XRCC1 Gene Polymorphism, Clinicopathological Characteristics and Stomach Cancer Survival in Thailand

Background: Stomach cancer is one of leading causes of death worldwide. In Thailand, the incidence and mortality of stomach cancer are in the top ten for cancers. Effects of DNA repair gene X-ray repair cross complementary protein 1 ( XRCC1 ) polymorphisms and clinicopathological characteristics on survival of stomach cancer in Thailand have not been previously reported. The aim of this study was to investigate the effects of XRCC1 gene and clinicopathological characteristics on survival of stomach cancer patients in Thailand. Materials and Methods: Data and blood samples were collected from 101 newly diagnosed stomach cancer cases pathologically confirmed and recruited during 2002 to 2006 and followed-up for vital status until 31 October 2012. Genotype analysis was performed using real-time PCR-HRM. The data were analyzed using the Kaplan-Meier method to yield cumulative survival curve, log-rank test to assess statistical difference of survival and Cox proportional hazard models to estimate adjusted hazard ratio. Results: The total followed-up times were 2,070 person-months, and the mortality rate was 4.3 per 100 person-months. The median survival time after diagnosis was 8.07 months. The cumulative 1-, 3-, 5-years survival rates were 40.4%, 15.2 % and 10.1 % respectively. After adjustment, tumour stage were associated with an increased risk of death (p= 0.036). The XRCC1 Gln339Arg, Arg/Arg homozygote was also associated with increased risk but statistically this was non-significant. Conclusions: In addition to tumour stage, which is an important prognostic factor affecting to the survival of stomach cancer patients, the genetic variant Gln339Arg in XRCC1 may non-significantly contribute to risk of stomach cancer death among Thai people. Larger studies with different populations are need to verify ours findings. case-control studies found XRCC1 Arg399Gln increases risk for overall cancer (Yi et al., 2013) and many studies suggest that the XRCC1 gene is one of the most important genetic risk factors for stomach cancer (Hong et al., 2009; Engin et al., 2011; Yuan et al., 2011; Chen et., 2012; Pan et al., 2012; Qiao et al., 2013) gene


Introduction
case-control studies found XRCC1 Arg399Gln increases risk for overall cancer (Yi et al., 2013) and many studies suggest that the XRCC1 gene is one of the most important genetic risk factors for stomach cancer (Hong et al., 2009;Engin et al., 2011;Yuan et al., 2011;Pan et al., 2012;Qiao et al., 2013) and previous studies have pointed to XRCC1 polymorphism as an important prognostic factor for survival of gastric cancer (Shim et al., 2010;Tahara et al., 2011;Deng et al., 2014;Zhang et al., 2014).
In terms of clinicopathological characteristics, Previously studies have reported tumor site, tumor size, lymph node ratio, staging of diseases, lymph node metastasis, tumor invasion, distant of metastases, Borrmann type, depth of invasion and surgical margin status all related to survival of stomach cancer patients (Yin et al., 2012;Li et al., 2013;Okholm et al., 2014;Posteraro et al., 2014;Deng et al., 2015). However, few studies have conducted in Thailand and none have been investigated the effects of clinicopathological characteristics or XRCC1 polymorphism on survival of stomach cancer patients. It is quite possible that genetic and lifestyle differences between Thai and other population (particularly western population). This study investigates the effects of XRCC1 gene and clinicopathological characteristics on survival of stomach cancer patients in Thailand.

Study subjects
In total, 101 newly diagnosis stomach cancer patients were included in this study. All cases were histologically confirmed and diagnosed according to the International Classification of Diseases for Oncology . Subjects were recruited from Srinagarind Hospital and Khon Kaen Regional Hospital, Khon Kaen Province, Northeast Thailand, during 2002 to 2006. All of patients were followed-up until death or the end of the study (31 October, 2012). Factors of interest were retrieved from medical records including age at diagnosis, gender, site of diseases, surgery type, histological type, histological grading and stage of disease. XRCC1 genotyping was the performed (described below). The classical endpoint in this study is survival time of stomach cancer. Status of each patient was checked from medical records and by linkage with the death registry of the Thai national statistics database.

Laboratory methods
Specimen collection and DNA extraction: Blood samples were taken from all stomach cancer patients diagnosis in the study period (n=101). Whole blood samples of 3-5 ml were collected and centrifuged at 3,000 rpm for 15 minute to separate plasma, buffy coat and red blood cells. All specimens were stored at -20 o C at the cancer unit, Faculty of Medicine, Khon Kaen University. The genomic DNA was extracted from the buffy coat at Nagoya city university medical school, Japan.

PCR amplification and genetic polymorphisms detection
The DNA analyses were performed by using real-time polymerase chain reaction with high resolution melting technique (Real-time PCR-HRM). DNA amplification was performed in a 96-well plate in the light Cycler® 480 Real-Time PCR System. The amplification of XRCC1 Gln399Arg gene was used two primers, [Forward]: 5´-AGT GGG TGC TGG ACT GTC-3´ and [Reverse]:5´-TTG CCC AGC ACA GGA TAA-3´. The HRM data were analyzed using the light Cycler® 480 Gene Scanning software version 1.5(Roche) and was performed at Department of Microbiology, Faculty of Medicine, Khon Kaen University.

