Study On Early Diagnosis And Treatment Of Esophageal And Cardia Cancer Among Population In High Incidence Areas

Objective: Esophageal cancer is one of the most common malignant tumors in the world. A very remarkable feature of the cancer is the highly centralized occurrence in the high risk regions, with the difference in incidence and mortality rates as large as 500 times exist between the high and low risk areas. The South Taihang mountain region of our country has been well recognized as the highest incidence area for esophageal cancer in the world. Recently it is know that this area not only bears high risk for esophageal cancer, but also for cardia cancer. However, early diagnosis of the cancer is hard to achieve because patients in the early stage have no obvious symptoms, and majority of patients treated in tumor hospital are in the late stage, so the five year survival rates after operation were no more than 25 percent despite the huge cost . So early diagnosis and treatment is the key to improve the survival. Presently, endoscopy with bioposy is the most effective screen method to discover early carcinoma and precancerous lesion. The present study performed a populational screen in the high incidence county of Cixian, and also evaluated the value of some potential biomarkers for early diagnosis.Our study selected the high incidence region of Cixian and Shexian counties in the area, established cohort and data bank through epidemiological investigation, then endoscopically screened the 40 to 69 age groups and treated early carcinoma and precancerous lesion with mucosal resection. At the same time, observed and compared the detection rates of precancerous lesions, and investigated the value of biomarkers for early diagnosis. The detailed are as followings:①In our study, we analyzed endoscopic screening rates and epidemiology data, calculated specificity and sensitivity of iodine staining under endoscopy to diagnoze carcinoma and precancerous lesions, identified distribution of cardia lesions, investigated the correlation between the screening rate and high risk factors. The above measure can provide help for prevention and cure in high risk region;②Analyzed the results of periodic screening with shorter intervals, investigate sojourn time and natural history of precancerous lesions, to look for the optimal screening schema;③In order to improve the diagnosis rate of cardiac diseases , we estimated the epidemic strength of esophageal and cardiac cancer in the high incidence of Shexian, and watched the microstructures of mucosa with different cardiac diseases using magnifying endoscope and scanning electron microscope, evaluated the diagnostic value,and at the same time treat the early cardia cancer and precancerous lesion by EMR or APC;④Investigated the association of interleukin(IL)-8 single nucleotide polymorphism(SNP) with the susceptibility to esophageal and cardia cancer to clarify the relationship of genetic predisposition and environmental factor with the risk of tumor, search the high risk individuals and defend them;⑤In order to improve the efficency of endoscopic screening, we established finger printing model of serum protein by SELDI-TOF-MS and array to look for more convenient, sensitive and specific early biomarker for preliminary screening of the symptomless people in the high incidence areas.MethodsPart 1 Endoscopic screening and analysis of risk factors in high incidence area of esophageal carcinomaFrom 2005 through 2006, our study selected 21 villages with higher incidence rate in Guyi community, established cohort and data bank through epidemiological investigation, then endoscopically screened the 40 to 69 age group. 3660 cases were examinated from esophagus to duodenal ampulla by using electronic gastroscope with mucosal iodine staining. All visible understained and unstained lesions were judged positive. And then, biopsy were obtained from above-mentioned and suspicious negative areas. Bioposying from the cardia were performed with the exact clock number recorded. All the histological diagnoses were double-blindly made. In our study, we analyzed endoscopic screening rates, calculated specificity and sensitivity of iodine staining under endoscopy to diagnoze carcinoma and precancerous lesions, identified distribution of cardia lesions, investigated the correlation between the screening rate and high risk factors. Part 2 The intermittent time of Endoscopic screening in high incidence area and the natural history of precancerous lesions from esophagus and cardiaShexian county locates southwest of hebei province. It is junction of three province in south taihang mountain. Endoscopic screening was carried out among the 40 to 69 age group in 10 villages of Guyi and Shentou community. 