Insights into the Basis of Chromosomal Imbalances during the Clonal Evolution of Multiple Myeloma Using SNP Array Analysis.

On the basis of hyperdiploidy, often involving trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21, and chromosomal translocations multiple myeloma (MM) can be separated into two broad groups. Neither the mechanism leading to the development of these trisomies nor their effect on myeloma pathogenesis is fully understood. With the aim of understanding these two questions further we have analysed the zygosity status and chromosomal copy number of 39 samples from the MRC Myeloma IX trial (9 t(4;14), 15 t(11;14), 15 hyperdiploid) and two cell lines using the Affymetrix 50K SNP mapping arrays. Plasma cells were selected using CD138+ MACS beads to purity greater than 95%. There was a good correlation between the FISH based detection of copy number change and that defined using SNP mapping arrays indicating that the SNP array methodology was robust. In the 24 non-hyperdiploid cases there are examples of chromosomes that are diploid by FISH and by copy number analysis, using a WGSA programme, however in some of these chromosomes there is loss of heterozygosity (LOH) across the whole or part of the chromosome indicating uniparental disomy (UPD). In addition, we demonstrate where there are large regions of UPD on one chromosome there will often be regions of UPD on other chromosomes. In the hyperdiploidy cases we find that large regions of UPD are rare on the trisomic chromosomes, but small regions of LOH exist in all 15 hyperdiploid samples indicating uniparental trisomy which is in agreement to MM cell lines where uniparental trisomy can also be found. In four out of 15 hyperdiploid samples LOH is also seen on tetraploid chromosomes. UPD, therefore, represents an important new mechanism for gene inactivation in myeloma whereby tumour suppressor genes can be inactivated even in the presence of increased copy number. The mechanism underlying this gene activation relies on the loss of one functional allele leaving only a single allele which may be defective. Duplication of the defective allele gives a normal chromosomal number but an abnormal phenotype, which would not be detected by FISH or conventional CGH. Such regions of LOH may arise through gene conversion events and somatic recombination during mitosis. The order in which these events occur in hyperdiploid cells can help in the understanding of the clonal evolution of myeloma. We demonstrate that gene inactivation methods such as deletions or UPD occur prior to chromosome copy number gain as areas of LOH are present on diploid and trisomic chromosomes. In order to understand how such regions of LOH contribute to the pathogenesis of MM and to define critically deregulated genes within these regions, where mutation and methylation may be important, we are currently comparing the SNP mapping arrays with expression patterns using the Affymetrix U133 plus 2 expression microarrays.