WHOLE EXOME SEQUENCING DEFINES CLONAL ARCHITECTURE AND GENOMIC EVOLUTION IN MULTIPLE MYELOMA

Multiple myeloma (MM) is an incurable malignancy of post-germinal centre B- cells whose pathogenesis is only partially understood. Chromosomal abnormal- ities, such as hyperdiploidy and recurrent immunoglobulin gene locus rearrangements are frequent, but are thought to be insufficient for malignant transformation because they are also observed in the pre-malignant syndrome called monoclonal gammopathy of uncertain significance (MGUS), while malig- nant progression is often associated with additional events, like MYC activation. This implies that the genetic landscape of MM changes over time as result of additional events that define its emergence and progression.To investigate genomic event underlying MM pathogenesis and evolution, we used targeted capture and massively parallel sequencing of exomes of CD138 purified plas- ma cells from 68 patients with MM. For 17 patients, serial samples (range 5-18 months apart) were available. Exome reads were used to call substitutions and insertions/deletions. We used single nucleotide polymorphisms (SNPs) to infer chromosomal copy number and allelic ratio of the tumor. Lastly, mutation bur- den was used to estimate the clonal architecture of each sample and its evo- lution over time (where available). All data were produced by in-house algo- rithms developed at the Wellcome Trust Sanger Institute.Our analysis con- firmed mutation recurrence in some of the genes previously identified (Chap- man et al, Nature 2011): KRAS 25%, BRAF 13%, NRAS 13%, FAM46C 9%, TP53 9%, CCND1 1%. Interestingly, 3/9 BRAF mutations co-occurred with acti- vating KRAS mutations in the same patient, raising important therapeutic impli- cations. We also described several genes previously unreported in MM. So far, 1803 variants have been validated in the cohort (range 0-140 per patient). 292 genes are recurrent, suggesting a role in MM pathogenesis, and among these are genes involved in the NF-kB pathway, histone-modifying enzymes, cyclins and cyclin-dependent kinases. We also report mutations in deubiquitinating enzymes, protein tyrosine phosphatases, cytoplasmic proteins involved in Gol- gi, endoplasmic reticulum and vesicle protein trafficking, and known tumor sup- pressors such as PTEN and APC.Analysis of the clonal structure showed at least two subclones in 66/68 (97%) patients at diagnosis, suggesting that myelo- ma is a heterogeneous disease at presentation. The burden of at least some of the mutations changed over time in 12/15 patients (80%) with serial samples, highlighting ongoing clonal evolution. Interestingly, 2/2 NRAS mutations only appeared in the late sample, and 3/6 KRAS mutations increased their burden over time. Furthermore, copy number analysis showed that 9/15 (60%) patients also acquired additional chromosomal gain/deletions, with loss of 17p in 4/9 (44%). This suggests a role for RAS activation and TP53 loss in disease pro- gression.In conclusion, in our cohort of MM samples we show: 1) a comprehen- sive list of previously unreported variants, many of which are recurrent; 2) evi- dence of tumor heterogeneity at the time of diagnosis; 3) discernable genetic changes and shifts in the clonal structure of disease at the time of progression. Our study provides new insights into the molecular pathogenesis of MM, and will help identify molecular alterations associated with progression of disease and development of drug resistance.