Serum Proteomics Analysis On Monozygotic Twins Esophageal Squamous Cell Carcinoma And Precarcinoma Lesions In High-incidence Areas Of Esophageal Cancer Of Henan Province

1.Background and objectiveEsophageal squamous cell carcinoma(ESCC),comprising the predominant histological subtype of esophageal cancer(EC),ranks as one of the six most common malignancies and still represents a great health concern in China.EC is characterized by the striking geographic variation:Linzhou city(formerly Linxian county),Weihui city(formerly Huixian county) and Anyang city have the highest incidence of EC in China and the world as well.Apparently distributed difference of areas is the extrusive epidemiological feature of esophageal cancer.This finding indicates that the environment plays the principal role in causing esophageal cancer,but it is worth mentioning more apparent features of EC familial aggregation phenomenon in the region of Linzhou.After 100 years the prevalence of ESCC in the migrant subjects is similar to that in Linzhou,suggesting the hereditary factor may be involved in ESCC. However,the contribution of hereditary factors of the ESCC is unknown,and candidate gene and key protein are not found interrelated with esophageal hereditary factors.Another main characteristic of ESCC is the high mortality rate,most of the EC patients can not survive more than one year after being diagnosed at healthcare centers the first time and the five-year survival rate for EC remains as low as 10%. However,if the EC patient could be diagnosed and treated in early period,five-year survival rate for the EC at early stage could be as high as 90%.Although studies have documented a lot of EC-associated molecules,such as oncogene,tumor suppressor gene,metastatic gene,apoptosis gene,etc.,high-specific and sensitive biomarkers for EC early detection,diagnosis and evaluation of treatment efficiency haven't been identified yet.Morphologically,ESCC originates from normal esophageal epithelia and evolves basal cell hyperplasia(BCH),dysplasia(DYS),carcinoma in situ(CIS) and finally forms malignant neoplasms.BCH,DYS and CIS have been recognized as precancerous lesions.Actually,high-risk subjects screening represents identification of subjects with precancerous lesions.In particular for lesions DYS and CIS,the patients can be cured by simple and economical treatment.Apparently,precancerous stages are relative to incidence and mortality of EC.Although esophagoscopy has the potential to detect the early EC,its widespread application is limited by its high-cost and discomforts.Although studies have documented a lot of EC-associated molecules, such as oncogene,tumor suppressor gene,metastatic gene,apoptosis gene,etc.,it is important to find high-specific and sensitive biomarkers for EC early detection, diagnosis and evaluation of treatment efficiency.Therefore screening for high-risk groups and the establishment of the early warning and early diagnosis of biological technology,methods and targets,and narrowing the scope of endoscopic examination, and then further examination are very important.Proteomics provides an effective approach to investigate disease pathogenesis by globally examining the entire protein component expressed by genome of cell,tissue or organism,it has a high-throughput and high sensitivity characteristics in particular for the selection of tumor marker research.In this study,we research the monozygotic twins because study of twins can not only point out the hereditary effects,but also estimate gene and environment interrelated relation in development of EC.The twins are the best model of esophageal cancer to study genes,gene-genetic factors. Furthermore,the use of twins makes it possible to estimate the magnitude of genetic environment effect on susceptibility to EC.The research includes monozygotic twins epidemiological investigation,endoscopy histopathological examination and immunohistochemical analysis,and monozygotic twins patients from the areas of high incidence of esophageal cancer,sporadic esophageal cancer(EC),sporadic gastric cardia adenocarcinoma(GCA),and magnetic beads proteomic control group.These studies have further comprehended the relation between hereditary and environment in esophageal cancer,but it may be informative for identifying biomarkers for ESCC as well.2.Materials and methods2.1 Objects of studyThe epidemiological investigation includes monozygotic twins,data integrity of the 108 pairs of twins.50 pairs of male,the median age of 45 years,58 pairs of female,the median age of 45 years.Voluntarily accepted gastroscopy contains 22 pairs of twins:sporadic EC(n=four cases),sporadic gastric cancer(n=one case).Sporadic SCC patients:5 patients in this study were from Linzhou Center Hospital,3 males,the median age of 55 years;2 females,the median age of 49 years.Sporadic GCA patients:5 patients in this study were from Anyang Tumor Hospital,4 males,the median age of 65 years;1 females,61 years.20 healthy twins cases were selected as control group from high incidence area, were confirmed no esophageal cancer by gastroscopy and biopsy.8 cases were not esophagitis and other benign esophageal