| 1. BackgroundIn China, cervical cancer is the second most common gynecologic malignancy. And seriously affects womens’ fertility and mortality. Cervical squamous cell carcinoma(CSCC) is the major histology type of cervical cancer, and the incidence of CSCC approximates 75% ~ 80%. The prognosis of cervical cancer is closely related to the clinical stage, the early detection and treatment is an effective way to improve the prognosis of patients. Epidemiological investigation shows that the related factors of cervical cancer mainly include: human papilloma virus(HPV) infection, sexual behavior and delivery times, etc. The high-risk population screening is an important means of improving cervical cancer early diagnosis. Correlated studies found that people in the first-degree relatives of family history of cervical cancer may increase the risk of CIN(Cervical intraepithelial neoplasia, CIN) III and invasive cervical cancer. With the development of cervical cancer molecular biology research, it showed that genetic factor may be an important risk factor of cervical cancer incidence.CSCC and Esophageal Squamous Cell Carcinoma(ESCC) are two kinds of the most common malignancies in China. They both have in common, such as the epidemiological characteristics, risk factors and especially histologic growth patterns. Epidemiological investigations showed that Henan, Shanxi and Shihezi of Xinjiang and other regions where existed high incidence of esophageal cancer simultaneously suffered from cervical cancer frequently. HPV infection has been proved closely associated with occurrence and development of cervical cancer; In recent years, a growing number of studies have supported that HPV infection can also increase the risk of esophageal cancer. The occurrence and development both of CSCC and ESCC are the gradual process according to basal cell damage, dysplasia, and carcinoma in situ and infiltrating carcinoma. Besides, the histological nomenclature of ESCC mostly depended on the early studies of CSCC. In recent years, several studies have found that there are many similar genetic susceptibility factors in the occurrence of cervical cancer and esophageal cancer.It should be pointed out that the recent tumor genetic and molecular biology research showed that there were lots of similar single nucleiotide polymorphism between ESCC and other tumors. Our previous study used GWAS and found some ESCC susceptibility genes which were proved to associate with many other tumors in subsequent researchs, such as gastric cancer, esophageal adenocarcinoma, lung cancer, gallbladder cancer and even gliomatosis cerebri. In recent years, GWAS studies on lung squamous carcinoma and gastric cancer showed some important susceptibility locus. In view of this, the present study is to investigate the relationship between clinical phenotypic characteristics and survival of the CSCC patients in Chinese Han population. Based on recently reported major susceptibility loci, which were found by GWAS studies on ESCC, lung and stomach cancer, we use the Mass Array molecular array technology to screen susceptibility loci of cervical cancer and identify the relationship with these susceptibility loci and clinical phenotypes of cervical cancer. This study will deepen our knowledge of the molecular mechanisms of cervical cancer.2. Materials and methods2.1 Analyses of risk and survival factors of CSCC2.1.1 Study objectThe cases of CSCC patients were from the third affiliated hospital of Zhengzhou university, Henan Cancer Hospital and Women & infants hospital of Zhengzhou from January 1, 2007 to April 30, 2014. The diagnosis of them was confirmed by histopathology. The average age of 809 CSCC cases were 50 ± 11(age range: 25 – 83). 795 control cases were medical examination patients of the third affiliated hospital of Zhengzhou university. The average age of 795 controls were 48 ± 11(age range: 24 – 88).2.1.2 The questionnaire surveyEpidemiological investigation was adopted by questionnaire survey. The informations of questionnaire included age, household registration, age of marriage, smoking history, pregnancy history, menarche age, menopause age, family historyand so on.2.1.3 Follow-up visitWe used the telephone or home visits methods to follow- up patients, and obtained the patients’ survival situations from the local doctors. All the data was inputed into computer using the EXCEL software.2.1.4 Statistical analysisAll the datas were analyzed by SPSS17.0 statistical software. The chi-square test and Logistic regression were used for comparisons among different groups(α = 0.05 was considered as the test standard). The survival rate was calculated by Kaplan-Meier method; the various factors influence on the survival were measured by Log-rank test; COX’s regression analysis model was used to analyze influencing factors. α = 0.05 was considered as the test standard.2.2 Study of CSCC susceptibility loci2.2.1 Study objectThe cases of CSCC patients were from the third affiliated hospital of Zhengzhou University, Henan Cancer Hospital and Women & infants hospital of Zhengzhou Henan Cancer Hospital from October 23, 2011 to May 5, 2012. The diagnosis of CSCC was also confirmed by histopathology. The average age of 376 cases with CSCC was 53 ± 11(age range: 25 – 83). 