Statistical analysis
Survival times of patient were calculated for each patient and were started from the date of diagnosis until the date of death or the end of follow-up (31 October, 2012). Percentages were used to describe categorical data and means with standard deviations or medians with ranges were used to describe continuous data. The observed survival rate was calculated and summarized using Kaplan-meier survival curves. The statistics used to compare survival between groups was performed by using the log-rank test. The univariate and multivariate Cox proportional hazard regression models were used to estimate the association between explanatory variable and survival experience, presented crude hazard ratios (HR) and adjusted HRs and their 95% confidence interval (CI). All analysis was conducted using the SAS statistical package (version 9.3; SAS institute, Cary, NC) and significance level of 0.05 was used for all analysis.

The Ethics Consideration
The study was approved by the Khon Kaen University Ethics Committee for Human Research. The reference number is HE561259.

Demographic characteristics of stomach cancer
The results of the descriptive analysis were summarized in Table 1. Of the 101 patients with stomach cancer, 57 (56.4%) were males. The mean age was 52.7 years. Most of the cancer patients were married 78.2 %, had only a primary school education were 74.3% and farmers or agricultural worker were 69.3%. Table 2 shows the frequencies and the contribution of pathological characteristics of cases. The most commonly specified anatomical sites of stomach cancer were the antrum (45.6% of all cases) and the cardia (16.8% of all cases). The most common type of surgery was subtotal gastrectomy (49.5%). Regarding histopathology, the most frequently specified histological type of malignancy was signet ring cells carcinoma (24.7% of all cases), and in most patients histological grade was assessed as poorly differentiated (58.4%) or unable to be assessed (28.8%). Stage IV cancers (53.5%) preponderated the majority of the patients. The allele frequencies of XRCC1 Gln399Arg polymorphisms for Gln/Gln, Gln/Arg and Arg/Arg genotypes were 47.5%, 40.6% and 11.9 %, respectively.

Survival rate of stomach cancer
The total follow-up person time was 2,070 personmonths, and the overall mortality rate was 4.3 per 100 person-months (95%CI: 3.49 to 5.35). Table 3 presents the survival rates. The cumulative 3-, 6-and 9 months, 1-, 3-and 5-years survival rates were 86.9 %, 63.7 % , 46.5% 40.4 %, 15.2 % and 10.1 %, respectively. The median survival time of stomach cancer after diagnosis was 8.07 months (95%CI: 6.00 to 10.23; Figure 1) DOI:http://dx.doi.org/10.7314/APJCP.2015.16.14.6111 XRCC1 Gene Polymorphism, and Stomach Cancer Survival in Thailand The associated of Clinicopathological and XRCC1 gene Polymorphisms with survival of stomach cancer Table 4 shows after adjusting for lymph node metastasis, comorbidity and complication. Tumour stage IV and Unknown stage lead to increased risked of death (HR: 3.6; 95%CI: 1.35 to 9.43; HR: 3.0; 95%CI: 1.08       were associated increased risked of death with Arg/ Arg homozygote but we can't demonstrated statistically significant. Table 5 show the final multivariate model of significant factors independently associated with hazard of death base on stepwise Cox proportional hazards regression and found a tumour stage IV was associated with hazard of death 1.7-fold (95%CI: 1.09 to 2.59).

Discussion
Our study investigated the factors associated with mortality among stomach cancer patients. This is firstly reported on the effected of the XRCC1 gene and clinicopathological characteristics on the survival of stomach cancer patients among Thai peoples. Our resulted found the stage of diseases was the factors affected to survival of patients, which is consistent with previous studies have been reported. They found out that the staging of diseases were impotent factors affected to survival of gastric cancer patients especially advance stage of diseases (Choi et al., 2011;Kwon et al., 2014). DOI:http://dx.doi.org/10.7314/APJCP.2015.16.14.6111 XRCC1 Gene Polymorphism, and Stomach Cancer Survival in Thailand The study on the effected of the XRCC1 gene to survival of stomach cancer patients, many studies have been explored much on the associated of XRCC1 gene and clinical outcome to survival of patients under treatment by chemotherapy (Liu et al., 2007;Wang et al., 2012;Zou and Yang, 2012;Xu et al., 2014) but non studies have been conducted in Thailand. Our results have found out that the XRCC1 Gln399Arg, Arg/Arg homozygote was affected to survival of stomach cancer patients but statistically non significant.
The tumor location and type of surgeries has important factors affected to survival of stomach cancer patients. Ours study found the tumor location and type of surgeries were not increases risked of death. This is inconsistent with preciously studies done in Korea, France and China they found out that the location of cancer in the stomach and type of surgeries were important factor that effected to survival of stomach cancer patients (Choi et al., 2011;Deng et al., 2014;Son et al., 2014;Herbreteau et al., 2015). The histology grading and histology type, our study found not increases risked of death, Similar findings have been previously reported elsewhere (Kwon et al., 2014;Son et al., 2014).
In conclusion, our study suggests the stage of diseases is the factors affecting to survival of stomach cancer in Thai population. We did not find any effects of XRCC1polymorphisms, tumor location, surgeries type, histology grading and histology type were associated with an increase risk of death of stomach cancer patients. It would be necessary to confirm these findings in the larger sample size.