Multiplex genotyping and gene expression assays for colorectal cancer treatment selection markers.

3651 Background: Molecular markers that predict inherent resistance or sensitivity to chemotherapy would greatly assist oncologists in selecting drugs with the best therapeutic index. Genetic alterations in TP, TS, DPD, UGT1A1,ERCC1, ERCC2 and GSTP1 have been reported to predict response of colorectal cancer (CRC) to 5FU, irinotecan and oxaliplatin as have the expression levels of some of these markers. Additionally, the increasing use of Avastin and Erbitux in CRC treatment suggests that further studies of VEGF and EGFR may also be warranted.,Two multiplex TaqMan panels have been developed to simultaneously quantify expression of a) TS, TP, DPD and b) EGFR, ERCC1, VEGF. A novel housekeeping gene, NUP214, is incorporated into each panel and is used to normalize expression of the other genes. Each TaqMan probe within a panel is labeled with a different fluorescent reporter molecule that is readily differentiated. The multiplex genotyping assay simultaneously amplifies 6 SNPs, 2 repeat sequences and 1 ins/del. Genotyping of the SNPs is accomplished post-PCR with an oligonucleotide ligation assay (OLA) that uses fluorescent-labeled probes. The repeats and ins/dels are amplified with fluorescent-labeled primers and detected directly after PCR. Products of the PCR and the ligation assays are identified by specific fragment lengths on the ABI Prism 3100.,The TaqMan assays simultaneously quantifies each of the 6 targeted genes and the expression levels are normalized to NUP214. The assays have been successfully used on RNA extracted from colorectal FFPE. The genotyping assays show that the 6 SNPs can be simultaneously amplified and detected by OLA. The genotypes determined by this approach showed 100% concordance with individual allele-specific genotyping assays.,The multiplex assays allow several markers to be interrogated simultaneously and for some genes, provide direct comparison of genotyping and gene expression results from the same tissue. These assays will aid in assessing the predictive value of these markers for CRC treatment and should contribute to improved patient management. [Table: see text].