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Genome Analysis of CLL by Representational Oligonucleotide Microarray Analysis (ROMA).

Vladimir D. Grubor, Jennifer E. Troge, Jennifer L. Meth, B. Lakshmi, Boris Yamron, Lisa A. Hufnagel, Yoon-Ha Lee, Jude T. Kendall, Deepa Pai, Annette Lee, Peter Gregersen, Sophia Yancopoulos, Steven Allen, Kanti R. Rai, Nicholas Chiorazzi, Michael H. Wigler and Diane Esposito

Abstract

The transforming events that lead to B-cell chronic lymphocytic leukemia (B-CLL) are unknown, and although genetic abnormalities appear to promote disease progression, none of these is seen in every patient. Therefore, we have applied a highly sensitive and specific method to probe, in greater depth, for genetic changes that can occur in B-CLL, and to explore if these correlate with disease development or progression. Utilizing ROMA to measure changes in gene copy number, we screened the entire genome of over two dozen B-CLL cases to identify characteristic and novel chromosomal changes. DNA from leukemic cells of patients was compared to the normal DNA from their unaffected neutrophils and to an unrelated, normal human control. By comparing normal DNA from each patient to an unrelated normal, we avoid inadvertently mistaking common copy number variation between people as possible candidates for lesions in B-CLL. ROMA was performed on both 85,000 and 390,000 probe microarrays enabling us to screen for far smaller lesions than are detectable by routine techniques, as well as resolve regions of interest, possibly pinpointing important genes in the etiology and progression of B-CLL. Data were also obtained by using the Illumina platform. These two methods (ROMA and Illumina), while not identical, are largely in agreement. We have observed virtually all the major cytogenetic imbalances previously reported, and in many cases to a higher resolution. Although there are fewer lesions present in B-CLL than in more advanced stages of solid cancers, many early stage cancers exhibit only a few common lesions. The most frequent deletion and often the sole abnormality found in B-CLL occurs at chromosome 13q13.4. Two micro-RNAs, miR-15a and miR-16-1, have been implicated in the smallest region of this deletion. ROMA analysis has further delineated the deletions on 13q, suggesting that there may be two epicenters, one encompassing these micro-RNA genes and one encompassing a neighboring region that does not span miR-15a or miR-16-1. A novel finding is the existence in certain patients (5/26) of numerous single probe deletions or amplifications. Since we compare B-CLL to the normal DNA from the same patient, and to a known control, even single probe events can be relevant. Many B-CLLs display single probe aberrations and these events are currently under investigation. In addition, we have observed two patients with major genomic instability, as evidenced by large and multiple regions that change copy number. The analysis of large numbers of B-CLL cases is essential to identifying and understanding the ongoing progression of genetic lesions and the role they play in clinical outcome. Initial results clearly indicate the need to obtain more data to identify critically mutated genes within the leukemic cells that cause them to become more aggressive. The identification and characterization of these genetic changes may be used as stratification tools for new diagnostic and therapeutic approaches to this currently incurable disease.