disease.2.2 Collection and processing of serum and tissue samples2.2.1 Serum:Five ml.fasting venous blood from each subject is collected into centrifuge tube in the field.Sera and blood clot are separated and stored under -80℃for further use.Before hemospasia every case is excluded with the disease of the acute or chronic inflammation(hepatitis,fever,etc).2.2.2 Esophageal endoscopy and mucosal biopsyEsophageal endoscopy is applied to the twins pairs(≥35 years old),and two biopsy specimens are collected at esophageal middle(25cm from fore-teeth) and lower segment(esophagus and gastric cardia junction,2cm from dentate line), gastric cardia,and stomach-antrum.The biopsy specimens are taken macroscopic randomly at lesion(unstained areas by iodine).One piece of biopsies is processed for histology,and another one is reserved under -80℃.Twins esophageal cancer,gastric cancer and adjacent tissues were from Anyang Tumor Hospital,Linzhou Central Hospital,Xinxiang City Central Hospital,twin esophageal tissues from the gastroscopy biopsy.2.3 Method2.3.1 Epidemiological surveys:The epidemiological surveys questionnaire is adopted door-to-door,main contents of the survey include:general state(age,sex,means of communication), twins family history,esophageal cancer family history,medical history,dietary habits of life,the general medical examination(height,weight,pulse,blood pressure,etc.). Informed consents are obtained from all the participants who would like to accept blood test or gastrocopy.2.3.2 Monozygotic identificationThe use of questionnaire combined with similar diagnosis,dizygotic twins or twins with incomplete data are excluded.2.3.3 Twins' esophageal mucosa and gastric cardia pathology diagnosis:The histopathological criteria established in laboratory for esophagus and gastric cardia is used in this study.Based on the epithelial cell morphology,tissue architecture and cell differentiation,the esophageal epithelia of the adjacent tissues are classified as normal(NOR),basal cell hyperplasia(BCH),dysplasia(DYS)as three stages;the gastric cardia epithelia are classified as normal,chronic atrophy gastritis of gastric-cardia(CAG),intestinal metaplasia(IM),gastric-cardia dysplasia (DYS)as three stages.These results are diagnosed by two experts of pathology.2.3.4 Microflex & ClinProTools protein microarray detection of serum proteome2.3.4.1 Sample preparation:(1) Take an ampula of copper chelating beads suspension out of 4℃refrigerator, shaking it up and down by hand,completely blending beads suspension,for one minute. (2) Draw 5ul beads adding it into a 200ul sample tube,add 50ul copper chelating beads with buffer(BS).(3) Add into the sample tube 20ul copper chelating beads with buffer,plus 5ul serum.(4) Remove peptide sample solution into a clean 0.5ml sample tube,used directly for mass spectrometry analysis.2.3.4.2 Preparing standardPrepare 10 mM ammonium acetate(77mg ammonium acetate dissolved in 100ml Milli-Q water.Mix as follows:5ul(25ul) Peptide Calibration Standard;20ul(100ul) 10mM ammonium acetate,25ul(125ul) Protein Calibration StandardⅠ.Fully mix 1 min,pack 5ul for each tube,and preserve under -20 degrees.2.3.4.3 Prepare matrix:(1) Prepare(1 g/L) HCCA,acetone solution.(2) Prepare end matrix solution 0.3 g/L of ethanol/acetone=2/1.(3) Prepare matrix solution on the same day configuration,fresh use.2.3.4.4 The generation of standard spectrum:(1)Acquire the scope of 1～13 kDa,error±5Da.(2)Laser energy in the laboratory mass spectrometry may be generally high-energy laser crystallization point to boom,and then below the high-energy laser 10～20%of the energy acquisition map.(3)The same standard crystallization target points,different crystallization point, multi-point acquisition,accumulation of 5 to 10 crystallization point data accumulated by the standard map,molecular weight deviation of less than 100 ppm.2.4 ImmunohistochemistryAvidin-biotin perosidase complexes(ABC) method is used for p53,PCNA protein expression examination in esophageal mucosa,adjacent dysplastic and esophageal cancer tissue from the twin patients.3.Results3.1 Twins Epidemiological survey and Histopathologic examination results in 22 pairs of twins Investigate a total of 479 pairs of twins,of which there are complete data of 108 pairs.50 male pairs,age ranging from 27 to 63 years,with a median of 45 years.58 female pairs,age ranging from 19 to 72 years,with a median of 45 years.Results: there are 13 cases of malignant tumors,including seven cases of esophageal cancer, one case of hepatocellular carcinoma,one case of gastric cancer,cholangiocarcinoma two cases,one case of the lymphoma and one case of osteosarcoma.Apart from one pair of twins at the same time suffering from gallbladder cancer, esophageal cancer and other tumors are all single.Esophageal cancer family history and found 11 pairs of twins esophageal family positive,seven cases of twins,five cases of esophageal cancer patients positive family history of esophageal cancer.108 pairs of twins have weight mass index BMI,height information integrity.There are 60 pairs of twins' BMI values on the line(BMI±1),no difference between men and women.Among twenty two pairs