Samples were taken from colorectal cancers in surgically resected specimens in 35 colorectal cancer patients. The expression profiles were determined using Affymetrix Human Genome U133 Plus 2.0 arrays. Comparison between the sample groups allow to identify a set of discriminating genes that can be used for molecular markers for CIN phynotype.
Overall design
Specimens from 35 consecutive stage II and stage III patients who had undergone surgical resection of CRC were studied. Patients with familial adenomatous polyposis and HNPCC were excluded from the study. Specimens from tumors and corresponding normal tissues in surgically resected specimens were snap-frozen in liquid nitrogen and stored at -80 ºC until use. Parallel tumor specimens were formalin fixed and paraffin embedded for histological examination. DNA and RNA was extracted from paired tumor and normal tissue using frozen samples. The patients provided written, informed consent to the collection of specimens, and the local Ethics Committee approved the study protocol. MSI and LOH phenotype analysis were done as follows; Using DNA, we performed the polymerase chain reaction (PCR) and determined the MSI status. In addition to five microsatellite markers recommended by the National Cancer Institute workshop, we also used TP53, D18S46, D18S363 and D18S474.9 We determined LOH status in non–MSI-high tumors, because LOH is rare in MSI-high tumors and also interpretation of LOH is difficult in MSI-high tumors. We defined LOH at each locus as a 50% reduction in the height of one of two allele peaks in tumor DNA relative to non-neoplastic control DNA. LOH was defined as present if any of the markers on the same chromosome show LOH. In order to evaluate the severity of chromosomal instability based on the actual frequency of LOH among evaluable loci, we defined the LOH Ratio (%) as: LOH Ratio (%) = Total number of chromosomes with LOH / Total number of chromosomes that could be evaluated for LOH × 100. Depending on the LOH Ratio, we classified tumors as having high levels of chromosomal instability (CIN-high) (33% < LOH Ratio < 100%) or low levels of chromosomal instability (CIN-low) (0% < LOH Ratio <33%). Furthermore, CIN-high tumors were divided into two subgroups; CIN-high (mild type) (33% < LOH Ratio < 75%) and CIN-high (severe type) (75% < LOH Ratio <100%).
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