1514 cases residents were examined by using endoscopy. The screening rate was more than 70%. Periodic endoscopic screening was performed among 301 residents within one to fifty months after the first screening. The method was same as above. We compared the periodic screening results, then investigated the sojourn time of rapid developing esophageal and cardiac precancerous lesions, multifocal carcinogenesis, and false negative results, in order to guide the screening.Part 3 Epidemic strength, early diagnose and treatment of cardia cancer in high incidence region of esophageal cancerIn this part, firstly, We estimated the epidemic strength of cardiac cancer in the high incidence region of Shexian county from 2000 to 2004, through comparing the morbidity and mortality of esophageal, cardia and stomach cancer in Shexian county with in Cixian and Linxian county. The detection rates of endoscopy were estimated in cardia cancer and its precancerous lesions. Secondly, we watched the microstructures of mucosa with different cardia lesions by using magnifying endoscope with 3 percent acetic acid spraying, and analysed the coincidence of mucosa pit patterns and histophathological results. At the same time, observed the changes of mucosa ultrastructure with scanning electron microscope. We also detected the infection of H.pylori in cardia lesions by 14C-urea breath test and histopathology , analysed the infection rate of 12 and 6 clock bit repectively, to estimate the relationship between H.pylori infection and its distribution of cardia cancer and precancerous lesions. Finally, treated the early cardia cancer and precancerous lesions by EMR and APC, regularly follow up the post-treatment patients, and estimated therapeutic effect.Part 4 A study on the association between IL-8 polymorphisms and susceptibilities to cardia and esophageal squamous cell carcinomaAll the patients, including 320 cases esophageal squamous carcinoma, 340 cases gastric cardiac adenocarcinoma and 404 cases healthy controls ,came from high incidence region of Hebei province. The total patients were diagnosed by histopathology, and detected by 14C-urea breath test. Whereafter, we collected the disease history, personal history, family history,and so on. Genomic DNA was extracted by tiangen kit. IL-8 was genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Random sampling of DNA sequencing analysis was used to confirm the results of IL-8 genotyping. Hardy-Weinberg analysis was performed to compare the observed and expected genotype frequencies using Chi-square test. Comparison of the IL-8 genotype and allelotype distribution in the study groups was performed by means of two-sided contingency tables using Chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model and adjusted by age and gender accordingly.Part 5 Application of serum protein fingerprint model in diagnosis of esophageal cancer and gastric cardiac adenocarcinoma and their precancerous lesions in high incidence areaThe patients came from natural population in high incidence area of EC--Ci county and She county from Sep 2004 to Dec 2005. The total patients consisted of 26 cases Esophageal mild dysplasia (EDYSⅠ),26 cases Esophageal moderate dysplasia (EDYSⅡ),11 cases Esophageal severe dysplasia (EDYSⅢ),15 cases Early esophageal cancer (EEC),36 cases advanced esophageal carcinoma (AEC),21 cases Gastric-cardia mild dysplasia (GDYSⅠ),10 cases Gastric-cardia moderate or severe dysplasia (GDYSⅡ~Ⅲ),34 cases Gastric cardiac adenocarcinoma (GCA) and 38 cases of NOR. All cases were checked by endoscopy with iodine stainning,and confirmed by superior pathologists. Esophageal precancerous lesion and early cancer were all further consultation to confirm the diagnosis. SELDI-TOF-MS and CM10 ProteinChip were used to detect the serum proteomic patterns of the total patients. The Data was analyzed by Zhejiang University Cancer Institute ProteinChip Data Analysis System (ZUCI-PDAS, www.zlzx.net). Each peak in experiment data was estimated by the P value of Wilcoxson T-test. The top ten peaks with the smallest P value were selected for further analysis. Combinations with the highest accuracy in distinguishing different groups of data were selected as potential biomarkers. The SVM model with the highest Youden's index was selected as the model for detecting esophageal carcinoma and precancerous lesions. The diagnostic model was evaluated and validated by leave one cross validation.ResultsPart 1 Endoscopic screening and analysis of risk factors in high incidence area of esophageal carcinoma1. 