Abstract: Multiple myeloma (MM) is accompanied by heterogeneous somatic alterations. The overall goal of this study was to describe the genomic landscape of myeloma using deep whole-genome sequencing (WGS) and develop a model that identifies patients with long survival. METHODS We analyzed deep WGS data from 183 newly diagnosed patients with MM treated with lenalidomide, bortezomib, and dexamethasone (RVD) alone or RVD + autologous stem cell transplant (ASCT) in the IFM/DFCI 2009 study (ClinicalTrials.gov identifier: NCT01191060 ). We integrated genomic markers with clinical data. RESULTS We report significant variability in mutational load and processes within MM subgroups. The timeline of observed activation of mutational processes provides the basis for 2 distinct models of acquisition of mutational changes detected at the time of diagnosis of myeloma. Virtually all MM subgroups have activated DNA repair–associated signature as a prominent late mutational process, whereas APOBEC signature targeting C 〉 G is activated in the intermediate phase of disease progression in high-risk MM. Importantly, we identify a genomically defined MM subgroup (17% of newly diagnosed patients) with low DNA damage (low genomic scar score with chromosome 9 gain) and a superior outcome (100% overall survival at 69 months), which was validated in a large independent cohort. This subgroup allowed us to distinguish patients with low- and high-risk hyperdiploid MM and identify patients with prolongation of progression-free survival. Genomic characteristics of this subgroup included lower mutational load with significant contribution from age-related mutations as well as frequent NRAS mutation. Surprisingly, their overall survival was independent of International Staging System and minimal residual disease status. CONCLUSION This is a comprehensive study identifying genomic markers of a good-risk group with prolonged survival. Identification of this patient subgroup will affect future therapeutic algorithms and research planning.

Abstract: Multiple myeloma (MM) and its precursor condition MGUS are characterized by chromosomal aberrations. Here, we comprehensively characterize the order of occurrence of these complex genomic events underlying MM development using 500 MGUS, and MM samples. We identify hyperdiploid MM (HMM) and non-HMM as genomically distinct entities with different evolution of the copy number alterations. In HMM, gains of 9,15 or 19 are the first and clonal events observed as clonal even at MGUS stage. These events are thus early and may underlie initial transformation of normal plasma cells to MGUS cells. However, CNAs may not be adequate for progression to MM except in 15% of the patients in whom the complex subclonal deletion events are observed in MM but not MGUS. In NHMM, besides the driver translocations, clonal deletion of 13 and 1q gain are early events also observed in MGUS. We combined this information to propose a timeline for copy number alteration.

Abstract: High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

Abstract: Multiple myeloma (MM) is a hematological malignancy characterized by various genetic abnormalities including translocations involving the IgH gene at 14q32. Amongst these, t(11;14) is one of the most common translocations. Recent clinical data suggests a significant impact of Venetoclax, a small molecule inhibitor of BCL2, in this subgroup of MM patients, representing the first example of personalized medicine in MM and opening a wide range of research aiming at elucidating its mechanism of action. However, despite the initial positive response to the drug, a significant proportion of patients eventually develop resistance and relapse. To delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistance phenotype, we modeled the response to Venetoclax in 2 MM cell lines (KMS27 and KMS-12PE with IC50 of 35.47nM and 3.64nM, respectively). Whereas the vast majority of cells plated into 96-well plates were killed within a few days of exposure to a high dose of drug concentration, we detected a small fraction of viable, largely quiescent cells, which were expanded by culturing them in high doses of Venetoclax. We successfully generated 4 independent clones from each cell line, that were single cell-cloned with continued growth in the presence of high doses of Venetoclax. These clones labelled as drug-tolerant expanded persisters (DTEP) were investigated for the mechanisms driving drug tolerance and resistance against Venetoclax. First, we observed that altered expression of apoptotic regulators were associated with Venetoclax resistance in DTEP cells. We indeed observe a significant increase in the anti-apoptotic proteins MCL1 and BCL-XL in DTEP clones, which translated in our observation of improved sensitivity to MCL1 and BCL-xL inhibitors (S63845 and A-1155463 respectively). We performed both whole genome sequencing (WGS) and RNA-seq to evaluate if DTEP cells undergo transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprograming during development of acquired drug resistance. While, WGS analysis didn't show any significant differences between parental and resistant clones, transcriptomic analysis showed both shared and unique transcriptome signatures in the DTEP clones. Gene set enrichment analysis (GSEA) of the common significantly modulated genes in the resistant clones revealed that the genes belonging to the PKA-ERK-CREB pathway were significantly upregulated in resistant clones, while apoptotic genes were downregulated compared to parental