| Purpose: There's a high incidence of gastric cancer in China. Prognosis differents from patients to patients after standard surgery, while controversies are still remain regarding the prognostic factors. The role of adjuvant therapy in gastric cancer has been controversial given the lack of significant survival benefit in many randomized studies and meta-analysis. Polymorphisms in the DNA repair genes JWA and XRCC1 have recently been reported to increase susceptibility for gastric cancer, but the expression pattern of these genes in gastric cancer is unknown. Herein,we chose resectable gastric cancer patients to evaluate the effect of base excision repair protein JWA and XRCC1 as potential prognostic and preditictive markers of chemotherapy.Patients and methods: Three independent retrospective patient cohorts were studied. The training cohort and the first validation cohort were recruited in Nantong Cancer Hospital, Nantong City, in the North of Jiangsu Province, China. The second cohort was recruited in Yixing People's Hospital, Yixing City, in the South of Jiangsu Province, China. The tissues were obtained from the respective pathology departments.Overall survival (OS) was the primary end-point of this analysis. Date of death was obtained from inpatient and outpatient records or patients'families through follow-up telephone calls. The cause of death for each case was double-verified by local civil affairs department and public security department. Survival time was calculated from the date of surgery to the date of death or to the last follow-up. The gastric cancer TMAs were created by contract service in National Engineering Center for Biochip at Shanghai (Shanghai, China) and designed with duplicate 1.0 mm diameter cores of tissue from each sample was punched from paraffin tumor block and corresponding non-tumoural tissues in the training cohort or from cores of primary tumor biopsies in the validation cohorts. As a tissue control, the biopsies of normal gastric epithelium tissues were inserted in the four angles and the center of each slide.A standard protocol was used for the immunostaining of the TMAs (Supplementary Appendix I). The polyclonal rabbit anti-JWA antibody (1:200 dilution; Research Genetics Inc., Huntsville, Alabama, USA), and monoclonal mouse anti-XRCC1 antibody (1:300 dilution; Abcam, Cambridge, UK) were used. The omission of the primary antibody served as negative control. The staining scores of the tissue controls in each microarray slide were pre-evaluated as a quality control of the immunostaining.Samples with IRS 0-6 were classified as low expression of JWA or XRCC1 and samples with IRS of 8-12 were classified as high expression of JWA or XRCC1. After we established the immunohistochemical assessment criteria in the training cohort, the expression of JWA and XRCC1 in the two validation cohorts were scored by the exact same procedure.Expression of JWA and XRCC1 were assessed by immunohistochemistry in three independent tissue microarrays, which were generated from paired tumoral and adjacent nontumoral gastric tissues collected from 142 gastric cancer patients in the training cohort and tumors taken from 578 and 998 patients in the two validation cohort group, respectively. Prognostic value of JWA and XRCC1 and other clinicopathologic factors were evaluated in patients with surgery alone in three cohorts. Univariate and multivariate analysis were used to identify the prognosis and predictive value of each of them.The benefit for adjuvant chemotherapy compared with surgery alone was assessed by JWA and XRCC1 expression status in two validation cohorts. All tests were performed at the 0.05 level of significances. Statistical analysis was completed using the SAS 9.1.3 statistic software.Results: JWA and XRCC1 protein levels were downregulated in gastric cancer lesions compared with adjacent noncancerous tissues in the training cohort. JWA expression was significantly correlated with XRCC1 expression not only in the nontumors taken from patients in the training cohort (P=0.006 for correlation), but in the tumors taken from all three cohort patients (P≤0.001 for all correlations). Low tumoral JWA or XRCC1 expression, which was associated with depth of invasion, presence of lymph node metastasis, high TNM stages and poor differentiation, was significantly correlated with shorter overall survival (OS) in all three cohorts. However, neither nontumoral JWA nor XRCC1 expression was associated with OS in the training cohort. Multivariate COX regression analysis showed that JWA and XRCC1 were significantly associated with OS (JWA: HR= 0.22, 95% CI = 0.13–0.36; XRCC1: HR= 0.22, 95% CI = 0.13–0.36) in the training cohort, which was confirmed in both two validation cohorts. It is shown that the patients received fluorouracil, leucovorin, and oxaliplatin (FLO) regimen was associated with improved survival compared with surgery alone (log-rank test, P=0.003), and this effect was similar in JWA negative group (HR=0.49; 95% CI=0.31-0.76; Log-rank test, P =0.004), however, among patients with JWA positive tumors, overall survival rate was not different in those treated with or without FLO regimen (HR=0.54; 95% CI=0.28-1.06; Log-rank test, P =0.282).Conclusions: JWA and XRCC1 both had lower expressions in cancer tissue, which suggested they may play an important role in the process of tumer developing. It is showed that JWA and XRCC1 levels were relative to the depth of tumor invasion, lymph node metastasis, distant metastasis and pathologic–TNM stage and differentiation. JWA and XRCC1 may be the novel biomarkers for human gastric cancer progression and prognosis after gastrectomy as well as the potential chemotherapeutic targets. Moreover, patients with JWA positive gastric tumors do not appear to benefit from postoperative adjuvant chemotherapy with FLO regimen to surgery alone.
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