Acquisition of New In Vivo Genomic Change over Time in Multiple Myeloma.

Abstract

Multiple myeloma (MM) cells present with significant genetic abnormality. This genetic instability is considered responsible for not only development of malignant phenotype but also progression of myeloma as well as development of drug resistance. We have previously demonstrated that MM cell lines have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood 2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here, we have performed a genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci, and identify areas of amplification and losses to determine ongoing development of new changes that may reflect instability. We have evaluated 17 MM patients with purified myeloma cells obtained at two time points, at least six months apart. CD138–expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points or more were compared with each other using the dChip software for LOH and copy number analysis. During the LOH analysis, we have adapted a tumor vs. tumor comparison to track all the new SNPs affected. We have observed that myeloma cells acquired new LOH loci in the subsequent samples. These new areas of genetic changes were recurrent; for example on chromosome 13, an average area of recurrent LOH of 87 Mb was found to be present among three patients. We were able to observe recurrence of significant copy changes on several chromosomes with/without LOH. The reproducible area of new acquisition of LOH and/or significant copy changes interestingly involved areas on chromosomes 1p, 1q, 8p, 9q, 13q, 15, 16q, 20p, 21q, and X that may have significant role in the pathogenesis and progression of the disease. We are currently analyzing the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary we demonstrate continued acquisition of new genomic changes in vivo in MM over time and provide a molecular basis for evolution of more aggressive disease able to escape therapeutic interventions. The mechanism governing such evolution is an important target to not only inhibit progression of the disease but also development of drug resistance.