cells. Western blot analysis confirmed activation of ERK and the downstream target cAMP response element-binding (CREB) gene in resistant clones; and importantly treatment with the ERK inhibitor U0126 rescued the resistance to Venetoclax, providing a synergistic activity in resistant clones but not in parental cells, with decreased cell viability and increased apoptotic cell death. To evaluate if the ERK pathway was also associated with intrinsic resistance to Venetoclax, we assessed a panel of 24 MM cell lines and then calculated Pearson correlation coefficients between the measured drug activity and individual gene expression levels (by RNA-seq) across all cell lines and subjected the resulting rank-ordered gene list to GSEA. This analysis showed that mechanisms driving the DTEP phenotype are different from those associated with the intrinsic resistance to Venetoclax. RNA processing and splicing pathways were strongly enriched, with high expression of these genes correlating with increased sensitivity. Moreover, among the genes correlated with a resistant phenotype, we observed that the gene G0S2 was significantly downregulated in the resistant cell lines. G0S2 is a tumor suppressor gene that binds and inhibits BCL2. Interestingly, we observed that while G0S2 is downregulated in MM compared to normal plasma cells, t(11:14) patients have a higher expression. We are now in the process of validating G0S2 in MM and its contribution to Venetoclax sensitivity in MM. In conclusion, we here provide evidences of molecular mechanisms of acquired resistance to Venetoclax with activation of the ERK pathway as one of the prime targets. Combining Venetoclax with ERK inhibitor may therefore prevent or overcome the acquired resistance to Venetoclax observed in MM patients. Disclosures Fulciniti: NIH: Research Funding. Munshi:C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract: Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, 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; Amgen: Consultancy, 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. Munshi:OncoPep: Other: Board of director.

Abstract: Multiple Myeloma (MM) is a complex disease with distinct molecular and clinical characteristics. Recent large collaborative efforts have identified number of driver genes. However over 95% of all somatic alterations occur in non-coding regions and very little is known about how they affect the disease. We performed a deep (average coverage 〉 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively analyze recurrent somatic alterations in non-coding regions. We detected median 11,852 (Range 4,802-87,396) mutations and indels per sample with overall more than 3.9M total somatic mutations. Introns (3.6 mutations/per Mb) and intergenic regions (4.06 mutations/per Mb) had significantly higher number of mutations per megabase compared to Exons (2.7 mutations/per Mb) (p 〈 1e-5). Mutations in coding regions in our data was similar to published whole exome sequencing studies. We observed 46 [range 7 - 219] structural variants (SVs) per sample with 98% involving non-coding regions. We found that number of SVs significantly correlated with overall survival (p value = 1.7e-5). We detected chromothripsis ( 〉 =7 oscillating copy number change and significant clustered SVs and/or clustered translocations) in 24% of newly diagnosed samples; and kataegis hotspots on chromosome 3q27-3q28 (24%), 11q13 (5.8%) and 12q24 (5.3%). By clustering SV breakpoints across the genome we have identified 3 SV hotspots on chromosome 17q21, 7q34, and 11q13. We next interrogated the non-coding regions to identify genomic loci with higher than expected mutation count compared to background mutation rate. We have identified 456 loci that are significantly enriched in non-coding regions (5' UTR, 3'UTR, promoter, intergenic, intronic, and distal regulatory regions) [adjusted p value 〈 1e-5 and observed in 〉 =10% newly diagnosed MM]. These loci are then assigned to genes or gene neighborhoods to evaluate their potential impact. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. Of these the most prominent examples are 1.) 3'UTR mutations are enriched in CD93 gene, which plays critical role in B cell development with loss of expression in CD138+ MM cells compared to normal plasma cells (p value 〈 1e-5); 2.) Promoter region - we have identified 635 mutations in 2kb region in BCL6 coming from 76% of all newly diagnosed samples. BCL6 (p value 〈 1e-5) has significantly downregulated expression in MM. Interestingly, but not surprisingly this hypermutated region showed high intensity of H3K27Ac activity in normal cells; 3.) 5'UTR - BCL7A (27.9%) and LPP (11.7%) were top two 5' UTR mutated target genes and RNA-seq data confirmed significant downregulation of their expression (p values 〈 1e-5 and 0.0048 respectively) in the MM cells. Additionally, BCL7A (48%) also showed significant enrichment of intronic mutations. A similar mutational hotspots were observed within the vicinity of additional functionally important genes in myeloma including ROBO1/2, ILF2, IRF8 and BCL2A1. Our data also showed that these frequent mutations have higher cancer cell fraction (CCF) [median CCF 〉 0.75] suggesting their occurrence earlier in the disease development. To validate the function of these mutations, we have started to carry out gain/loss of function studies. Our analysis with BCL7A shows that BCL7A knockdown increases the cell viability while its overexpression decreased growth, colony formation and increased apoptosis. This