| 1. BACKGROUDEsophageal cancer (EC) ranks as the sixth most common malignanct disease and still represents a great health concern in China. EC is characterized by the striking geographic variation throughout the world (the ratio for incidence rates between the high and low incidence areas could be as high as 500:1), and another epidemiologic pronounced feature of EC is the familial aggregation phenomenon, which suggesting that both environmental and hereditary factors may be involved in esophageal oncogenesis. However, there is not a clear conclusion on the significancy of these factors in EC carcinogenesis.We demonstreated EC family history positive (FHP) patients ( = 2 EC patients within generations) account for 32% in all the EC patients based on the investigation in Linzhou (formerly Linxian) and nearby counties Hebi, and Huixian, the high incidence area in Henan, Which suggest that 1/3 of EC patients correlate with hereditary factor directly. At present, key candidate genes involved in EC have not been identified, one of primary reasons is because of the difficultyes in pedigree data collection. Through the mass survey and follow-up studies in high incidence area in Henan, we have demonstreated the high frequency of positive EC family history, and EC familial aggregation in these area named as "high risk family for EC" ( = 2 EC patients within generations), The unique for these population is that all the people are living in the same countyside for several generations with similar environment and genetic background. Furthermore, this population has many family memebers. All this features are much desirable to elucidate key genes related with EC (candidate susceptibility genes). Obviously, it is crucial foundation to collect and sort clinical and genetic background data, and to establish EC core pedigree to elucidate. Just based on the above mentioned studies, the present study was undertaken to establish core pedigree and to analyze the risk factors related with EC by interviewing with family members, collecting clinical and histopathological data, drawing pedigree maps, and other materials including blood sample before and after operation, EC specimen and endoscopic biopsies, which is fundermental to identify key susceptibility genes related with EC family.2. SUBJECTS AND METHODS2.1 SubjectsAll the subjects were from the large scale of mass survey and follow-up in Linzhou and Anyang City, the high incidence area for EC, where we had established a research field from 1995 to 2006. According the investigation of family history, we selected investigated subjects and identified the case group and control group.2.2 Epidemiological investigationThe epidemiologic survey questionnaire was adopted and family members were interviewed at their home by the investigators, including age, gender, family history, family personnel construction, dietary habits and mental factors, upper gastrointestinal tract disease, drug history, circumjacent environment, and so on. All the data of investigation to identify the candidate high risk family for EC were introduced and reserved into computer by using professional software EPIdata (http://www.epidata.dk/download.php)2.3 Collecting and sorting of case informationsAccording the results of epidemiological investigation for the high risk family for EC, by retrospective analysis from representative hospitals in this area or other correlated hospitals (the hospital where the patient was diagnosed and received the therapy), the data from each case of EC patients from the high risk family for EC,were identified the clinical diagnosis and collected including clinical background information, paraffin tissue specimen, pathological diagnosis and pathological type etc.2.4 Identification of the core pedigree of EC and drawing pedigree chartsAccording to the analysis results of the epidemiological investigation and the datafrom clinical cases, the family members were re-interviewed to make clear data. Meanwhile we drew the pedigree charts by handwork. And then the investigation data and the relative information of genealogy were recorded into the data bank of genealogy. Professional software Cyrillic2.02 (http://www.cvrillicsoftware.corn) was used to draw the standard pedigree charts.2.5 Collection and processing of blood specimensThe fasting peripheral venous blood sample was collected twice from the family members (=15 years old), 5ml from each person one time. Serum/blood clots were separated, the whole blood, serum and blood clots were preserved in liquid nitrogen. Then the samples were packed apartly in small quantity, and reserved in -80℃refrigerator.2.6 Esophageal endoscopy and mucosal biopsyEsophageal endoscopy was applied to the family members (=30 years old), and two biopsy specimens were collected at esophageal middle (25cm from fore-teeth) and lower segment (esophagus and gastric cardia junction, 2cm from dentate line). The biopsy specimens were tacken macroscopic randomly at lesion (Unstained areas by iodine). One piece of biopsies was processed for histology, and another one was reserved in -80°C refrigerator.2.7 Collection and processing of blood specimens and surgically resected specimens from the patients in the core pedigree of ECThe fasting peripheral venous blood sample was collected twice from the patients of the core pedigree of EC, 5ml from each person one time, respectively 3 days before operation and 10 days after operation, and the samples were processed using the methods described at 2.5; the surgically resected specimens were processed follow the standard of our laboratory(half fixed with ethanol, half preserved in liquid nitrogen). The fixed half received standard histological processed for histopathological diagnosis and TNM stage, and another freezed half was reserved in -80°C refrigerator.2.8 Analysis on genetic epidemiology dataBalanced test was used to case group and control group, to determine if there was selectivity difference between case and control group. The data were analysed by SPSS12.0 software, using single factor and multiple factors non-condition Logistic regression analysis to ditermine different factors' effect on esophageal carcinogenesis. Falconer method was used to calculate heritability, and Li-Mantel-Gart method was used to calculate segregation ration, binomial distribution fitting distribution of EC, and x~2 test was used for frequency distribution goodness of fittest