Chromosomal Deletions and Amplifications in Multiple Myeloma Detected by 500K Single Nucleotide Polymorphism Array Analysis.

Abstract

Genetic aberrations, such as deletions and amplifications are among the major pathogenetic mechanisms underlying malignant transformation and progression. Analysis of chromosomal aberrations is particularly important as amplifications of oncogenes and deletions of tumor suppressor genes are major steps in the “multi-hit” process of tumorigenesis. Genome-wide molecular analyses, such as loss of heterozygosity (LOH) profiling and comparative genomic hybridization (CGH) have significantly enhanced our ability to detect chromosomal aberrations in cancer cells and assess their role in tumorigenesis. The recent introduction of high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci is able to analyze small areas of gains or losses. Here we describe the use of high-density array representing upto 500K SNPs to not only characterize SNPs but also evaluate genome-wide areas of gains and losses in myeloma. We purified CD138+ myeloma cells from 5 patients and also obtained their peripheral blood mononuclear cells. Purified genomic DNA from these cells was digested with Sty and Nsp restriction enzymes, PCR amplified and hybridized to 500K SNP array. Results from DNA obtained from CD138+ myeloma cells were compared with DNA from normal peripheral blood mononuclear cells from the same patient using the dChip software for LOH and copy number analysis. Areas of deletions and amplifications were identified and correlated with gene expression profile obtained from CD138+ myeloma cells of the same patient. The preliminary analysis identified various areas of amplifications and deletions. The significant areas of structural gains or losses included Chromosome 1, 6, 8, 11, 12, 13, 14, 15, 22, X among others and ranged in size from 1 Mb to the loss of entire chromosome (13q−). In this small sample we have identified recurrent LOH shared by 2 or more samples including 1p21, 6q, 12p13 and 13q. The derived CGH results correlated with known cytogenetic abnormalities in the patients, with identification of additional areas of abnormality not detected by conventional cytogenetics or FISH. These structural changes also corresponded with the results from gene expression profiling of MM cells from same patients, thus validating the technique and its application. A large series of patient samples are now under investigation using 500K SNP chip to define regions of homozygous deletions as well as amplification. In conclusion, these results demonstrate that SNP array analysis provides a powerful new tool for the analysis of allelic imbalance in myeloma. Furthermore, this technique can identify novel candidate loci to study in pathogenesis of myeloma and advance identification of potential therapeutic targets for this fetal disease.