tumor suppressor function of BCL7A is being further analyzed in light of our mutational data in the nearby non-coding region. In conclusion, this large deep whole genome sequencing data from newly-diagnosed MM patients identifies a vast majority of non-coding mutations with potentially significant functional and biological role in MM. Our integrative approach using both WGS and RNA-seq data from the patients now provides us important tools to further characterize the impact of these mutations and develop opportunities for targeted therapeutics. Disclosures Richardson: Celgene: 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; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: 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; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Moreau:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thakurta:Celgene Corporation: Employment, Equity Ownership. Anderson:Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Avet-Loiseau:Amgen: Consultancy, 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; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.

Abstract: Multiple Myeloma (MM) is a plasma cells malignancy with number of recent therapeutic options that has improved outcomes with median survival now stretching beyond 8 years. There has been an intense search to identify genomic and laboratory correlates of outcome for high risk patients. However, a subgroup of patients have a long survival but genomic segmentation of this important group which can be potentially cured has not been identified. We here described first such attempt at identifying a subset of patients with long survival. We have analyzed data from 205 newly-diagnosed uniformly-treated MM patients using both deep whole genome (WGS) (80x) and whole transcriptome sequencing (RNAseq). Median number of SNVs, small insertion and deletion per megabase were 2.18 [0.49 - 14.52], 0.077 [0.024 - 0.17] and 0.12 [0.03 - 0.26] respectively. MM subgroups defined by FISH or copy number alterations (CNAs) had significantly different mutational load (Kruskal-Wallis p-value = 0.003). In general, Hyperdiploid MM (HMM) patients had lower SNV load compared to other subgroups and t(14;16) had the highest mutational load per megabase (Dunn test FDR 〈 0.25). These subgroups also showed significant differences for mutational process utilization. Mutational processes associated spontaneous deamination of 5-methylcytosine to thymine was significantly high in HMM, APOBEC activity was very strong in t(14;16) MM and DNA repair was significantly different in t(6;14), del17p and t(11;14). Importantly, the mutations associated with clonal cell population and thus in early phases of MM cell progression were driven by spontaneous or enzymatic deamination (Signature 1), somatic hypermutation in lymphoid cells (Signature 9) and Signature 17 (ANOVA p value 〈 1e-16) and surprisingly APOBEC activity was constant among the clones (ANOVA p value 〉 0.05). However, mutations associated with homologous recombination (HR) and nucleotide excision repair (NER) activity were significantly enriched in the subclonal mutations (ANOVA p value 〈 1e-16) suggesting their role in later stages of MM progression. Based on this information, we next calculated the homologous recombination repair defects using copy number alterations from WGS data. We identified that HR score generated using WGS copy number information predicted outcome in newly diagnosed myeloma. Further analysis of this data identified that newly diagnosed MM patients with hyperdiploid MM (HMM) and no HR- repair deficiency have superior outcome with 6 year overall survival (OS) probability for this of 100% in IFM/DFCI 2009 cohort (Logrank test p value = 0.0012). We next validated this finding using MMRF CoMMpass dataset and we confirmed 6-yr OS probability of 95% (Logrank test p value = 0.0016) in this independent dataset (Figure 1). We further investigated RNAseq data between these genomically defined groups and identified that chromosome and telomere maintenance pathways (FDR 〈 0.01) and low bone disease associated genes described by Zhan et al. (FDR = 0.004) were up regulated in low-risk group. Finally, patients who were able to maintain HR, had lower mutation rate for TP53 (0% vs 9%, FDR 〈 0.05), CSMD1 (0% vs 6%, FDR 〈 0.05), FAT3/FAT4 mutations (1.7% vs 15%, FDR 〈 0.05), however surprisingly higher mutation rate for NRAS (42% vs 14%, p value 〈 0.001). In addition, DNA damage associated processes predicted by SNVs trinucleotides were significantly lower in the low-risk group (12% vs. 20%, p value 〈 1e-05) providing further support to this data. In conclusion, we report a detailed genomic profile using deep DNA and RNA sequencing and identify a genomically-defined sub group that is predicted to have a long survival. This study also identifies the role of HR in myeloma with potential for its translational application in both prognosis as well as therapy. Figure 1 Disclosures Richardson: Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: 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; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: 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. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Thakurta:Celgene: Employment, Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Avet-Loiseau:celgene: Consultancy, Other: travel fees, lecture fees, Research Funding; takeda: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Takeda: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy.

