Abstract

Among B-CLPD, characterized primarily by morphology and expression of cell surface markers, is is important to identify patients with CD5+ atypical B-CLL that is regarded as clinically more aggressive than typical B-CLL. However these cases are not well defined. To better characterize the genetic lesion observed in atypical B-CLL we have analysed a cohort of 40 patients by CGH microarrays.Study was made on 5 typical B-CLL and on 35 atypical B-CLL patients with either CD20dim or bright expression that do not express cyclin D1. All the patients with atypical B-CLL will be defined as the presence of an absolute B-CD5-positive lymphocytosis > 4 x 109/l and a RMH score < 4. DNA was extracted using QuiAmp kit according to manufacturer recommendations. 2μg of DNA was used in each hybridization experiment. DNA labeling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumoral sample respectively. CGH-arrays was performed using the 1 Mb Human genome micro-array from “arraygenomics” that contains 3400 BAC clones fully FISH mapped and end sequenced all printed in triplicate. Each experiment was made using two slides in dye swap method. Cy5 and Cy3 fluorescence intensities spot were quantified using Axon Scanner 4100 and Acuity Software. Data were imported into SpectralWare 2.0 software and Normalise Suite, version 2.0, Profiler from Squire lab.

Results: Identification of known and previously uncharacterized copy number alterations (CNAs) in the a-B-CLL cells genome was made in all the cases. The CGH profiles revealed that a-B-CLL genome is highly rearranged harbouring large numbers of distinct copy-number aberrations (75 CNAs among 31 chromosomal regions were found). Some of these CNAs are recurrent across different samples, allowing the definition of minimal common regions (MCR) of amplification or deletion. The size of the CNAs was extremely variable from one Bac probe to complete chromosome gains or losses. Specifically, our dataset included the known gains of chromosome 12 (14 cases), and the known deletion at 11q23, 13q14.3, 17p region but also new region of interest as +3p, 3q22 to 3qter, 4pter to 4q35.2, 5p15, 6p25.3 to 6p22, 8q22 to 8q24, 15q15.3 to 15q26, 17q11 to 17qter, +18 and +19 for gains and 1p35,1, 1p33, 2q22.3, 3p26.3 to 3p21.3, 5q34, 6p25.3, 6q16, 6q25.3 to 6q27, 7q31.3 to 7q32.2, 8p23.3 to 8p12, 10q11.2 to 10q21.1, 10q21.3, 10q23.2 to 10q24, 11q22.3 to 11q24.2, 15q14 to 15q21, 16p11.2 to 16q21, 21q22.1 for the deletions.To further corroborate the above finding, we had performed conventional FISH analysis using known probes for del 13q, del 11q, trisomy 12, del 17p and correlate our results with conventional cytogenetic findings when they were available. In all the cases CGH-arrays findings were confirmed by fish analysis or karyotype. The above findings were confirmed also in few cases using the Agilent’s Human Genome CGH Microarray 44K that contains over 40,000 probes. Our first results confirmed BAC arrays results. CGH-arrays appears to be very informative to detect lesion in B-CLPD and show the high frequency of genetic lesions in a-CLL. The biological impact of this lesions by transcriptome analysis on the same sample and the prognosis impact is in progress.

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