Donat Alpar1, Richard Kiss1, Szabolcs Kosztolanyi2, Lilit Atanesyan3, Ambrus Gango1, Karel de Groot3, Maryvonne Steenkamer3, Pal Jakso4, Andras Matolcsy1, Bela Kajtar4, Laszlo Pajor4, Karoly Szuhai5, Suvi Savola3, Csaba Bodor1
1MTA-SE Lendulet Molecular Oncohematology Research Group, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary
21st Department of Internal Medicine, Clinical Center, University of Pecs, Pecs, Hungary
3MRC-Holland, Amsterdam, The Netherlands
4Department of Pathology, University of Pecs Medical School, Pecs, Hungary
5Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
Introduction: Multiple myeloma (MM) has a heterogeneous genomic landscape with diverse clinical outcome. Copy number alterations (CNAs) including whole chromosome and subchromosomal gains and losses are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. Genome-wide analysis of recurrently altered regions with short turn-around time is therefore highly warranted.
Aims: To establish a rapid, high-resolution and comprehensive copy number profiling method to characterize disease relevant CNAs in MM.
Methods: A novel digital multiplex ligation-dependent probe amplification (digitalMLPA) assay has been developed for the molecular characterization of MM by combining conventional MLPA and next-generation sequencing (NGS). The method allows for screening for all chromosomal and focal CNAs recurrently occurring in MM as well as for specific detection of BRAF V600E mutation. Diagnostic bone marrow samples from 56 patients were analyzed by digitalMLPA and results were compared to conventional MLPA, fluorescence in situ hybridization (iFISH), pyrosequencing and droplet digital PCR data.
Results: Copy number profiling at 371 genomic loci identified on average 4.4 subchromosomal CNAs per patient and revealed numerical chromosome alterations, predominantly monosomy 13 and trisomies of odd-number chromosomes, characteristic features of MM. IFISH, MLPA and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). Additionally, we provide proof-of-principle that digitalMLPA is able to detect known point mutations, in this case the BRAF V600E.
Conclusion: Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen specific point mutations in MM. The method could efficiently be included in the methodological repertoire of myeloma diagnostics, thus facilitating the stratification of patients into risk groups and predict their responses to various treatment strategies.
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Supporting grants: NKFIH K119950 and NVKP_16-1-2016-0004; UNKP-17-4-III-SE-9; HAS LP95021.