731 controls came from health check-up crowd of the first affiliated hospital of Zhengzhou university. The average age of 731 controls was 51 ± 10(age range: 18 – 80), and 389 of them were female(mean age: 51 ± 10; age range: 18 – 77) and 342 were male(mean age: 51 ± 11; age range: 19 – 80).2.2.2 Blood sample collection and DNA extraction5 ml venous blood was collected into vacuum tube with EDTAK2. Each of them was averagely subdivided into 5 1.5ml tubes and stored at-80 oC. Genomic DNA was extracted by using Qiagen Gene DNA kits(Qiagen, Germany) according to instructions. The concentration was measured and normalized to 15-20 ng/μl with OD between 1.8-2.0.2.2.3 Genotyping detectionIn this study, we detected 18 SNP that were recently published and were closely associated with ESCC, lung cancer and gastric cancer by using GWAS technology. The 18 SNP respectively are 4 SNP of ESCC, 12 SNP of lung cancers and 2 SNP of gastric cancers. We designed the primers for each SNP and detected the difference of genotyping between cases and controls by using Sequenom Mass Array.2.2.4 Statistical analysisMinor allele frequency(MAF), Hardy-Weinberg equilibrium rates(HWE) were calculated, respectively. Quality controlled data were analyzed and outputted with Plink l.03 software. P value, odds ratio(OR) and 95% confidence interval(95%Cl) were calculated with Cochran-Armitage trend test. The chi-square test with Logistic regression analysis was applied to compare the gene frequency of each SNP loci between cases and controls. α = 0.05 was considered as the test standard.3. Results3.1 Analyses of risk factors for CSCC3.1.1 The proportion of CSCC patients in different age groups were 7.17%(≤35,58/809), 30.41%(36 ~ 45,246/809), 33.87%(46 ~ 55,274/809),20.52%(56 ~65,166/809) and 8.03%(≥66,65/809). The results indicated that the highest incidence of CSCC in women aged from 36 to 55.3.1.2 The proportion of CSCC patients with rural residents was higher than that in urban residents(510/809, 63% VS. 299/809, 37%). It indicated that the lower socioeconomic status was a risk factor of CSCC.3.1.3 The proportion of CSCC patients whose menarche age under 14 was lower than control group(195/804, 24% VS. 437/795, 55%). It indicated that the age at menarche had a significant effect on the morbidity of CSCC(OR=0.262, 95% CI 0.212-0.325, P=0.000).3.1.4 The proportion of CSCC patients in the group with marriageable age under 22 was higher than control group(315/767, 41% VS.109/795, 14%). It indicated that early marriage was an important factor of CSCC(OR=4.386, 95% CI 3.422-5.621, P=0.000).3.1.5 The proportion of CSCC patients with the time gap less than 8 years from menarche to marriage was higher than control group(368/767, 48% VS. 149/795, 19%) which indicated that shorter interval time between menarche and marriage could increase the risk of CSCC(OR=3.999, 95% CI 3.185-5.021, P=0.000).3.1.6 The proportion of CSCC patients in the group with more than 3 times pregnancy was higher than that in control group(621/805, 77% VS. 349/795, 44%). It indicated that much more pregnancies can enhance the risk of CSCC(OR=4.313, 95% CI3.475-5.353, P=0.000).3.1.7 The proportion of CSCC patients with more than two children was higher than that in control group(643/805, 75% VS.210/795, 26%). It indicated that the numbers of children have relation with the risk of CSCC(OR=11.057, 95% CI 8.754-13.966, P=0.000).3.1.8 Logistic regression analysis showed that the age of menarche, early marriage, much more pregnancy and production are the independent factors affecting the risk of CSCC.3.2 The influence of clinical characteristics on survival of CSCC patients3.2.1 In univariate survival analysis, pregnancy, clinical stage and treatment were closely related to prognosis(P<0.05). In COX regression analysis showed that different clinical stages are the independent factors affecting the risk of CSCC(P<0.05).3.2.2 For stage Ⅰb~Ⅱa cervical cancer, both univariate survival analysis and COX regression analysis found that Lymph node metastasis is independent factor affecting the prognosis of CSCC(P<0.05).3.3 Genetic susceptibility of CSCC3.3.1 By comparing the analysis of 18 SNP loci between CSCC patients and healthy controls, we found that the rs36600 SNP sites located in MTMR3 gene can increase the risk of CSCC(OR=0.68, 95% CI 0.53~0.88, P=3.40×10-3). In order to identify whether the men can be as control group in the studies of women specific disease, CSCC patients were compared with healthy men control, No significant differences of this SNP site were found between CSCC patients and men control group(OR=0.78, 95% CI 0.60~1.03, P=0.08). However, it was found differences between CSCC patients and a mixed group of men and women(OR = 0.73, 95% CI 0.58~0.92, P = 6.68 × 10-3).3.3.2 The correlation analysis between the rs36600 SNP loci and prognosis of CSCC patients found that the carriers of different genotype have no significant difference(X2 = 0.000, P = 0.985).4. Conclusion4.1 The younger of menarche was protective factors to CSCC. However, early marriage, more pregnant and production frequency may be the risk factors of CSCC.4.2 Late clinical stage was independent risk factor affecting the survival for prognosis of CSCC. For stage Ⅰb~Ⅱa cervical cancer, lymph node metastasis is closely related to the prognosis of CSCC.4.3 This is the largest study in Chinese population and one loci is identified with high-risk susceptibility for CSCC: rs36600. One gene at locus is implicated: MTMR3 at 22q12.2. |
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