Multiplex ligációfüggő szondaamplifikáció alkalmazásának lehetőségei onkohematológiai kórképek DNS kópiaszám eltéréseinek vizsgálatában

Abstract

Analysis of DNA copy number aberrations (CNAs) plays an important role in the routine diagnostic workup of hematological malignancies. Since these alterations are frequently of clinical significance there is a need for their fast and cost-efficient detection. The multiplex ligation-dependent probe amplification (MLPA) is a PCR based method enabling assessment of CNAs at 50-60 different loci and is also capable of the simultaneous detection of point mutations. The next generation sequencing based digital MLPA (dMLPA) can be even more efficient with analyzing the CNA status of 500 to 1000 loci in one run. In our study, we examined the efficacy of the conventional MLPA in samples from 18 newly diagnosed and 6 ibrutinib treated CLL patients. We also tested a novel dMLPA assay developed for MM in bone marrow samples of 56 MM patients. Additionally, we utilized a dMLPA assay in pediatric ALL for the detection of clinically relevant aberrations and dissection of mechanisms leading to disease relapse. We paid special attention to the identification of clinically relevant abnormalities and the dissection of the genetic alterations leading to relapse. Using conventional MLPA in CLL, we identified prognostic aberrations in several cases and we also successfully applied the method to reveal patterns of clonal evolution induced by ibrutinib treatment. In MM, we were the first in the international literature to demonstrate the effectiveness dMLPA in detailed analysis of CNAs with prognostic and predictive value in the disease. In childhood ALL, in addition to mapping clinically relevant CNAs using dMLPA, three different clonal mechanisms were discovered based on the CNA profiles in paired diagnostic and relapsed samples. Furthermore, we introduced a new risk classification system by combining dMLPA-defined CNAs and cytogenetic abnormalities. Based on our study, MLPA-based techniques represent useful complementary approaches to methods used in the routine diagnostic setting and can also be successfully applied in research of malignant hematological diseases.