Genomic Evolution of Multiple Myeloma In Vivo over Time.

Abstract Genetic instability is an important feature of malignant cells, specifically in multiple myeloma (MM). This instability, by activation of oncogene or deletion of tumor suppressor genes, plays an important role in oncogenesis. The ongoing genetic instability is responsible for tumor progression, acquisition of invasiveness, and development of drug resistance, as well as eventual mortality. We have previously demonstrated that MM cells 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 genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 250K single nucleotide polymorphisms (SNP) loci and able to analyze small areas of gains or losses as an indicator of genomic instability to determine ongoing development of new changes that may reflect instability. We have evaluated nine MM patients with purified myeloma cells obtained at two time points, atleast 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 were compared with each other as well as with sample from normal peripheral blood mononuclear cells using the dChip software for LOH and copy number analysis and areas of deletions and amplifications. The changes at the first time point were considered as base line, and the subsequent sample was considered as test sample where acquisition of new changes was evaluated compared to base line. In two patients we had three samples available on a serial basis. We have observed that myeloma cells from the second time point demonstrated significant new areas of genomic change with acquisition of mean 4467 (range 79 to 18998) new LOH loci from base line sample. Of theses new areas of change, 41 SNP loci were found to be present in more than three patient samples indicating recurrent loci of interest in regards to progression and/or development of drug resistance. In 2 patients with 3 serial samples, we observed serial increase in acquisition of new SNPs, 4947 and 18998 at first follow up and 531 and 5275 at second follow up suggesting cumulative accumulation of new genetic change. The reproducible area of new acquisition interestingly involves the area with chromosome 1 and 8 that may have significant role in the pathogenesis and progression of the disease. We are currently obtaining the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary our results confirm genomic evolution of myeloma in vivo over time and provide means to identify molecular targets associated with progression of MM that can be utilized to inhibit progression of the disease and possibly development of drug resistance.