Simultaneous Analysis of Multiple Chromosomal Aberrations in Chronic Lymphocytic Leukemia (CLL) by Multiplex Ligation-Dependent Probe Amplification (MLPA).

B cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course. Characteristic genomic abnormalities have shown to provide highly significant prognostic information (Dohner NEJM 2000). However, karyotyping and/or FISH are laborious and expensive techniques. Although other prognostic markers (like mutation status of VH-genes and expression of CD38 and ZAP70) have been noted, these fail to identify all patients with a cytogenetic high-risk profile. We have performed multiplex ligation-dependent probe amplification (MLPA) analysis on 37 CLL patients, using the P037 and P038 kits (mrc-holland, http://www.mlpa.com/pages/indexpag.html), which allow the simultaneous identification of 56 genomic CLL-specific targets. The MLPA data obtained were compared with interphase FISH data in a blinded fashion using the ATM, centromere 12, RB1, D13S319, and TP53 specific probes (Vysis). By doing so, a good correlation was revealed in those cases in which the percentage of abnormal cells was at least 27%. We diagnosed six cases with loss of the ATM locus, six cases with trisomy 12, twenty cases with loss of chromosome 13q14 (three of them with a biallelic deletion), and three cases with loss of the TP53 locus. In three cases the genetic abnormality was detected by interphase FISH only. In these latter cases, the percentages of abnormal cells were 13%, 20% and 20%, respectively, which may explain the false negative results of the MLPA analyses. Since multiple probes were used for the 13q14 loci, a critical region of genomic loss could be identified which excluded the RB1 gene but included the KCNRG, DLEU2 and DLEU1 genes and miRNA15A. Also for the TP53 and ATM genes multiple probes were tested and, based on these results, one patient is suspected to have an intragenic ATM deletion. Also, additional abnormalities were detected by MLPA as e.g. trisomy 19.

In conclusion, MLPA appears to be a useful technique for the identification of clinically relevant genetic abnormalities in a diagnostic setting.