Hypoxia Signature-Based Tumor Stratification

The epithelial hypoxia signature gene list consists of 253 unique image clones on the cDNA Stanford array by selecting a gene cluster showing induction in the tested epithelial cells (HMECs and RPTECs). For gene expression analysis of renal cell carcinoma [28 (link)], breast cancers [29 (link)], and ovarian cancer, the expression value of these clones was extracted and genes were selected for further analysis for which the corresponding array elements had fluorescent hybridization signals at least 2.5-fold greater than the local background fluorescence. We further restricted our analysis to genes for which adequate data were obtained in at least 80% of experiments. The image clones in the epithelial hypoxia signature that satisfied all the above criteria were used to stratify tumors in different datasets based on their hypoxia response with hierarchical clustering. For the analysis of breast cancer samples of Netherlands Cancer Institute (NKI), 35 of 253 unique image clones could not be mapped to a Unigene cluster. The 218 remaining clones were mapped to 168 unique Unigene clusters. The 168 Unigene cluster represented 180 unique sequences on the Rosetta/NKI oligo array. Cross-checking gene names revealed 22 probes that could not confidently be contributed to genes in the original hypoxia signature. These were removed, resulting in 158 matched probes. These 158 were the matching probes to 123 unique Unigene clusters. In order to overcome possible overestimation of Unigene clones that were matched to more than one probe on the NKI array, the probes that were not uniquely match to one Unigene cluster were averaged. Genes were mean centered and clustered and visualized in TreeView. Based on genes highly expressed in the hypoxia response, two groups of patients were identified. These were called, respectively, high- and low-hypoxia response. Overall survival was defined by death from any cause. Distant metastasis-free survival was defined by a distant metastasis as a first recurrence event; data on all patients were censored on the date of the last follow-up visit, death from causes other than breast cancer, the recurrence of local or regional disease, or the development of a second primary cancer, including contralateral breast cancer.
Kaplan-Meier survival curves were compared by the Cox-Mantel log-rank test in Winstat for Excel (R. Fitch Software, Staufen, Germany). Multivariate analysis by the Cox proportional hazard method was performed using the software package SPSS 11.5 (SPSS, Chicago, Illinois, United States).