Multiplex ligation‐dependent probe amplification and fluorescence in situ hybridization to detect chromosomal abnormalities in Chronic lymphocytic leukemia: A comparative study

Abstract

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by recurrent chromosomal aberrations of prognostic significance. We aimed to evaluate the potential of the multiplex ligation-dependent probe amplification (MLPA) assay to detect genomic alterations in CLL. Highly purified (>90%) peripheral mononuclear CD19+ cell populations from 100 untreated CLL patients (pts) in early stage disease (Binet stage A) were included in this study. All samples were investigated by fluorescence in situ hybridization (FISH) for the presence of trisomy 12 and 17p13.1, 11q22.3, and 13q14.3 deletions. For MPLA analysis, DNA was amplified by means of two commercially available probes sets allowing the simultaneous screening of 56 genomic sequences. Overall, a high degree of concordance (95%) between MPLA and FISH results was found, if the abnormal clone was present in more than 30% of the leukemic cell population. The use of multiple MPLA probes allowed the fine-mapping of the 13q14 deletion and the identification of intragenic or small alterations undetected by FISH. Moreover, additional alterations in 2p24 (MYCN) (3 pts), 8q24 (MYC) (1 pt), 9p21 (CDKN2A2B) (1 pt), 1q21 (LMNA) (1 pt), and 6q25-26 (1 pt) regions not covered by a standard FISH assay were detected and all confirmed by FISH. Our data extend previously limited evidence that MLPA may represent a useful technique for the characterization of well-known lesions as well as the investigation of additional genomic changes in CLL.

Detection of Clonal Aberrations by Cytogenetic Analysis after Different Culture Methods and by FISH in 129 Patients with Chronic Lymphocytic Leukemia

“…Biallelic deletion of 13q14 was observed in 17% of the cases with both DNA probes D13S25 and D13S319, which is in the range reported previously [Garg et al, 2012]. The occurrence of trisomy 12, del (11) [Fabris et al, 2011;Ouilette et al, 2011;Véronèse et al, 2013;Xu et al, 2013;Puiggros et al, 2014;Stevens-Kroef et al, 2014]. However, these new assays share several limitations: the inability to detect balanced rearrangements, low-level mosaicism, and a clonal evolution of neoplastic B cells.…”

Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review

“…So far, over 300 probe sets specific for a very large range of genetic disorders are commercially available. MLPA is a multiplex polymerase chain reaction (PCR)-based technique that can quantify up to 50 different genomic targets simultaneously in a single experiment through amplification of specific hybridizing probes 42 . As the sequence recognized by an MLPA probe is only 50-70 nucleotides long, this assay is very useful for detection of deletions or amplifications of single exons 14 .…”

Specific Associations Between Clinical Signs, Immune Cells, Disease Genetic Background and Burden in a Group of Patients with B-Cell Chronic Lymphocytic Leukemia

“…DNA was extracted from PBMCs (5x10 6 cells) using a Promega kit-Wizard Genomic DNA Purification kit, then diluted to a final concentration of 40 ng/ µL. Five µL of DNA were used for each reaction following the standard protocol indicated by MRC Holland and previously described in detail [9]. In each of the runs at least one normal (non B-CLL) DNA specimen was tested.…”