High throughput screening of genomic copy number changes in chronic lymphocytic leukaemia using array comparative genomic hybridisation
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is characterised by a highly variable clinical course. The genetic basis of CLL has been widely investigated over the last two decades using techniques such as chromosome banding, fluorescence in situ hybridisation (FISH), and metaphase comparative genomic hybridisation (CGH). By these different approaches, chromosomal regions with recurring aberrations have been well documented and shown to be important indicators for clinical care. The recent development of array CGH provides new opportunities to study genetic aberrations at high resolution. We are applying array CGH to further investigate the genetic basis of CLL and to identify previously unknown genomic aberrations that may have diagnostic or prognostic relevance.
For this analysis, leukaemic DNA is isolated from mononuclear cells separated from whole blood using Ficoll-Hypaque gradient centrifugation. Samples that contain a low proportion of leukaemic cells are further purified using magnetic beads selective for CD19-positive cells. Leukaemic DNA is then co-hybridised with a non-leukaemic female reference DNA onto BAC arrays. The BAC arrays are sourced from the University of California at San Francisco, and printed with triplicates of 2464 clones providing 1.4Mb resolution for the entire human genome. For some of the samples, the integrity of array CGH findings has been validated using FISH and metaphase CGH.
Known recurrent copy number changes, including deletions of 13q14, 11q22-23, 17p13, 6q21 and trisomy 12, have been identified in the 12 patients analysed so far. Preliminary screening has also identified novel copy number alterations affecting other regions. Our results show that array CGH provides considerable potential for identification of sub-microscopic genomic aberrations. Our experience to date supports potential utilisation of array CGH as a diagnostic tool to provide risk-adapted management of CLL.