3660 cases residents accepted the endoscopic screening. The screening rate was seventy one percent. From above people, we detected 11.9 percent mild dysplasia, 2.1 percent moderate dysplasia, 0.4 percent severe dysplasia, 0.8 percent early carcinoma (carcinoma in situ and intramucosal ), 0.3 percent invasive cancer respectively. In contrast, detection rates of precancerous lesions and cancer from cardia were 1.1, 0.4, 0.2, 0.2 and 0.2 percent respectively.2.With age increasing, the detection rates of precancerous lesions and caner in esophageal and cardia group became gradually higher respectively. There was an association of ascensus trend between the detection rates and age.(esophageal lesions group:χ~2 =130.9,P=0.00;cardia lesions group:χ~2 =40.0, P=0.00)3. One hundred percent early esophageal cancer, 93.8 percent severe dysplasia, 19.9 percent normal and inflammation were positive with iodine staining. There was remarkable difference of positive rate between the ahead and the later two groups.(χ~2=169.8,P<0.01)4. 62 cardiac precancerous lesion are detected by endoscope. In them, 64.9 percent lesions distributed in 12 to 3 clock bits of cardia, and 30.6 percent lesions distributed in other clock bits. There was remarkable difference between the above two groups. Same as above, there was significant difference of clock bits distribution in early cancer groups (P<0.01).5. The detection rates of precursors and carcinomas from the esophagus were higher in male, positive family history of upper gastrointestinal cancer, and smoking group than the opposite groups(χ~2=17.0,22.7,15.8,γ=5, P=0.005,0.000,0.007). There is no correlation between the detection rates and drinking group(χ~2 =4.5,P=0.5).6.The detection rates of precursors and carcinomas from cardia were higher in male, positive family history than the opposite groups(χ~2=34.6,14.3, P=0.000和0.0014). There is no correlation between the detection rates and smoking or drinking(χ~2=8.2,8.0,P=0.16,0.19).Part 2 The intermittent time of Endoscopic screening in high incidence area and the natural history of precancerous lesions from esophagus and cardia1. Periodic screening with shorter interval was performed among 301 cases residents in high incidence area. In above all, 7 cases of SD, 6 cases of cancer in situ and intramucosal carcinoma, 3 cases of invasive cancer were detected by using electronic gastroscope. The longest interval time was 50 months.2. Sojoum time for severe dysplasia(SD) was 13 months after a baseline diagnosis of normal epithelium in 1 subject,7 months after a baseline diagnosis of base cell hyperplasia(BCH) in 1 subject,3 months, 4 months, 4 months, and 10.5 months after baseline diagnoses of mind dysplasia(mD) in 4 subjects,and 12.5 months after baseline diagnosis of moderate dysplasia(MD) in 1 subject.3. Sojoum time for cancer in situ or intramucosal carcinoma was 48 months after a baseline diagnosis of mD in 1 subject,4 months and 13 months after baseline diagnoses of MD in 2 subjects, and 3.5 months, 9 months, and 17.5 months after baseline diagnoses of SD in 3 subjects.4. Sojoum time for invasive cancer was 50 months after a baseline diagnosis of MD in 1 subject, 14 months and 19 months after baseline diagnoses of SD in 2 subjects.Part 3 Epidemic strength, early diagnose and treatment of cardia cancer in high incidence region of esophageal cancer1 Epidemiological investigation of GCA1.1 Incidence and mortality rates of GCA in Shexian countyThe incidence rates of cardia cancer for man and woman in Shexian county from 2000 through 2004 were 69.9 and 41.5, and the mortality rates were 54.3 and 33.2 per 100,000 respectively. Esophageal, cardia and distant stomach cancers put together make up 79.8, 78.7 percent for man, and 68.6, 71.9 percent for woman of the incidence rate or mortality figure for cancer of the whole body.1.2 Compared the detection rates of upper gastrointestinal cancer and precancerous lesions in Shexian , Linxian and Cixian countyEndoscopic survey with iodine staining and biopsy for people aged 40-69 years old could detect 0.6, 0.7 and 0.1 percent intramucosal carcinoma of cardia in Shexian, Linxian and Cixian county respectively. The aboved rates resulted in a ratio of 1:4 to the detection rate of cancer in situ from esophagus. Similar to cancer in situ, detection rate of severe dysplasia from the cardia was also much lower than that from the esophagus, the ratio was 1:4 in Shexian and Cixian