Complementary Molecular Biology Techniques for the Detection of Unbalanced Chromosomal Abnormalities in Acute Myeloid Leukemias and Myelodysplastic Syndromes: Comparison of Fluorescence In Situ Hybridization (FISH) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background: The accurate detection of cytogenetic abnormalities in Acute Myeloid Leukemias (AML) and Myelodysplastic Syndromes (MDS) play an important role in prognostic value and often assist in therapeutic choice. The most commonly used genetic test for the diagnostics of these conditions is karyotype (KT), which is able to detect frequent non-random chromosomal abnormalities. However, this technique requires metaphases and offers low resolution (5Mb), which may lead to the loss of clinically important smaller genomic rearrangements. Therefore, a complementary technique is sometimes needed. FISH, for example, is a highly reliable technique, it can be performed in interphases, has higher resolution, and can detect balanced translocations (important for prognosis mainly in AML), although it is very expensive. As an alternative method, MLPA is usually less costly, is able to evaluate more genomic regions at once and is performed with genomic DNA, which is convenient for biological material availability. On the other hand, it is not able to detect balanced translocations due to its semi-quantitative PCR-based method. The aim of this study was to evaluate MLPA as a complementary technique to KT, and to compare the outcomes of MLPA and FISH panels for the high resolution detection of common unbalanced genetic alterations in MDS and AML.

Methods: A total of 23 samples from patients diagnosed with MDS (n=16)/AML(n=6) were tested for KT and MLPA. 20 metaphases were analyzed in G-banding KT. MLPA (Kits P144 and P145, MRC-Holland) was performed with genomic DNA extracted from bone marrows. A subset of 10 samples was tested for FISH MDS/AML panel (PML/RARA, CBFB/MYH11, RUNX1/RUNX1T1, MLL rearrangement, del5q, del7q, de17p and del20q, Cytocell), and the outcomes were compared to MLPA. Reagent costs were also compared between MLPA and FISH techniques.

Results: Outcomes from KT and MLPA were mostly concordant: 91.3% of samples had either concordant or partially concordant results (considering "partially concordant" when MLPA could detect smaller genetic alterations not achieved by KT). Discordance between KT and MLPA occurred in two cases: (i) MLPA detected a deletion of RUNX1 gene (chr 21) in a previously normal KT, and (ii) MLPA showed different abnormalities than those in a complex KT case, with low mitotic index. When comparing regions that are notoriously related to MDS/AML (-5/del5q, -7/del7q, +8, del11q, del13q, del17p, del20q and del21q), all results were concordant between MLPA and KT. Comparison of MLPA and FISH was performed with 10 samples, of which 4 were negative for genetic alterations and 6 were positive. 90% of samples had concordant results, and 10% (one sample) showed extra genetic alterations in MLPA (del7q; del11q and del20q). This is expected since MLPA analyzes more genetic regions then FISH MDS/AML panel at once. Genomic regions accessed both by MLPA and FISH included -7, +8, del17p, del21q.

Regarding financial expenses (cost of commercial MDS/AML panels and additional reagents), MLPA costs approximately U$280.00 (considering test sample plus internal controls), while FISH costs U$555.00 (values were converted from quotes provided by regional distributors and converted to dollars).

Conclusions: MLPA results are equivalent to FISH for the detection of MDS/AML frequent unbalanced genetic alterations. Both techniques are reliable as complementary molecular biology tests for KT. Moreover, MLPA is a friendly and less expensive technique for the detection of high resolution genomic unbalanced abnormalities, frequently adding extra information to KT results. Nevertheless, it is important to notice that only FISH can detect balanced translocations with significant prognostic value in AML.