Multiplex ligation‐dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma

Abstract

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i-FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation-dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32-31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i-FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy-chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i-FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra-chromosomal and intra-clonal rates of loss or gain. Our results suggest that MLPA is a reliable high-throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i-FISH should be applied as complementary techniques in diagnostic pathology.

Detection of recurrent cytogenetic aberrations in multiple myeloma: A comparison between MLPA and iFISH

“…The X046-A1 MM probemix contained 53 MLPA probes (42 diagnostic probes and 11 reference probes) with amplified products between 122 and 499 nt, indicating that the PCR reaction efficiency was different among probes. As a result, it was inappropriate to use an arbitrary ratio range as the sole cutoff value for all probes, which was applied in most MLPA studies [ 10 , 11 ]. Therefore, we established a normal range for each diagnostic probe (to be prognostically relevant) to improve the accuracy of MLPA analysis.…”

Coexistent hyperdiploidy does not abrogate poor prognosis in myeloma with adverse cytogenetics and may precede IGH translocations

“…28 Copy-number abnormalities including gains and hyperdiploidy, as well as losses, were assessed by multiplex ligation-dependent probe amplification. 29,30 HRD-Tx samples were selected for single-cell genetic analysis (supplemental Figure 1).…”

High-Throughput Copy Number Profiling by Digital Multiplex Ligation-Dependent Probe Amplification in Multiple Myeloma

“…MLPA reactions were performed as previously described. 23 Briefly, 150 ng input genomic DNA was denatured and hybridized using the SALSA P425 version A1 probemix (MRC-Holland) containing 42 probes targeting regions affected by recurrent CNAs of prognostic significance in MM: 1p32 (FAF1, CDKN2C, PLPP3, and DAB1), 1p21, 1q21.3 (CKS1B), 1q23.3, 5q31.3, 12p13.31, 13q14 (RB1 and DLEU1/DLEU2), 16q12 (CYLD), 16q23 (WWOX ), and 17p13 (TP53) (Supplemental Table S2). The reactions, including negative control samples, were performed according to the vendor's instructions.…”

“…For the cell-based detection of loss(1p) and gain(1q), bacterial arteficial chromosome clones specific for regions 1p32, 1p21, and 1q21 were selected, labeled, and validated, as described previously. 23 FISH signal patterns were evaluated by following the European Myeloma Network's recommendations. 28 All patient samples were screened by two independent investigators (B.K.…”