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  • The study was approved by the Institutional Review Board and the Ethics Committee of the Changhua Christian Hospital, Changhua, Taiwan (IRB no. 121228). The data were analyzed anonymously, and informed consent from the participants was waived by the Institutional Review Board and the Ethics Committee of the Changhua Christian Hospital. A total of 93 patients with lung adenocarcinoma were examined in this study. Surgically resected tumor tissues from patients with confirmed histological diagnosis were collected at Changhua Christian Hospital between 1998 and 2010. Cancers were staged according to the AJCC Cancer Staging Manual (7th edition). Clinical data including gender, age, stage, T, N, and M stages, and follow-up information were obtained from medical records and the cancer registry. Immunohistochemistry staining and evaluation of STEAP1 immunoreactivity: IHC staining was performed at department of pathology, Changhua Christian Hospital. Tumor tissue was taken from paraffin blocks and used to construct tissue microarrays composed of tumor tissue and peri-tumoral lung tissue. Antibodies for 24 biomarkers using 22 different biomarkers selected from a literature search were used for the IHC study of tumor tissue (S1 Table). A mouse monoclonal anti-FEN1 (Flap endonuclease 1) antibody (1: 400 dilution, ab462, Abcam Ltd.) and a mouse monoclonal RAD54B (1:60 dilution, sc-101234, Santa Cruz) were used for IHC staining according to the manufacturer’s instructions, the specificity of these antibodies was also confirmed [11]. Each tissue microarray core on the slides was interpreted by 2 pathologists. Staining localized to the cell membrane, cytoplasm, and nucleus was graded on a 0 to 3 intensity scale (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive). Positivity was defined as more than 5% of the tumor cells stained by the antibody. Survival curves were estimated by the Kaplan-Meier product-limit method, and survival distributions were compared across FEN1 and RAD54B expression groups using the log-rank test. We used time-dependent Cox proportional hazards modeling by univariate and multivariate analysis to estimate the hazard ratio (HR) of incident lung cancer associated with the FEN1 and RAD54B expression during the cohort follow-up. Results were presented as hazard ratio (HR) and 95% confidence intervals (95% CI). Differences between categories of FEN1 and RAD54B expression and lung cancer death patients with respect to continuous variables like age were tested by one-way analysis of variance (ANOVA) or linear correlation. Categorical variables were analyzed by a chi-square test. The Cochran-Armitage test for trend was applied for linear correlation between categories of FEN1 and RAD54B expression and categorical variables. Statistical analysis was performed employing the commercial software packages SAS 9.3 (SAS Institute Inc., Cary, North Carolina, USA) and Statistical Package for the Social Sciences, Version 19.0 (SPSS Inc., Chicago, IL, USA).
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