county. The detection rates of moderate and mild dysplasia from cardia were similarly much lower than from esophagus.2 H.pylori infection rates and observation about microstructures and ultrastructures of mucosa from cardia lesions2.1 The microstructure of cardiac mucosa with magnifying endoscopeAfter analyzed the relationship between four kinds of mucosal pit patterns and superficial, active and atrophic inflammations, the results indicated that there was significant correlation between these pit patterns and the degree of chronic inflammation. The type of B,C and D displayed in the mucosa of intestinal metaplasia, and type C (66.13 percent) was significantly higher than type B and D in this group. Type C, D and E in 23 cases dysplasia were 48.0, 21.7 and 30.4 percent respectively. The frequencies of type E (64.28 percent) in severe dysplasia group was significantly higher than that in mild and dysplasia dysplasia (P<0.05). All pit patterns of early cardiac cancers were type of E. Analyzed the correlation of dysplasia groups and the pit pattern of muscoal by using rows and column related test (χ~2=10.3, pearson =0.644,P <0.05).2.2The ultrastructure of mucosa with scanning electron microscopeFrom type A to E, the ultrastructure of mucosa observed in low power scanning electron microscope was that the pit was extend and incompleted gradually, from regular punctiform, rod-shape, and en plaque to irregular or disappeard. In high power, we observed that type A was regular circular, made up of two to four cells, all cells which had microvillus were regular and lined up in order. Type B was short rod-shape, there were more cells in the pit, the shape of cells regular and changed little. Type C was arborization, made up of cells more than type B, the pit bent and appeard long slit-shaped ,cells were irregular. Type D was en plaque, the cells were chaotic and degeneration little. Type E was disorganization, cells were degenerate obviously, the microvillus of it were disappeared. Cellula columnoepithelialis of the cryptae like brick wall in sectional view, cells desquamated easily.2.3 Infection rates of H.pylori in cardia lesionsThere was a close relationship between infection rate of H.pylori and cardia cancer and precancerous lesions (χ~2=32.35, P<0.05). The infection rate of H.pylori was the highest in active cardia inflammation. Infection rates of H.pylori in 12 and 6 clock bit were 96.48 and 65.10 percent respectively. There was significant difference in above distribution. (χ~2=108.18, P <0.05).3 Results of the endoscopic therapy in early cardia cancer and precancerous lesions Forty three (78.18 percent) lesions located root of the mucosal fold in cardia (12 to 3 clock bit), twelve (21.82 percent) lesions located other sites. There was statistical significance in above groups. Eleven (73.3 percent) lesions were cut completely with EMR, other four cases remnant lesions were cauterized with APC. The achievement ration of APC were 87.5 and 100 percent respectively. Comparing the pathology of pretherapy and post-treatment, one case intramucosal carcinoma was confirmed postop among four cases preoperative moderate dysplasia, and two cases intramucosal carcinoma were confirmed postop among seven cases preoperative severe dysplasia, and nine cases were bleed, no perforation in all the cases. Regular follow up were performed according to 1,3,6,12 months after treatment.Part 4 A study on the association between IL-8 polymorphisms and susceptibilities to cardia and esophageal squamous cell carcinoma1 Results of PCR-RFLP: The allele gene frequency of IL-8 was 49.6 percent for A in GCA , and 43.1 percent in controls respectively. The differences between the two groups were significantly (χ~2=6.260,P=0.012). However, there were not significantly differences among ESCC and controls (χ~2=6.299 ,P=0.043).2 The genotype distribution of IL-8 in cases and controls: The mutant-type homozygote AA genotype frequency of IL-8 was significant different between GCA and controls [ age, gender, smoking status, family history of UGIC and infection of H.pylori adjusted odds ratio (OR)= 2.014, 95 percent confidence interval(CI)=1.017-3.990]. In ESCC, AA genotype frequency was lower than controls, (15.6percent VS 18.3percent), nevertheless, the differency was not significant(OR=1.901, 95percent, CI=0.952-3.794).3 The stratification analysis of IL-8 Single nucleotide polymorphism and risk factors of cancer3.1 In the smoking status, the AA genotype frequency of positive smoking in ESCC and GCA patients all were not significantly differences than that in controls , and