ALBERT

Description: INTRODUCTION: Multiple myeloma (MM) is a biologically and clinically heterogeneous disease. Different recurrent driver genomic events have been reported, but to date no unifying feature has been identified in MM evolution. The recent interest in signatures of mutational processes through analysis of whole-exome sequencing data has led to initial insights into what generates MM mutational repertoire (Bolli et al, Nat Com 2014). Here, taking advantage of the increased power provided by whole genome sequencing (WGS), we analyzed 22 paired samples from 11 patients first at the smoldering (SMM)/MGUS stage and subsequently at the time of progression to symptomatic MM to gain a deeper understanding of the full landscape of mutational processes operative in MM, especially during their evolution over time. MATERIAL AND METHODS: DNA from bone marrow CD138+ cells underwent WGS along with a matched normal sample using HiSeq X Ten machines (Illumina, Inc.). Mutational signature extraction was performed running non-negative matrix factorization (NMF) as previously described (Alexandrov et al, Nature 2013). RESULTS: We have observed and utilized a median number of 5780 (range 2599-7760) substitutions per patient at the asymptomatic stage and 5954 (ranges 2824-8227) at progression to MM stage to extract mutational signatures. NMF extracted 5 main signatures (http://cancer.sanger.ac.uk/cosmic/signatures). Specifically, APOBEC- (signature #2) and age-related signatures (signatures #1 and #5) accounted for 13% (1-21%) and 23% (3.2-40%) of all substitutions, respectively. In addition, we found two known signatures that were not implicated in MM so far: non-canonical AID (Signature #9), contributing to 28% of all substitutions (17-55%); and signature #8, accounting for 28% of all substitutions (13-45%) and pertaining to a yet unknown mutational process. Finally, the fifth signature did not match any of the previously described ones, representing a potential novel process which we defined as MM-1 (7%, range (1-16%). Interestingly, we found a differential contribution of processes in non-coding and intronic regions where AID was more prevalent, while exonic regions where APOBEC and age signatures were more prevalent. In intronic regions we found widespread regions of kataegis (9/11 patients), reflective of localized hypermutation. In our patients, kataegis was associated with rearrangements in 60% of cases, and was present in both the SMM and MM sample in 84% of cases, suggestive of an early event during tumor development. Contrary to what is observed in solid cancers, APOBEC signature was only responsible for 25% of kataegis variants, vs 70% for AID, suggesting a causative role of aberrant AID activity in shaping the early mutational repertoire of neoplastic plasma cells. To confirm this, we looked at serial samples in our cohort. While the percent contribution of each signature varied in each patient, confirming genomic heterogeneity of MM, it did not change when paired SMM and MM samples from the same patient were compared. This shows that mutational processes required for the development of symptomatic MM act early, and have been already operative at the SMM stage. However, by clustering substitutions as clonal (early variants present at the time of tumor initiation) or subclonal (late variants arisen closer to the time of sampling) using a Bayesian hierarchical Dirichlet process (Bolli et al, Nature Comms 2014), we could analyze processes operative before SMM was diagnosed. NMF analysis of these clusters reported striking differences. Specifically, AID and age were the predominant mutational processes in early substitutions in all patients, contributing to a median of 35% (25-54%) and 30% (15-43%) of variants respectively. Conversely the contribution of AID was minimal among late substitutions (5%, 1-22%), where instead APOBEC, Signature #8 and MM-1 activity was prominent [19% (1-43), 38% (8-73%), 16% (2-50%) respectively]. CONCLUSION: WGS data allowed the identification of mutational processes operative well before MM becomes clinically evident. Our observation that all samples have signs of aberrant AID at the time of tumor initiation supports a unifying model where MM precursors are initially transformed with the contribution of AID, providing a fertile ground for other later processes (i.e APOBEC and signature #8) to act and shape the final genomic landscape of overt multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a malignancy of post-germinal centre B-cells whose pathogenesis is only partially understood. Chromosomal hyperdiploidy and recurrent immunoglobulin gene locus rearrangements are frequent, but are insufficient for malignant transformation, which is associated with additional events such as somatic mutations, epigenomic aberrations, and chromosomal copy-number changes. To investigate genomic event underlying MM pathogenesis and evolution, we used whole exome sequencing, copy number profiling and cytogenetics in 67 patients and 84 samples. For 15 patients, 2 or 3 serial samples (median 299 days apart) were available. Exome reads were used to call substitutions and indels. We used the Genome-Wide SNP Array 6.0 or exome reads to estimate the allele-specific copy number of the tumor. To cluster variants and estimate the clonal architecture of each sample and its evolution over time, we used the mutation burden, corrected for copy number and normal cell contamination. Analysis of the clonal structure of the tumors showed at least one subclone in 94% of patients at diagnosis, suggesting that myeloma is a heterogeneous disease at presentation. Interestingly, many mutations of known MM driver genes (KRAS, NRAS, BRAF, TP53, FAM46C) were subclonal at diagnosis. In 5/67 patients, BRAF and KRAS/NRAS mutations co-existed in the same sample, raising therapeutic implications given the paradoxical ERK-activating effect of BRAF inhibitors in RAS-mutated cells. Furthermore, only 3/10 BRAF variants were V600E, the current target of most inhibitors. Altogether, only the 5 previously known genes were significantly enriched in our cohort, highlighting marked heterogeneity of the spectrum of candidate driver gene mutations across MM patients. Nevertheless, we identified several new recurrent gene lesions: inactivating mutations of SP140 (7.5%), a gene previously linked to germline susceptibility to CLL, and in ROBO1 (7.5%), a gene recently implicated in pancreatic cancer; clustered missense substitutions in EGR1 (6%), a gene previously implicated in plasma cell apoptosis; clustered truncating mutations in LTB (4.5%), a TNF-family protein implicated in lymphoid development. The subclonal structure of the sample changed over time in 72% paired samples, highlighting genomic evolution at relapse. We described 4 different scenarios with striking concordance between mutations and chromosomal copy number changes: no change, linear evolution (a new clone appears in the later sample), differential clonal response (the relative proportions of the subclones change over time), and branching evolution (new clones emerge, while others decline in frequency or disappear). All subclonal variants in known driver myeloma genes increased their clonal fraction at the later time-point, consistent with the expected positive selection for the subclones harboring them. To investigate mutational processes responsible for the generation of the mutational repertoire in MM, we extracted the variant context and analyzed the mutational signatures. We found two signatures in our samples. The most represented one is enriched for spontaneous deamination of methylated cytosines, a common process in cancer and aged cells. The second signature was more represented in samples showing extremely high numbers of variants, sometimes clustered in small regions of ∼200 bp (kataegis). We hypothesize that it results from aberrant activity of the APOBEC family of cytosine deaminases, recently described in breast cancer. Interestingly, cases of extramedulary relapse were always associated with branching evolution and showed increased contribution from this APOBEC signature. In conclusion, in our cohort of MM samples we show: 1) evidence of tumor heterogeneity at the time of diagnosis; 2) discernable genetic changes and shifts in the clonal structure of disease at the time of progression; 3) different mutational processes responsible for an heterogeneous mutational repertoire across patients, and over time in the same patient; 4) a comprehensive list of recurrent variants, many of which are previously unreported. Our study provides new insights into the genomic architecture of MM, and will help identify molecular alterations associated with progression of disease and development of drug resistance. Disclosures: Tai: Onyx: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Moreau:Celgene: Honoraria, Speakers Bureau. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity’s Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder , Scientific Founder Other.