (n=223) and from non-tumor tissue (peripheral blood or saliva) was processed using the HyperCap workflow (KAPA Biosystems). Of the 223, 48 samples were processed in a clinical diagnostic laboratory. Adapter ligated DNA was hybridized with a mixture of the mutation and translocation panel and purified, amplified libraries were sequenced using 75 bp paired end reads. Sequences were aligned to hg19 and mutations and translocations identified using Strelka and Manta. Copy number was determined using the ratio of non-tumor to tumor reads in each targeted region. Data were validated using clinical FISH (translocations, n=116), MLPA (copy number, n=101), known standards (mutations), ddPCR (mutations), and whole genome sequencing (WGS; translocations and copy number, n=122). Results: Canonical IgH translocations were detected in 43.2% of patients by the panel, and all agreed with WGS. FISH detected one additional "variant" t(4;14), but did not detect 4 translocations detected by both sequencing methods. In the remainder of the samples no canonical IgH translocation was detected, agreeing with FISH results. Non-canonical translocations were detected in 14.5% of samples, 43% of which were to the MYC locus. MYC translocations were detected in 37.3% of samples with copy number abnormalities occurring surrounding MYC in 32.7% of samples. Overall, MYC abnormalities were detected in 46.4% of samples. Copy number was determined by panel sequencing and MLPA for 22 regions that were directly comparable between the technologies in 101 patient samples and 13 myeloma cell lines. The copy number concordance between the technologies was 96.9% and 99.6% in patient samples and cell lines, respectively. For the important prognostic regions, the concordance was R 2=0.962 (CDKN2C), R 2=0.986 (CKS1B), and R 2=0.973 (TP53). Panel copy number data were also compared to WGS data and showed complete concordance across the three prognostic regions, which the exception of 2 samples. In these 2 samples a homozygous deletion was detected by the panel but not by WGS. The deletions were 6.2 and 8.0 kb in size, one encompassing the coding sequencing of TP53 and the other exons 1-4 of TP53. A larger homozygous deletion of 36.3 kb was detected by both sequencing methods. Mutation detection validation was performed using Horizon Discovery samples with known variant allele frequencies (VAF) for common mutations. We were able to determine the sequencing VAF for 74 mutations across 5 samples which had a concordance of R 2=0.9849 between the expected and observed frequencies. The minimum detected VAF was 1.3% at an average depth of 891x. We also performed ddPCR on 6 patient samples with the common KRAS, NRAS and BRAF mutations which resulted in a VAF concordance of R 2=0.9983. Conclusion: We have developed a targeted sequencing panel for MM patient samples that is as robust or better than both FISH and WGS. A full protocol for sample processing and analysis is available, and has been used in a clinical diagnostic laboratory. Disclosures Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abu Zaid: Pieris: Current equity holder in publicly-traded company; Incyte: Research Funding; Pharamcyclic: Research Funding; Syndax: Consultancy, Research Funding. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yong: GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; Autolus: Research Funding; Janssen: Honoraria, Research Funding. Morgan: Takeda: Honoraria. Abonour: Celgene-BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Jensen: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Flynt: Bristol Myers Squibb: Current Employment. Ansari-Pour: Bristol Myers Squibb: Consultancy. Gooding: Bristol Myers Squibb: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

Abstract: Introduction Identification of the causes of, and biomarkers for, drug resistance in myeloma is important for understanding treatment failures, and for future instigation of targeted therapeutics for myeloma. Using the largest set of whole genome sequencing (WGS) of advanced and drug resistant multiple myelomas to date, we reported that even heterozygous loss of the 3p region, which harbours immunomodulatory drug (IMiD) and CRBN E3 ligase modulator drug (CELMoD)-binding protein Cereblon (CRBN), undergoes strong therapeutic selection on lenalidomide (LEN) and/or pomalidomide (POM) treatment (Gooding et al 2021, PMC7893409). We hypothesized that copy loss of other genes required for IMiD activity may also have clinical relevance. Several groups have reported pharmacogenetic screens identifying genes essential for IMiD sensitivity in vitro, particularly genes required for the maintenance of the CUL4-DDB1-CRBN E3 Ubiquitin Ligase, such as members of the COP9 signalosome complex, function of which prevents CRBN protein degradation. However, loss of these genes has hitherto not been reported in myeloma. Methods and results We identified candidate genes whose loss may favor IMiD drug resistance from published pharmacogenetic screens (n=5), and shortlisted genes consistently identified as essential for LEN or POM function in ≥2 screens (n=23). In our WGS dataset of 455 patients (cohorts: newly diagnosed (ND) n = 198, LEN-refractory n = 203; and LEN-then-POM-refractory n = 54), the incidence of mutation of shortlisted LEN/POM-essential genes in drug-refractory cohorts was rare ( & lt;5% combined), as found with CRBN. We next identified all those with overall incidence of & gt;10% copy loss at the LEN-then-POM-refractory state, plus incidence of copy loss that increased from ND to LEN-then-POM-refractory states by ≥1.5-fold. This delivered 3 copy loss regions for further investigation: a) 3p, which we had already reported; b) 17p, loss of which is known to be strongly selected in myeloma as the site of TP53; and c) 2q, previously unidentified as relevant in myeloma, but whose minimal common region contained two members of the COP9 signalosome (COPS7B, COPS8). Proportion of loss of this region increased between ND (5.5%), LEN-refractory (9.8%) and LEN-then-POM-refractory states (16.6%), p=0.009. Those patients who had lost a copy of these genes also demonstrated a significant reduction in COPS7B/COPS8 gene expression (p & lt;0.01 both genes). In a separate cohort of myeloma patients (n=24) with sequential sample WGS analysis before and after LEN and/or POM resistance acquisition, we traced acquisition of CNA-defined subclones. 5/24 (21%) patients had acquired either clonal or subclonal loss of the 2q region containing COPS7B and COPS8 at IMID resistance, which had been either absent or below limit of detection pre-IMiD exposure. No other CNA newly-emerged in such a high proportion during IMiD treatment. Relative decrease in even one COP9 signalosome gene has been shown to cause CRBN protein level to fall, and reduce LEN efficacy (Sievers et al 2018, PMC6148446). We are now analysing CRBN protein levels in sequential biopsies from these cases. Conclusion Copy number aberrations have not previously been shown to drive a therapy-specific clonal advantage in myeloma in the clinic. We have now identified a second novel CNA, 2q loss, which increases in incidence through LEN- and POM-refractory states to emerge as a marker of dominant clones in advanced, IMiD-resistant disease. Whether these CNAs will mark resistance to novel CELMoDs remains to be seen. The CRBN protein is key to the function of these drugs, and many novel proteolysis targeting chimeras (PROTACs) in development, but whether the kinetics of their CRBN binding are as sensitive to relative CRBN protein loss remains a key question. CNAs may be easily and cost-effectively detected in the clinic by targeted sequencing approaches, and may prove valuable in future therapeutic decision making. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Ansari-Pour: Bristol Myers Squibb: Consultancy. Karagoz: h.: Research Funding. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding. Vyas: Astellas: Consultancy, Honoraria; Takeda: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Abstract: We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. Experimental Design: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). Results: Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. Conclusions: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.