also in the negative groups(P>0.05).3.2 The AA genotype frequency of positive family history of upper gastrointestinal cancer (UGIC) in ESCC (23.1 percent) and GCA (19.8 percent) patients were significantly higher than that in healthy controls (13.8 percent) (P<0.00). Compared with the wild-type homozygote TT, individuals with IL-8-251AA, especially among positive family history of upper gastrointestinal cancer (UGIC), have a higher probability of of ESCC and GCA[the age, gender, smoking stutas and infection of H.pylori ( in GCA groups) adjusted odds ratio (OR)= 2.378 and 14.895, 95percent confidence interval(CI)=1.075-5.258 and 2.889-76.516], but in the negative groups, all the genotype frequencies were not significantly different from that in healthy controls.3.3 Genotype frequency of AA in the group of H.pylori infection positive was significantly higher than that in negative group compared with TT genotype (P=0.017). After adjusted with age, gender, smoking and family history, OR value and 95%CI were 3.520 and 1.249-9.918, respectively. There were no statistics differents in the group of H.pylori infection negative groups.Part 5 Application of serum protein fingerprint model in diagnosis of esophageal cancer and gastric cardiac adenocarcinoma and their precancerous lesions in high incidence area1 Establishment of serum protein fingerprint models in esophageal lesions1.1 We employed SELDI-TOF-MS and support vector machine(SVM)classifier to screen and build nine diagnostic models of EC and precancerous lesions in high incidence area. These diagnostic models are EDYSⅠvs NOR,EDYSⅡvs NOR,EDYSⅢvs NOR,EEC vs NOR,AEC vs NOR,EDYSⅠvs AEC,EDYSⅡvs AEC,EDYSⅢvs AEC and EEC vs AEC.1.2 Among the total, the diagnostic models of EDYSⅠvs NOR,EDYSⅡvs NOR,EDYSⅢvs NOR,EEC vs NOR and AEC vs NOR have specificity (Spe) of 92.11,84.21,81.58,81.58 and 89.47 percent, and have sensitivity (Sen) of 42.31,73.08,45.45,80.00 and 83.33 percent respectively.1.3 The diagnostic models of EDYSⅠvs AEC,EDYSⅡvs AEC,EDYSⅢvs AEC and EEC vs AEC have respectively Spe of 92.31,80.77, 90.91 and 73.33 percent, and have Sen of 80.56,83.33,94.44 and 91.67 percent.1.4 In above-mentioned diagnostic models, the protein peaks (M/Z 4291,4975,5644,5664,and 8775Da) appeared again.2 Establishment of serum protein fingerprint models in cardiac lesions2.1 We employed SELDI-TOF-MS and SVM classifier to screen and build six diagnostic models of GCA and precancerous lesions in high incidence area. These diagnostic models are GDYSⅠvs NOR,GDYSⅡ~Ⅲvs NOR,GCA vs NOR,GDYSⅠvs GCA,GDYSⅡ~Ⅲvs GCA and GDYSⅠvs GDYSⅡ~Ⅲ.2.2 Among the total, the diagnostic models of GDYSⅠvs NOR,GDYSⅡ~Ⅲvs NOR,GCA vs NOR have respectively Spe of 86.84,100 and 94.74 percent, and have Sen of 61.90,90.00 and 88.24 percent.2.3 The diagnostic models of GDYSⅠvs GCA,GDYSⅡ~Ⅲvs GCA have respectively Spe of 90.48 and 80.00 percent, and have Sen of 88.24 and 91.18 percent.2.4 The diagnostic model could differentiate GDYSⅡ~Ⅲfrom GDYSⅠwith a Spe of 100 percent and a Sen of 80.00 percent.2.5 In above mentioned diagnostic models, the protein peaks (M/Z 3271,6891 and 8571Da) appeared again. ConlusionsPart 1 Endoscopic screening and analysis of risk factors in high incidence area of esophageal carcinoma1 The results demonstrated that the screening with endoscopic staining examination could improve early detection of esophageal carcinoma and precancerous lesions.2 There was significant difference about detection rates of early cardia cancer and precancerous lesions between in 12 to 3 clock bits than in other bits. It hinted that we could raise the detection of precances lesion by observing and biopsy in 12-3 sites.3 The results indicated that esophageal and cardia carcinoma developing had gender difference. The people with positive family history of upper gastrointestinal cancer had higher incidence rates than the others with negative history group . There was no remarkable correlation between incidence rate and smoking and drinking.4 Enhancing the compliance and cognition about edoscopic screening in high incidence region, made the high risk people into screening range, could be helpful to improve the detection rates of early carcinoma and precancerous lesions.Part 2 The intermittent time of Endoscopic screening in high incidence area and the natural history of precancerous lesions from esophagus and cardia1 One reasons for interal cases was that significant individual variation existed in the sojourn time, some of them develop rapidly; and the other reason is multifocal carcinogenesis; and the last reason is the false negative results in