Sample selection strategy using microarray-based CGH to identify a recurrent gene dosage alteration common to colorectal carcinomas and adenomas.

B138 Advanced colorectal cancer (CRC), widely known for genomic instability, often shows alterations of large chromosomal segments in a clonal manner. Previous reports by conventional comparative genomic hybridization (CGH) show recurrent changes in 13q and 18q. Clonal alterations are thought to confer a proliferative or anti-apoptotic advantage in neoplastic progression. Sensitive microarray measures of gene dosage and expression at the gene level are best performed on optimal nucleic acids from fresh frozen samples, but availability of clinical outcomes data for such samples is limited. We have developed protocols to extract and process DNA from formalin fixed paraffin embedded (FFPE) tissue sections for downstream genome analyses including copy number assessment by the Agilent 44 K feature 60-mer oligonucleotide microarray for CGH. We performed hybridizations of 14 advanced CRC and 10 advanced colorectal adenomas using a standard genomic reference. Samples analyzed include colorectal adenomas collected in the Phase III Wheat-Bran-Fiber clinical trial. DNA was extracted from archival FFPE blocks with variable fragment length in the 500 base pair to 12 kb range by Sybr-Gold gel electrophoresis. In order to obtain uniform DNA template, the sample and sex-mismatched genomic reference DNA samples were DNase digested prior to application to the Agilent 44 K feature 60-mer oligonucleotide array for CGH. Our strategy for sample selection was to first hybridize advanced CRCs to visualize maximal extent of gene copy number changes. As expected we noted genomic instability with gains and losses of entire chromosome arms in a majority of the CRCs; however, several CRC samples showed remarkably stable genomes. One such advanced CRC with a stable genome showed a distinct gain of 150 kilobase size on 7p. Upon visual analysis by CGH Analytics 3.324 software and quantitative algorithm of ADM, Agilent Inc, Palo Alto, CA this gain was annotated as a gene family that encodes a transcription factor critical in embryological gastrulation in a specific temporal-spatio pattern. Stringent algorithm criteria identified this alteration in 5 of 14 CRCs and an additional 4 CRCs showed significant increase in hybe signal over baseline. Hybridization of 10 advanced adenomas, 6 by villous morphology and 4 by size > 10 mm, in contrast to the CRCs, showed more stable genomes with occasional partial loss of 18q present. Of most significance, however, was that 60% of the adenomas shared the common discrete gains in the gene family first detected in the CRCs. Here we demonstrate that sensitive and specific gene dosage measures by array CGH can be used to detect recurrent and common gene dosage alterations in colorectal adenomas and carcinomas. We are currently validating these findings on CRC / colorectal adenoma tissue microarrays to correlate protein expression patterns by immunohistochemistry.