| 1. Background and purposeEsophageal squamous cell carcinoma (ESCC) is the sixth most common malignant diseases wordwide. Rural regions around Taihang Mountain in the junction of Henan, Hebei and Shanxi provinces in northern China have been well documented as the highest incidence area for esophageal cancer (EC), not only in China but also in the world. EC remains the leading cause of cancer-related death in these areas. Although the accumulated evidences indicated that the nutritional deficiencies, intake of pickled vegetables, nitrosamine-rich or mycotoxin-contaminated foods, smoking and alcohol consumption are major risk factors for EC. But the exact etiological mechanism to EC is still unclear. Our primary investigation in high-risk region for EC in China was demonstrated that familial aggregating for EC is obvious suggesting that the hereditary factors may play an important role in EC pathogenesis. The susceptibility loci at PLCE1 and C20orf54 (RFT2) for EC have been identified by our study team recently.It is controversy on the prognosis effect by familial and sporadic factors. Familial EC have a better prognosis than sporadic EC. It is noteworthy that the sample size for EC family history analysis is usually too small to make a conclusion.Apparently, it is much desirable to correlate the familial EC and SNPs identified from GWAS, which will provide important clues for elucidating the genetic mechanisms involved in EC and screening useful biomarkers to high-risk subject prediction.Thus, the present study was undertaken to characterize the familial and sporadic EC in clinicopathology, survival in large-seal EC patients, and to correlate those changes with SNP varients idendified from the same population.2. Materials and Methods2.1 Patients and questionnairesAll the 61781 patients diagnosed with ESCC from 1973 to 2009 in northern China including Henan, Hebei and Shanxi were enrolled in this study. There were 38283 males with a mean age of 58.75±9.41 and 23498 females with a mean age of 59.27±9.38. The sex ratio is 1.63. The clinicopathological data include name, onset age, gender, ethnicity, residence location, family history of EC, tumor location, histology type, pathology type, grade of differentiation, TNM stage of cancer, and survival after medical interventions. All the patients were confirmed as ESCC by at least 2 histopathologists. The clinical pathology information was collected by consulting medical record of hospitals where the patients were diagnosed and treated. Questionaires were performed face to face at patients' home or hospitals. A follow-up study was carried on and the information was collected. The data was analyzed by SPSS 17.0. Descriptive statistics were used to describe patient baseline characteristics. To evaluate a potential association between the genotype (PLCE1 and RFT2) and clinicopathological parameters the chi-square test was used. Survival curves of the patients were plotted using the Kaplan-Meier method and analysed using the Log rank test. For multi factors unconditional regression analysis, Cox survival analysis was used. Significan atements refer to Pvalues of two-tailed tests that were<0.05. The study was app(?) ed by each institutional and hospital ethical committee and conducted according to Declaration of Helsinki principles.2.2 Blood sample c(?)lection, DNA extracting and genotyping on 2 SNPs PLCE1 (rs2274223) and (?)FT2 (rs6140125) Blood samples were collected by draw blood from antecubital vein after questionnaires were finished. Blood drawing was operated under sterile conditions to maintain blood samples were not contaminated and put them into the -80℃refrigerator for preservation immediately. Genomic DNA was extracted from peripheral blood leukocytes by standard procedures using Flexi Gene DNA kits (QIAGEN, Germany). Approximately 200 ng of genomic DNA was used for genotyping analysis. Briefly, each sample was wholegenome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared Illumina Human 610-Quad BeadChips. After hybridization, the BeadChips oligonucleotides were extended by a single labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina BeadStudio 3.2 software, which converted fluorescence intensities into SNP genotypes.3. Results3.1 EC family history, clinicopathologic and survival analyses3.1.1 The ratio of the patients with positive family history (FH+) for EC was 34% in all the patients with ESCC; In Taihang Mountain area in China, the FH+ rate was as high as 40%;3.1.2 Overall, familial patients with ESCC showed a significantly earlier onset age than the sporadic ESCC patients (the mean onset age:57.99±9.29 vs 59.45±9.42, Pt-test<0.001; the median onset age:58 and 60, respectively). The FH+ rate and sex ratio decreased gradually with aging;3.1.3 A significant difference was found between familial and sporadic patients with ESCC (χ2=12.600, Plog Rank=3.857×10-1) by using single factor analysis of Kaplan-Meier survival. Overall familial patients with ESCC have a better prognosis than sporadic patients with ESCC. According to the results of multi factors unconditional Cox regression, hazard risk of sporadic patients is 1.2 times of familial patients as other factors were adjusted. Family history of ESCC is a protect factor for the prognosis of patients with ESCC; Generally speaking, the onset age (OA) affects the prognosis (P=0.003). The hazard risk of patients (OA<45y) are higher than the patients (OA>65y) 1.2 times with other factors were adjusted (P=0.036). Early OA is a hazard factor for the prognosis;3.1.4 There was no significant difference between the familial and sporadic ESCC patients as respect for their tumor sites, tumor histology, pathology type, degree of differentiation, TNM stage. But, FH+ rate in patients with high-degree of differentiation was higher than those with moderate-degree and low-degree of differentiation (34% vs 30%, P=0.007; 34% vs 28%, P=0.0002). There was a significant difference of FH+ rate between early stage patients and progressivestage patients (39% vs 33%, P=1.866×10-4). There were more familial patients with ESCC in early stage patient group.3.2 Correlating of SNP varients with clinicopathology and survival in familial and sporadic ESCC patientsFor PLCE1 (rs2274223), the prevalence of GG allele genotype in FH+ patients was appearently higher than in sporadic patients (9% vs 7%, P=0.036). The prevalence of CC and AA genotype for RFT2(rs6140125) in ESCC patients at progressive stage was higher than in those at early stage (56% vs 47%,P=0.016; 10% vs 6%, P=0.003).4. Conclusions4.1 Familial aggregation for ESCC in Taihang Mountain high-risk area is very common with a FH+ rate of as high as 40% and the familial ESCC patients show earlier onset and better survival than sporadic patients, suggesting that genetic factors may play an important role in this high-incidence area for EC;4.2 Survival of Familial EC patients is better than sporadic patients; Survival of young patients (OA<45) is worse than elderly patients.4.3 Familial patients with PLCE1 (rs2274223) variant GG homozygote genotype are more prevalent than sporadic patients, suggesting that this variant may be a promising indicator for familial ESCC;4.4 The variants CC and GG homozygote genotype at the loci of RFT2 (rs6140125) may be promising biomarker for poor prognosis. |
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