Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia

Abstract

Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH , genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%) , trisomy 12 (7%) , ATM deletion (6%) , 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes , the most common being 19p13 (LDLR and CDKN2D). Moreover , the cost for MLPA analysis, including technical time and reagents , is 86% less than FISH. In conclusion , cytogenetic abnormalities are a common finding in CLL patients , and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.

Evaluation of multiplex ligation‐dependent probe amplification as a method for the detection of copy number abnormalities in B‐cell precursor acute lymphoblastic leukemia

“…Although MLPA is not suitable for whole genome analysis, it provides a low cost, simple alternative to array-based techniques for many routine applications (www.mrc-holland.com). It is now used in a number of laboratories to identify copy number alterations in hematological malignancies, such as chronic lymphocytic leukemia (CLL), for which specific copy number changes are clinically significant (Coll-Mulet et al, 2008;Stevens-Kroef et al, 2009;Al Zaabi et al, 2010).…”

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

“…Notably, some of the alterations (2p and 8q24 gains or 9p21 deletion) were found as the sole abnormality in only two patients, suggesting that they could be considered secondary changes to alterations commonly associated with CLL, as also suggested by Al Zaabi et al, (2010).…”

“…A similar reduced threshold was reported by Coll-Mulet et al, (2008) on a cohort of purified peripheral mononuclear CD19þ cells from 50 CLL cases investigated by MPLA. In contrast, a lower sensitivity was observed when DNA was extracted from whole blood, as described by Al Zaabi et al, (2010), although recently a protocol was developed to improve the MLPA assay in CLLs not subjected to B-cell purification (Abdool et al, 2010). In the second group, including five patients, MLPA provided additional results in the detection of 13q14 alterations.…”

“…Although the MPLA assay is limited in the identification of alterations occurring in small leukemic subclones, we showed that sensitivity could be enhanced by pre-enrichment of the B-cell population. Furthermore, differently from the few previously published studies (Al Zaabi et al, 2010;Coll-Mulet et al, 2008;Buijs et al, 2009), we performed an independent validation by FISH of additional altered regions detected by MLPA. This validation allowed us to define one probe exceeding the normal range as threshold sufficient to define the alteration of the considered region.…”

“…Notably, MLPA is a fast, relatively inexpensive and easy method allowing a large numbers of samples to be processed simultaneously. Since its development, it has proved to be accurate and reliable for the detection of deletions, duplications, and amplifications in several types of cancers (Koolen et al, 2004;Buijs et al, 2006;Jeuken et al, 2006;Scarciolla et al, 2006Scarciolla et al, , 2007 also including CLL (Coll-Mulet et al, 2008;Stevens-Kroef et al, 2009;Abdool et al, 2010;Al Zaabi et al, 2010). Concerning CLL, a few studies have been reported thus far but additional investigations are required to validate the sensitivity and specificity of the MLPA approach in a large series of highly purified CLL patients.…”