Previous G-banded karyotype analyses detected clonal chromosome abnormalities in 40-100 percent of B-CLL patients studied after appropriate in-vitro stimulation with polyclonal B-cell mitogens. It was unknown whether the wide variations in frequencies of abnormalities reported in different series were due to true differences in disease characteristics or merely to in-vitro culture conditions. Common recurring chromosomal abnormalities included trisomy 12, rearrangements of 14q32, translocations or deletions of 13q, deletions of 6q, and deletions of 11q. Recent studies have shown increased detection of trisomy 12, 13q deletions, 11q deletions, and 17p (p53) deletions with interphase fluorescence in-situ hybridization (FISH) techniques, suggesting these changes do indeed occur early in the course of the disease. Data regarding the clinical and prognostic significance of cytogenetics in B-CLL are emerging. The data suggest certain karyotypic and/or FISH abnormalities are associated with specific clinicopathologic subsets of B-CLL, and with the course of the disease. To address the questions of timing of karyotypic changes in B-CLL, and the possible specificity and significance of these changes, we have been conducting prospective studies of patients with sporadic or familial B-CLL referred to the NCI for evaluation and possible treatment. The cytogenetics specific aims of this project are: to determine the optimal culture conditions for obtaining karyotypically abnormal mitotic cells from peripheral blood of patients with B-CLL;to determine whether or not interphase FISH detects clonally abnormal cells missed by G-banded metaphase analysis;to determine whether or not comparative genomic hybridization (CGH) will detect gains or losses of chromosomal material not found by metaphase or FISH analyses;and to correlate cytogenetics results with clinical, morphologic and immunophenotypic features of the disease, with cDNA microarray analysis of gene expression, and with outcome with new therapies. Data from more than 200 patients entered on-study reveal clonal chromosome abnormalities in only 30 percent of cases using G-banded metaphase analysis, and demonstrate inferiority of e. coli lipopolysaccharide as a mitogen for the leukemic cells. Interphase FISH with a panel of five probes has confirmed or expanded the G-band findings in patients with abnormal clones, and has detected abnormalities in all but approximately 10% of patients tested to date. From 2002 through 2005 we performed CGH retrospectively on DNA isolated from patients previously entered on-study, and prospectively on DNA from new patients. CGH was less sensitive than interphase FISH in detecting submicroscopic deletions, but detected gains and losses of regions not probed by FISH. Combining G-banding, FISH, and CGH, we were able to find molecular cytogenetic abnormalities in more than 90% of B-CLL patients. Furthermore, dual hybridization with two probes and also sequential analyses in multiple patients have shown certain abnormalities present at diagnosis, and additional abnormalities at the time of transformation. The cytogenetics results are being correlated with clinical and other laboratory features at diagnosis and transformation, and with cDNA microarray analysis of gene expression and outcome with novel treatments. Interphase FISH results are currently being used to classify patients with regard to risk, and in assignment to treatment protocols. This project, which has the potential to yield new information regarding clinical cytogenetics diagnostic techniques, to demonstrate new clinicopathologic subsets of B-CLL patients, and to define genetic rearrangements critical to initiation or progression of B-CLL, is ongoing.
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