Egan, Jan B.

SummaryWe constructed a multiple myeloma (MM)‐specific gene panel for targeted sequencing and investigated 72 untreated high‐risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high‐risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Abstract Background: In order to understand the genetic events associated with disease progression and the development of drug resistance in multiple myeloma, we studied four longitudinally collected samples from a single, high risk, t(4;14) multiple myeloma patient. Samples over a four-year period were available from diagnosis, at time of first progression on lenalidomide, second progression while on bortezomib, at time of relapsed refractory disease and with end stage plasma cell leukemia. To better understand these genetic events, we utilized DNA extracted from germline tissue and CD138 purified bone marrow tumor cells to conduct whole genome sequencing (WGS) utilizing the Life Technologies SOLiD™ platform. Results: We have sequenced over 100 billion bases per sample with whole genome fragment library sequencing providing an average of 30X aligned coverage at all four time points studied. Each sample was aligned to the reference genome (NCBI36/hg18) with BFAST and post-alignment removal of duplicates and recalibration performed with Picard and Genome Analysis Toolkit (GATK) respectively. We then called point mutations with SolSNP and compared the germline sample to each tumor sample utilizing the Paired Mutation Walker. To date, we have identified 224 nonsynonymous point mutations within 217 genes. We then utilized the Sorting Intolerant from Tolerant (SIFT) algorithm and identified 109 potentially damaging point mutations which include 108 novel mutations in coding exons. Of the 224 somatic mutations observed, only 20 of these mutations are common to all tumor time points. Interestingly, the diagnostic and bortezomib refractory (second progression) samples had 11 mutations in common while the lenalidomide refractory (first progression) sample shared only 4 mutations in common with the bortezomib refractory sample. This suggests the presence of multiple clones at diagnosis that, due to treatment selection pressure, may emerge or regress as the dominant clone. Preliminary pathway analysis reveals the presence of mutations in genes regulating NFKB and Akt, whose pathways are often dysregulated in cancer. Conclusions: Whole genome sequencing has revealed evidence of mutational differences in exons between tumor and germline DNA indicative of the presence of multiple clones at diagnosis and evolution of these clones over time. This data supports the use of multiple drug therapies that target multiple clones and challenges the long held assumption that a patient who becomes refractory to a particular treatment will always be refractory to that treatment. Additional evaluation in a larger population is needed to confirm these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2011-4838

Abstract The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

Abstract Emerging evidence indicates that tumors can follow several evolutionary paths over a patient's disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.