A high-resolution allelotype of B-cell chronic lymphocytic leukemia (B-CLL)

Abstract

The most frequent chromosomal aberrations in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q, 11q, and 17p, and trisomy 12, all of which are of prognostic significance. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) are used for their detection, but cytogenetic analysis is hampered by the low mitotic index of B-CLL cells, and FISH depends on accurate information about candidate regions. We used a set of 400 highly informative microsatellite markers covering all chromosomal arms (allelotyping) and automated polymerase chain reaction (PCR) protocols to screen 46 patients with typical B-CLL for chromosomal aberrations. For validation, we compared data with our conventional karyotype results and fine mapping with conventional single-site PCR. All clonal cytogenetic abnormalities potentially detectable by our microsatellite PCR (eg, del13q14 and trisomy 12) were picked up. Allelotyping revealed additional complex aberrations in patients with both normal and abnormal B-CLL karyotypes. Aberrations detectable in the samples with our microsatellite panel were found on almost all chromosomal arms. We detected new aberrant loci in typical B-CLL, such as allelic losses on 1q, 9q, and 22q in up to 25% of our patients, and allelic imbalances mirroring chromosomal duplications, amplifications, or aneuploidies on 2q, 10p, and 22q in up to 27% of our patients. We conclude that allelotyping with our battery of informative microsatellites is suitable for molecular screening of B-CLL. The technique is well suited for analyses in clinical trials, it provides a comprehensive view of genetic alterations, and it may identify new loci with candidate genes relevant in the molecular biology of B-CLL.

Disparate expression of the PTEN gene: a novel finding in B‐cell chronic lymphocytic leukaemia (B‐CLL)

“…Our previous allelotype analysis of B-CLL (Novak et al, 2002) pointed to a preponderance of LOH at 10q23.3. Therefore, the PTEN gene located at this site would have been a candidate gene in the molecular pathology of CLL.…”

“…Western blot analysis of leukaemic cells with a polyclonal PTEN antibody (Cell Signalling Technology, Beverly, MA, USA) was performed as previously described (Novak et al, 2002). Blots were read by eye, and scored as -(no specific band), or graded from + (weak expression) to +++ (strong signal).…”

“…Complete absence of one allele in leukaemic DNA was defined as 'complete loss of heterozygosity' (LOH). Allelic imbalance was assessed as previously described (Novak et al, 2002).…”

“…At the genetic level, B-CLL cells often show chromosomal deletions, rather than translocations. In our B-CLL allelotype analysis, 22% of patients exhibited complete loss of heterozygosity (LOH) or allelic imbalances at 10q23.3 (Novak et al, 2002). This locus contains the tumour suppressor PTEN (for phosphatase and tensin homologue deleted on chromosome 10), the first phosphatase found to be mutated in an appreciable number of human solid tumours.…”

Multiplex ligation‐dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia

“…Throughput in FISH also remains low compared with other molecular genetic techniques. Finally, although many other chromosomal alterations have been described in CLL using various methodologies (Bentz et al , 1995; Odero et al , 2001; Novak et al , 2002; Stilgenbauer et al , 2002; Schwaenen et al , 2004; Dicker et al , 2006; Mayr et al , 2006), they are not usually analyzed in clinical practice.…”