of monozygotic(MZ) twins,only five pairs of MZ twins had concordant pathology:the chronic mild esophagitis(n=3 pairs),chronic superficial gastritis(CSG)(n=1 pairs),NOR(n=1 pairs).Patients with malignant tumor(n=5 cases):esophageal squamous cell carcinoma(n=4 cases),gastric antrum tubular adenocarcinoma(n=1 case);precancerous lesion(n=1 case,the esophageal middle dysplasia and lower esophageal mild BCH);gastric body severe intestinal metaplasia,cardiac intestinal metaplasia and dysplasia,esophageal middle dysplasia, upper esophagus mild dysplasia(n=1 case);chronic mild esophagitis,chronic superficial gastritis and mild atypical hyperplasia(n=1 case);barrett's esophagus(n=1 case);chronic mild esophagitis,chronic atrophic gastritis and intestinal metaplasia (n=1);chronic atrophic gastritis(n=1).All the patients with malignant tumor and precancerous lesion are sporadic cases.The others are chronic mild esophagitis or chronic superficial gastritis.3.2 Result comparison of Microflex twins esophageal cancer,ClinProTools serum protein group3.2.1 Compare twins of esophageal cancer and healthy controls:Proteomic spectra of twins ESCC patients with different stages of infiltration: Ninety-two different proteins are significant,in which 48 proteins are up-regulated and 44 are down-regulated in TW096B(ESCC) and TW096A.Eighty-four different proteins are significant,in which 49 proteins are up-regulated and 35 are down-regulated in TW097B(ESCC) and TW097A.Eighty-three different proteins are significant,in which 67 proteins are up-regulated and 16 are down-regulated in TW098B(ESCC) and TW098A.Sixteen different proteins are significant in three cases of esophageal cancer.And their molecule vulumes are:M1450.61 Da,M 1546.24 Da,M 4090.35Da,M 4192.84 Da,M 4209.20 Da,M 4673.53 Da,M 5398.55 Da,M 5904.17 Da,M 5965.86 Da,M 6033.29 Da,M 6089.22 Da,M 7767.12 Da,M 7829.23 Da,M 8143.70 Da,M 8936.12 Da,M 9293.30 Da.3.2.2 Sixteen different proteins are significant in three pairs of twins of esophageal cancer,the same molecular weight(±5 Da) compared with normal is up-regulated or down-regulated.The protein up-regulations include:M4090.35Da,M 4192.84Da, M4209.20Da,M4673.53Da,M5398.55Da,M5904.17Da,M 5965.86Da,M6033.29Da, M6089.22Da,M7767.12Da,M7829.23Da,M8143.70Da,M8936.12Da,M 9293.30Da; while the protein down-regulations include:M1450.61 Da,M1546.24Da.3.2.3 Compare with three twins of esophageal cancer(ES) and twenty healthy twins controls:Three different proteins(M1779.04 Da,M5905.29 Da,M5967.68 Da) are statistically significant(P＜0.05).Compare with three twins of ES and eight healthy twins controls:thirteen different proteins(M1692.12 Da,M 1779.03 Da,M 2951.75 Da,M 3241.47 Da,M 5905.03 Da,M 5966.85 Da,M 9294.42 Da,M 2660.77 Da,M 2932.58 Da,M 3192.02 Da,M 3263.02 Da,M 3882.19 Da,M 5807.33 Da.) are statistically significant,ROC/AUC:＞95%.Compare with five Sporadic SCC patients and twenty healthy twins controls:Five different proteins(M1779.1 Da,M1866.43Da, M1881.41Da,M1929.57Da,M4287.48 Da) are statistically significant(P＜0.05). Compare with five Sporadic GCA patients and eight healthy controls:four different proteins(M 2660.46Da,M 4963.83Da,M 5807.01Da,M6090.41Da.) are statistically significant,ROC/AUC:＞95%.3.3 Immunohistochemical analysisFrom the normal esophageal epithelium→basal cell hyperplasia(BCH)→change as DYS→squamous cell carcinoma(ESCC),with the lesions progression,an increasing tendency of p53 protein positive expression is observed,and the semi-quantitative also gradually increased.The positive detection for p53 diffuse type: 3 cases of esophageal cancer;1 case of gastric cancer in patients with positive expression of p53(papillary type);three cases of esophagitis with intestinal metaplasia(two cases are p53 positive);13 cases of chronic esophagitis(nine cases of p53 protein positive).P53 test results of all twins:seven pairs are positive in each of the twins,but the p53 semi-quantitative results are quite different,one pairs of twins are negative.PCNA is more than 50%in three cases of esophageal carcinoma tissues, and more than 25%in patients with gastric cancer tissues.The other patients' PCNA results are less than 25%,except two cases negative.4.Conclusions(1)All patients with eight pairs of monozygotic twins are ill with sporadic esophageal carcinoma.The conformity between each pair of the twins patients is low by histopathological examination.It is shown that environmental factors may play critical roles in esophageal carcinogenesis in Henan,a high-incidence area for EC.(2)Sixteen different proteins are significant in the results between three pairs of twins of esophageal cancer and healthy controls.These different proteins may become candidate screening of high-risk groups.(3)The expressions of p53,PCNA in twins patients of EC are higher than those in the control group and adjacent tissues.In our previous studies,the alteration of p53 and PCNA system has been observed as the earliest molecular events in the high-risk groups of the esophageal carcinogenesis.But there is great difference between the twins.It is shown that the environment may play an even greater role of the incidence of esophageal cancer.