Search

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.

Deletions on chromosome 13 are thought to be one of the most important prognostic features in Multiple Myeloma (MM). The biology underlying this is, however, uncertain. Chromosome 13 abnormalities have been evaluated conventionally by FISH using probes for 13q14, covering the retinoblastoma gene (RB1) region. Typically, for recurrent regions of loss of heterozygosity (LOH) it is possible to map a minimally deleted region within which an important gene may be located. This should be the case with 13q−, or alternatively there may be linkage with another genetic lesion, which could be contributing to the poor prognosis. Following the implementation of high-density single-nucleotide polymorphism (SNP) array, it is now possible to genotype the whole human genome with a mapping resolution of less than 50 Kb. Thus, the SNP array approach offers an opportunity to analyze both copy number abnormalities and LOH simultaneously. The aim of this study was to determine the numerical alterations, LOH and changes in the gene expression profile of the chromosome 13 in MM, and its possible association with other genetic events. For this purpose, we analyzed 17 patients included on the Myeloma IX trial with deletion of 13q14 compared with 22 samples without deletion, using Affymetrix 50K SNP arrays and Affymetrix U133 Plus 2 expression array. IGH translocations and 13q deletion were determined by FISH. dChipSNP and WGSA programs were used to analyze the data. With respect to 13q14, there was 100% correlation between FISH and SNP array results. 16 out of 17 cases with deletion of the RB1 gene by FISH analysis showed loss of 13q arm by SNP array, demonstrating that loss of the whole chromosome 13 is responsible for 13q deletions found in MM in >90% of cases, with only one case showing a defined region of deletion of chromosome 13 (13q14.11–13q21.2). Using gene expression arrays we could not define a specific pattern characteristic of expression loss in genes at 13q. Lower RB1 expression levels were not only restricted to cases with del(13). However, samples containing IGH translocations (t(11;14) and t(4;14)) without del(13) showed up to 4 times more RB1 expression, suggesting that MM evolution in cases containing IGH translocations is independent of RB1 expression. Interestingly, the hyperdiploid cases with and without del(13) expressed similar level of RB1. We also investigated whether other key cell cycle regulatory genes were associated with del(13); in particular, 4 cases showed 9p21 LOH by SNP array and no different gene expression levels, which suggest that LOH does not seem to be a mechanism of lost of expression of CDKN2A, CDKN2B and p14/ARF. We could not find any significant correlation with del(13) and expression of cell cycle regulatory genes, apart from 8/17 samples with del(13) that had low expression of p53 gene, including 6 t(4;14) cases and 2 t(11;14) cases. Also, 2 cases without monosomy 13 (1 with t(4;14) and 1 with t(11;14)), showed low p53 expression levels. However, SNP array data did not show any deletion at 17p in 38 cases, with the exception of a case with monosomy 13 and t(11;14) in which SNP array data showed loss at 17pter-17q21.2 and FISH detected p53 deletion. Further investigation between the association of p53 and del(13) are ongoing and maybe useful in defining the biology of this poor subgroup of patients.