Fresh-frozen tumor specimens from 293 consecutive CRC patients were retrieved from the tissue banks of the Royal Melbourne Hospital, Western Hospital and Peter MacCallum Cancer Center in Australia, and the H. Lee Moffitt Cancer Center in the United States; individuals who had received preoperative chemo- and/or radiotherapy or for whom tumor-derived total RNA was inadequate for microarray analysis (RIN < 6) were excluded. All patients gave informed consent, and this study was approved by the medical ethics committees of all sites. Patient median age at diagnosis was 67 years (range 26 to 92 years). All specimens were derived from primary carcinomas and were snap-frozen in liquid nitrogen immediately after surgery for storage at −80°C. Cases comprised 44 stage A, 95 stage B, 93 stage C and 61 stage D cancers; 252 were localized to the colon and 40 to the rectum, with one case missing this information. 22 of 94 patients who had stage B disease and 64 of 91 patients who had stage C disease had received standard adjuvant chemotherapy (either single agent 5-fluouracil/capecitabine or 5-fluouracil and oxaliplatin) or postoperative concurrent chemoradiotherapy (50.4 Gy in 28 fractions with concurrent 5-fluorouracil) according to hospital protocols. All patients were assessed annually. For stage B and C patients, follow-up and additional clinical data including patient gender and TNM staging were collected by Biogrid Australia 1 for Australian patients and the Moffitt Cancer Center Tumor Registry for US patients. The median duration of follow-up was 47.8 months (range 0.9 to 118.6 months) for the 140 patients without recurrence, and 19.1 months (range 1.6 to 93.7 months) for the 48 patients with local or distant recurrence. The median follow-up for all 188 patients was 37.2 months (range 0.9 to 118.6 months).
Total RNA was extracted using Trizol reagent (Invitrogen) from CRC samples containing >60% tumor cells. All samples included showed good integrity of 18S and 28S ribosomal bands (RIN > 6) using a 2100 Bioanalyzer (Agilent Technologies). Total RNA was labeled and hybridized to HG-U133Plus2.0 GeneChip arrays (Affymetrix) according to the manufacturer’s instructions. The microarray data on a subset of 174 tumors have been published previously (NCBI Gene Expression Omnibus, GSE5206 and GSE13067).
In addition, published gene expression data were retrieved for 42 stage A CRCs, 83 stage B, 73 stage C and 62 stage D CRCs analyzed as part of the Expression Project for Oncology (expO) 2 using HG-U133Plus2.0 GeneChip arrays (Affymetrix) (Supplementary Table S1). Of the 62 stage D CRCs, 32 were primary cancer and 30 were metastectomy specimens. None of the primary cancer patients had received preoperative therapy, but 17 metastectomy specimens were from patients who had received adjuvant chemotherapy treatment prior to resection. Data processing and analysis were performed using the statistical software package R (15 ) and appropriate Bioconductor packages (16 (link)).