Abstract: Alternative splicing (AS) is a critical post-transcriptional event, which affects number of cellular processes including cell growth and survival. Our group has previously shown that aberrant splicing of numerous genes in multiple myeloma (MM) affects the disease biology. Dysregulated expression or mutations in several splicing factors (SFs), including SF3B1, Fox2, SRSF1, NONO, in MM compared to normal plasma cells have been reported with impact on outcome, highlighting significant role of splicing factors in myeloma. We have previously reported global alternative splicing events in MM to understand biology. Here, we report, using a deep RNAseq data, a detailed analysis of the splicing events, including retained intron, cassette exon, Alternative 3' / 5' site, mutually exclusive exons, mutually alternative 3' / 5' exons and analyze significance of AS in predicting prognosis. We purified myeloma cells from 420 newly-diagnosed patients treated homogeneously with lenalidomide, bortezomib and dexamethsone (RVD) with or without high-dose melphalan followed by lenalidomide maintenance in the IFM/DFCI 2009 study. RNA isolated from these purified CD138+ MM cells and from 18 normal donor plasma cells were processed by RNAseq (80-100 million paired end reads on Illumina HiSeq) and analyzed using a custom computational and statistical pipeline. Our results showed 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). Although we observed 1288 (~84%) of these 1534 genes with only one type of AS, remaining 16% of the genes had 2 or more AS events. Within the AS types, we observed retained intron (495 - ~32%) and cassette exons (727 - ~47%) as the most frequent events, whereas mutually exclusive exons were observed in less than 1% of 1534 genes. We found that 158 genes had same type of AS in more than 50% of the patients and 445 genes showed same AS in more than 30% of patients suggesting significant biological role for the spliced isoforms. Importantly, we have evaluated impact of each AS event with clinical outcome and observed that 43 AS events showed significant correlation with event free survival (EFS) (p value 〈 0.01), 27 AS events predicted overall survival (OS) and 5 AS events predicting both EFS and OS. We have also observed that although overall expression of number of these genes does not predict overall survival outcome, an isoform level expression predicts survival. We are now developing a composite model using gene expression and alternative splicing events that in our preliminary analysis has provided increased prediction power compared to gene expression model only which shows that alternative splicing events may carry independent significance for outcome prediction over overall gene expression alone. In summary, this study for the first time reports a detailed splicing landscape in myeloma and highlights the biological and clinical importance of alternative splicing events. It suggests the need to further integrate study of spliced isoform in our biological studies, in understanding disease behaviors as well as develop targeted therapeutics. Disclosures Richardson: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Anderson:Acetylon: Other: Scientific Founder; Onyx: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: Scientific Founder; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Attal:celgene: Consultancy, Research Funding; sanofi: Consultancy; janssen: Consultancy, Research Funding; amgen: Consultancy, Research Funding. Moreau:Takeda: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau. Avet-Loiseau:celgene: Consultancy; amgen: Consultancy; sanofi: Consultancy; janssen: Consultancy.

Abstract: BCMA targeting chimeric antigen receptor (CAR) T cell therapy has shown deep and durable responses in multiple myeloma. However, relapse following therapy is frequently observed, and mechanisms of resistance remain ill-defined. Here, we perform single cell genomic characterization of longitudinal samples from a patient who relapsed after initial CAR T cell treatment with lack of response to retreatment. We report selection, following initial CAR T cell infusion, of a clone with biallelic loss of BCMA acquired by deletion of one allele and a mutation that creates an early stop codon on the second allele. This loss leads to lack of CAR T cell proliferation following the second infusion and is reflected by lack of soluble BCMA in patient serum. Our analysis suggests the need for careful detection of BCMA gene alterations in multiple myeloma cells from relapse following CAR T cell therapy.

