Dissecting genetic aberrations in multiple myeloma using aCGH and MLPA

Abstract

Development of modern genome-wide screening techniques along with improved understanding of multiple myeloma (MM) pathogenesis suggests that common prognostic FISH panels alone are insufficient for description of genome heterogeneity of malignant plasma cells. Low number of neoplastic cells and low mitotic potential is a major constraint for molecular evaluation in MM. Recent advent of new high-throughput techniques such as aCGH has allowed detection of genetic lesions in more than 90% of MM cases. Multiplex ligation-dependent probe amplification (MLPA) has simultaneously emerged as a fast and robust alternative method to analyze copy number changes at multiple loci across multiple patients in a single experiment based setting. The main objective of the present study was to evaluate the utility of aCGH and MLPA in identification of genetic aberrations in MM and to compare the results obtained from aCGH and MLPA. Genomic DNA isolated from purified CD138 cells from bone marrow of MM patients (n=108) was labeled using SureTag Complete DNA Labeling Kit and hybridized onto 4x180K sureprint G3 CGH+SNP array chip (Agilent technologies). Agilent Euro Female/Male was used as the normal reference in the hybridization experiments. aCGH data was screened for aberrations by CytoGenomics software ver. 4.0. DNA from patients (n=34) was subjected to SALSA MLPA kit P425 (MRC Holland, Amsterdam, The Netherlands). Data was analyzed with Coffalyser.NET Software (MRC Holland) using healthy donors as controls. Additional copies of odd numbered chromosomes were identified by aCGH, 47% cases were categorized into hyperdiploid subgroup and 53% cases were categorized into non-hyperdiploid subgroup. Most commonly observed copy number aberrations were: del13q14.2 (aCGH:38%, MLPA:50%) followed by gain 1q23.3 (aCGH:35%, MLPA:44%) and gain 1q21.3 (aCGH:32%; MLPA:35%). Del17p13.1 was identified in 15% of the cases by both aCGH and MLPA. The comparison of results obtained from aCGH and MLPA suggested a concordance range of 88-100% for the identified aberrations. The discordance observed in the results obtained from a CGH and MLPA related to ploidy status (12%), del1p32.3 (5%) del1p31.3(Nil), gain1q23.3 (9%), 1q21.3 (3%), del 13q (12%) and del 17p (Nil). This study has revealed that the frequency of hyperdiploid subtype and the genetic aberrations associated with prognosis such as del13q and del17p is similar to that reported in literature. The results showed high concordance between aCGH and MLPA for almost all the aberrations except ploidy status and del 13q14; this could be because of presence of only three chromosome probes for evaluation of ploidy status in MLPA. Taken together, both aCGH and MLPA are powerful tools to detect all the prognostically relevant molecular events except balanced translocations.