Whole Genome Paired End Sequencing Identifies Genomic Evolution in Myeloma.

A prominent feature of most cancers is striking genetic instability and ongoing accrual of mutational changes associated with tumor progression, including acquisition of invasiveness, drug resistance, and metastasis.

We first utilized single nucleotide polymorphism (SNP) arrays (Affymetrix) to evaluate genome-wide gains and losses in copy number and heterozygosity in CD138+ multiple myeloma (MM) cells collected from 14 patients at two time points at least 6 months apart. To estimate the extent of genomic instability in each patient, the number of events leading to copy number or heterozygosity changes throughout the genome were calculated. An event was defined as detectable change in copy number or heterozygosity in three or more consecutive SNPs. Two cases were also investigated for genome-wide rearrangements utilizing a paired-end approach on next generation sequencing.

In a period of six months, all MM patients analyzed acquired multiple new mutational events including changes in copy number and heterozygosity, ranging from 0.021 - 2.674 %, indicating a wide range of genetic instability. Although the rate of mutation varied, the majority (71%) of MM patients had acquired > 100 mutational events within the six months period, thus indicating a striking genetic instability. Chromosomes 1, 13, and X were unique with respect to copy number changes and showed large areas of change, spanning the entire length of a chromosome in several patient samples analyzed. Chromosomes 1 and 13 also showed large areas of loss or gain of heterozygosity in several patients, indicating areas of recurrent changes. We were also able to correlate genomic changes with changes in expression of corresponding genes. In two cases, we investigated genome-wide rearrangements utilizing a massively parallel sequencing approach. Short insert (400bp) libraries from two samples collected 6 months apart were constructed and subjected to paired-end sequencing utilizing 37bp readlengths on the Illumina GAII instrument. Approximately 80 million reads were generated for each of the 4 samples. Read pairs were mapped back to the reference genome, and those mapping aberrantly (incorrect orientation, different chromosomes, incorrect genomic distance) were further analyzed. Bespoke PCR assays defining each breakpoint were designed and used to verify the somatic nature of the mapped rearrangement. Further, PCR fragments spanning somatic genomic rearrangements were sequenced to generate base-pair resolution of breakpoints. To date, 29 somatic rearrangements have been sequenced, including three that were present only in the second sample. One of these was on chromosome 13. Breakpoint sequencing revealed a 64.9Kb homozygous (no wild-type readpairs found) deletion removing the first two exons of the RB1 gene. No reads spanning this breakpoint were found in the matching sample taken six months earlier.

This is the first study utilizing massively parallel sequencing to investigate the MM genome and provides important insight into the pathogenesis of disease progression .as well as confirms the potential of whole genome sequencing to inform biology of the disease that may affect the therapeutic approach in future.

Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Celgene: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Novartis : Consultancy, Honoraria, Research Funding.