Abstract: Multiple Myeloma (MM) is characterized by genomic heterogeneity with copy number alterations (CNA) as one of the most prominent genomic perturbation. Hyperdiploidy involving chromosomes 3,5,7,9,11,15,19, and 21 is observed in nearly half of the patients, however the chronological sequence of their occurrence during MM development remains unknown. Here, we have acquired one of the largest genomic datasets from 647 patients that combines data from monoclonal gammopathy of undermined significance (MGUS) to newly diagnosed MM (336 newly diagnosed MM, 147 smoldering MM (SMM) and 164 MGUS) to characterize when and in what sequence CNA occurs in MM development. We deduce the order of CNA events by identifying their pattern of clonality basically inferring that clonal genomic change suggest its origin at an earlier stage of the disease. In hyperdiploid MM (HMM), gains in chromosome 19 (95%), 15 (90%) and 9 (90%) are the most frequent events, followed by gains in 5,11,3,7 and 21. Based on the clonality assessment the gain of chromosome 15 is the first and most frequent clonal/near clonal event, observed in 95% of HMM patients followed by Chromosome 9. Surprisingly, although chromosome 19 gain is the most frequent event overall, its clonal occurrence was lower than clonal chromosome 15 gain. More than 96% of HMM samples had concurrent gains in at least 2 of the 3 most frequent chromosomes (9,15 and 19). Moreover, less frequent events such as chromosome 21 gain, 18p gain, and 1q gain showed higher frequency of clonal/near clonal occurrence compared to other events indicating that when these events occur they are early events. Majority of the deletions occur as late subclonal events with few or none observed as clonal events. In the nonhyperdiploid MM (NHMM), del13, gain of 1q and gain of 11 had the highest frequency of clonal occurrence. Most were clonal events signifying its importance in the early stages of the disease. As all MM originates from its precursor conditions, MGUS and SMM, clonal and likely early CNAs in MM, must also exist in MGUS and SMM. So, we next investigated genomic data from SMM and MGUS for the occurrence of clonal events observed in MM. We confirmed same patterns for top MM-related CNA events in SMM and MGUS and observed no significant difference (p=0.1) between the number of events in hyperdiploid groups in MM (median=10, IQR= [8-12]) and SMM (median=9, IQR= [7-11] ).To further confirm the analysis, we calculated an average clonality score for each chromosomal alteration using a 1 to 5 clonality index (1 being clonal 5 being low subclonal) in MM, SMM and MGUS and observed that similar clonal trisomies median=5, IQR=[4-6] are observed in both HMM and hyperdiploid MGUS; and that not all trisomies are required or occur at the same time. With occurrence in over 96% of cases trisomy involving chromosome 15 is central to the development of MGUS and later on MM. This is closely followed by trisomy of 9, and 19. Gain of chromosome 21 is also an early event. Major events like deletion 13 and 1q gain occur relatively later than first hyperdiploid events. NHMM on the other hand is well known to have clonal IgH-associated translocation as an initiating feature which is also observed in SMM and MGUS. However, different from HMM, it shows only few CNAs at an early stage and does not accumulate frequent additional alterations. The only exception to this rule is a deletion group observed in HMM, NHMM and SMM but not MGUS. In this deletion in over 10 whole chromosome or its p or q arm are involved as subclonal events. Its absence in MGUS suggests them to be a later event in MM development. On the other hand, number of deletions are observed at the same locations in both hyperdiploid and non-hyperdiploid groups with similar frequency. Moreover, similarity of events in this deletion groups strongly suggest that in sub group of both HMM and NHMM a similar process may be operative to induce such deletions. Our results also highlight that for both HMM and NHMM groups the major copy number events are not adequate for eventual malignant transformation since only a small fraction of MGUS patients progress to MM. Here, we describe the time line of initial copy number alterations observed in MM and confirm their early occurrence using data from a unique early stage plasma cell cases. Similarities between stages show that large scale DNA alterations happen early however some copy number hotspots are enriched over the time which could be important for disease progression. Disclosures Moreau: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.