High-Resolution Array-Based Comparative Genome Hybridization (CGH) Identifies Novel and Recurrent Regions in CLL.

Although we have some understanding of the genetic abnormalities occurring in B-cell chronic lymphocytic leukemia (CLL) and their association with clinical outcomes, there is an incomplete comprehension of all of the mutations contributing to disease development and progression. In most abnormalities previously observed, it has been difficult to pinpoint specific candidate genes, reflecting the inadequacy of present tools for assessing chromosomal damage.

We examined copy number changes in the genomes of B cells from 58 patients with CLL using a 390,000-probe microarray, enabling us to perform comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least one genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported were also observed, most of which were highly recurrent. We observed the boundaries of known events with greater clarity and identified lesions previously not described, some of which were also recurrent.

A newly identified 3.6 Mb deletion at 8p21.2-p12 includes the gene TRIM 35. A second novel deletion at 2q37.1 (587kb) encompasses the gene SP100/110/140. Of the refined regions, a 249kb region at 9p21.3 spanning the CDKN2A (p16^-INK4) and a 156kb region at 18q23 containing NFATC1 are particularly interesting. In the case of NFATC1, the minimal region of overlap spans that single gene.

Furthermore, we tilized our arrays to examine the clonal heterogeneity of CLL within the same patient from mixed sub-populations. The presence of greater than 30% of B-cells with the CD38 cell surface marker has been associated with poor outcome in CLL. It is an open question whether this reflects genetic heterogeneity and possibly clonal evolution. To investigate this possibility, we analyzed CD38⁺ and CD38⁻ fractions from individual patients and demonstrated that three out of the four patients examined had undergone clonal diversification leading to new subclones of appreciable size.

Additionally, we have utilized a 2.1 million probe ultra-high density (HD2) array from NimbleGen (Madison, WI), providing us with the capability to scan the human genome for copy number changes at a resolution of a probe every 1500 bp. Utilizing the HD2 array, we are presently re-analyzing all the CLL samples in our study, and in the process are discovering many more previously unpublished lesions, several of which pinpoint single genes. We will present the results of our findings and discuss the potential applications of high resolution CGH analysis in a clinical setting.