first time of endoscopic biopsy sampling.2 Periodic screening with shorter intervals should be considered to control the number of interval cases due to rapid development , multifocal carcinogenesis, and false negative results in endoscopic biopsy sampling, change the way of wide-intervaled screenings and then fall behind .3 Scheme of screening in this study was that two or three years to the cases with Normal ,BCH and mD, half or one year to the MD and above. However,this scheme should be further evaluated.Part 3 Epidemic strength, early diagnose and treatment of cardia cancer in high incidence region of esophageal cancer1 The south Taihang mountain region is a high risk area not only for esophagus cancer, but also for cardia cancer. To control upper gastrointestinal tract cancer as a whole in the place, special attention should be paid to the control of cardia cancer by endoscopic screening. The detection rates of early cardia cancer and precancerosis were low, so it is very important to improve the discrimination of above lesions.2 There was a close relationship between type of mucous pits and histopathology by the magnifying endoscope in cardia. The type of pits are useful for biopsy. Type C is a special phenomenon of intestinal metaplasia. The degree of dysplasia gradually aggravated from type C to type E. Type E indicated severe dysplasia and early cancer by magnifying endoscope.3 There was a close relationship between infection of H.pylori and cardia cancer and precancerosis. The infection rate of H.pylori was topmost in active cardia inflammation. It indicated that infection of H.pylori was a risk factor of it, infection rate of H.pylori were higher in 12 clock bit than in 6 clock bit.4 Scanning electron microscope can distinct the fine structure from normal to cardia cancer, and have practical value for the study of mechanism of pit patterns observed by magnifying endoscope.5 EMR and APC are safe and effective methods for treating early cardia cancer and precancerous lesions. The therapeutic patients range should include moderate dysplasia cases. The method of injecting drug in submucosa is one kind of useful method for therapeutic endoscopy.Part 4 A study on the association between IL-8 polymorphisms and susceptibilities to cardia and esophageal squamous cell carcinoma1 People who carry IL-8-251AA genotype have high risk of developing GCA, however, no evidence was found to support that the polymorphisms of IL-8 had relationship with ESCC.2 Family history of upper gastrointestinal cancer (UGIC) significantly enhanced the risk of developing ESCC and GCA, the AA genotype of positive family history of upper gastrointestinal cancer (UGIC) was significantly high risk of developing GCA, the polymorphism of IL-8 alleles may be not associated with the susceptibility of ESCC, but the AA genotype enhanced the susceptibility of ESCC among individuals with UGIC history.3 H.pylori infection positive was risk factor of GCA, the AA genotype of H.pylori infection positive was significantly high risk of developing GCA.4 Smoking was a risk factor of ESCC, but there was no relationship between smoking and GCA, no evidence was found to support that the polymorphisms of IL-8 had relationship with smoking status. Part 5 Application of serum protein fingerprint model in diagnosis of esophageal cancer and gastric cardiac adenocarcinoma and their precancerous lesions in high incidence area.1 In this study, we employed SELDI-TOF-MS to screen and build fifteen diagnostic models of EC, GCA, and their precancerous lesions. In above all, ten diagnostic models could differentiate the above lesions and NOR with high Spe and Sen.2 After further analysed the experimental data, we found that the five protein peaks (M/Z 4291,4975,5644,5664 and 8775Da) had alike function to classify EC and esophageal precancerous lesions from screening crowd, and the three protein peaks (M/Z 3271, 6891 and 8571Da) had similar classification to cardia lesions. The above peaks may be the important biomarkers, and play the significant roles in carcinomatous change.3 Compared with decision tree and artificial neural network classifier, the SVM classifier is a kind of algorithm with many merits. It could guarantee the accuracy and validity of experimental data.4 The above diagnostic models could differentiate EC, GCA, precancerous lesions and NOR well. Their reproducibility and validity should be authenticated through enlarging samples. The valuable protein peaks should be further separation, depuration and identification. It will be helpful to screening in high incidence area.