to determine if there was familial aggregation.2.9 Establish EC genetic correlation gene data bankEC genetic correlation gene data bank would be established by Utilizing MS Access and Visual foxPro6.0, matching software of Microsoft SQL Server 2000.3. RESULTS3.1 Balanced test to case and control groupThe ratio of case group and control group was 1:1, and control group were randomly chosen from each mass survey field. There were 265 subjects with a mean age of 60±11 yeas old in case group and 265 subjects with a mean age 61±9 yeas old in control group, respectively. No significant (P>0.05) in age, gender, culture degree, marriage state, family economy state, and family personnel construction. There was also no selected deflection between the case group and control group.3.2 Investigation by epidemiologic survey questionnairesTotal 4854 questionaires were distributed and 4804 were returned. Of the 4804 questionnaires, 2442 were from mass survey field, 2362 were from the patients and their relatives in several hospitals, 2520 were from patients and their first degree relatives, and 1728 were from the second degree relatives.3.3 Collection and sort of the data from medical recordsAccording to the family history results of the epidemiological investigation, 200 thousands of cases were investigated in 3 representative local hospitals from 1986 to 2006 (over 90% of the patients with EC investigated by us ever received diagnosis or therapy). Seven cases of related clinical informations and 2 paraffin tissue specimens of patients from EC high risk family were identified. The results of the clinical informations from Linzhou and Hebi showed the rate of the patients with positive family history in all EC patients investigated by us were 30% (352/1172) and 23% (217/922), respectively.3.4 The pedigree charts of core EC pedigreeOf the 264 households among the two natural villages (448 natural households, 1107 dwellers) investigated in high risk area, there were 153 households (58%) with positive family history (72 patients with EC) in 264 households. There were 5 households (2%) with more than 10 EC patients within three generations, 8 households (3%) with 7 EC patients within three generations, and followed by 13 (5%) with 6 case, 21 (8%) with 5 case, 28 (11%) with 4 case, 46 (17%) with 3 case households. Genealogies came from epidemiological investigation of EC patients in hospitals, 306 pedigree charts had been drawn, and 230 core genealogies of EC were identified base on epidemiological investigation. We also collected detailed family information of the 8 EC high-risk family, and drew detailed pedigree charts, identified the information such as whether alive, the hospital where received diagnosis and therapy, the age at EC developed, and so on.3.5 Collection of blood specimens from core EC family members Two hundred and forty two blood samples had been collected from 117 family members of 8 high risk families for EC. Whole blood was separated into serum and blood clots, and then was packed in EP tubes with 100μl apartly to prevent the damage by repeated freeze-thaw, and reserved in -80°C refrigerator. Thirty blood samples were collected at most in a family.Of the 117 family members, there were 79 males, 38 females, and 54 cases with the ages below 40 years old, 46 cases with ages 40～60,17 cases with ages over 70 years old.3.6 Collecting of esophageal endoscopy biopsy specimens from family membersEsophageal endoscopy and mucosal biopsy had been applied for 468 cases fromtwo natural villages in high incidence area for EC. There were 45 cases of family members from core genealogies for EC.3.7 Collection and processing of blood specimens and surgically resected specimens from the patients of the core EC genealogiesSo far, we had collected 35 surgically resected specimens from 35 patients within the core EC pedigree, 70 fasting peripheral venous blood samples (before and after operation). The blood samples were packed apartly in small quantity and reserved in -80°C refrigerator. The half of the EC speciments was fixed with 85% ethanol, paraffin embedded, serially sectioned, and HE staining for histopathological diagnosis. And the other half of the surgically resected specimen was reserved in -80°C refrigerator3.8 Analysis genetic epidemiological factors for EC3.8.1 Analysis of EC familial aggregationUsing SPSS 12.0 to do binomial distribution goodness of fittest x~2 test, among family that x~2=187.0124, P=0.0007, P<0.05, actual case numbers distribution was higher than possibility range of binomial distribution, thus it illustrated that EC among family was not followed probability equably to distribute, but it presented obvious family aggregation trend.3.8.2 Analysis of EC genetic epidemiology dataThere was no significant difference equilibrium test between case group and control group. Heritability of first degree relative and second degree relative was 51% and 31% respectively, and the weighted mean heritability was 47%, segregation ratio was P=17.23±2.81%, far less than 25%, Which was consistent with the features of multi-gene genetic diseases.3.8.3 Analysis of the risk factors for ECSooty, frying, hard and hot food, smoking, and upper gastrointestinal tract complaint were all correlated with EC for the first degree ralatives and cases. To second degree relatives: being sulky and stifle, having introverted characteristic, were risk factors for EC.3.9 The establishment of EC genetic correlation gene data bankWe have established EC genetic data bank successfully. After entering administrator interface of data bank, the data could be recorded, revised, stored, and classified, retrievaled and indexed for EC genetic correlation or epidemiology data conveniently by man-machine conversation.4. CONCLUSIONS4.1 We have identified 8 EC genealogies, 8 pedigree charts have been drewed, and part of the family members' blood samples, mucosal biopsies has been collected.4.2 Logistic regression analysis demonstreats that EC carcinogenesis risk factors include sooty, frying, hard and hot food, smoking, suggesting that environmental factors play important role in EC carcinogenesis.4.3 Analysis of EC genetic epidemiological data indicate that there are obvious family aggregation trend, base on the features of multi-gene genetic diseases and pedigree chart analysis, typical or concrete heredity mode could not be yield.
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