| 1Background and PurposeEsophageal squamous cell carcinoma (SCC) is one of the six most commom malignancies worldwide. The dramatic geographic difference is one of the striking epidermiological characteristics for SCC, the ratio for SCC incidence between the high-and low-incidence areas could be as great as500:1. Linzhou city in Henan province has been well recognized as the highest incidence area for SCC in the world. In addition, gastric cardia adenocarcinoma (GCA) seems to occur together with SCC in this area. That SCC and GCA occur in the same patient at the same time or successively are not uncommon, which has been termed as concurrent cancers (CC) in our previous studies. The genetic backgrounds and environmental factors of CC are same, thus, CC may be one of the promising models to address the molecular difference for SCC and GCA. Just because its hereditary similarity, it is precisely important meaning to explain the genetic factors, especially the epigenetic (methylation) role for SCC, comparing with single SCC and GCA.DNA methylation is a kind of epigenetic modification and DNA damage caused by environmental factors. DNA methylation refers to add methyl to the basic group of DNA molecule under the function of DNA methyltransferase. It usually means to use DNA cytosine C-5methyl trans ferase to recognize5-CG-3sequence, transfer methyl of S-Adenosylmethionine(SAM) to C-5position of cytosine and generate5-methylcytosine (5mC). In the5’end regulation section of structure gene, CpG dinucleoside often arrange in the form of clusters. This region rich in CpG dinucleoside is called CpG islands. Its size is500-100bp, approximately56%of encoding genes contain this structural. DNA methylation is one reason of gene inactivation that can influence gene expression. Methylation can be highly expressed and local low expressed when tumor happens. Studies have shown that gene promoter methylation and deletion of the corresponding gene expression are important molecular markers of human cancer. DNA methylation is related to many cancers. At present the overall analysis focuses on CpG methylation site of DNA in cells of tumor and normal tissues.Our laboratory have reported on a series of studies for CC, and investigated its clinicopathology characteristic and prognostic factors preliminary. This study aims to further explore the family history, clinical pathology and survival rate on the patients with CC and the patients with either single SCC or GCA, on the basis of original work of laboratory with expanding cases of CC patients. Epidemiological evidence was provided for clinical assessment and tumor early prevention, based on popular pathological features and prognosis anylysis. This study also aims to analyze the gene methylation result of CC patient, discuss the molecular change of cancer process of CC and provide molecular target index for clinical treatment of CC patient.2Materials and Methods2.1Subjects2.1.1Material sourceHome visit, questionnaire survey, hospital pathology check and telephone follow-up were performed on1,011patients with CC,2,095patients with single SCC and1,859patients with single GCA. These patients came from the high incidence area of SCC and GCA at the junction of Henan, Hebei and Shanxi provinces. Among them there were263patients with CC,796patients with single SCC and252patients with single GCA who had follow-up results.2.1.2Methylation experimentsOne patient with CC came from high incidence area of esophageal cancer, confirmed by histopathological examination. We obtained the surgical specimens of esophageal and gastric cardia and extraction3ml anticoagulant.2.2Methods2.2.1Clinical follow-upHome questionnaires were used to collect the information for patients with SCC, GCA and CC in high incidence area of esophageal cancer, including name, age, occupation, native place, disease time, disease type, treatment hospital, treatment time, family history, contact information and so on. The informations for clinical and pathological features with patients were checked in department of pathology, and living conditions of patient were abtained by telephone follow-up.2.2.2Methylation Bead Chip HybridizationGenomic DNA was extracted by standard procedures using Flexi Gene DNA kits (QIAGEN, Germany). Then we used Human Methylation450K Chip to detect DNA methylation changes of one patient with CC on blood, esophageal and gastric cardia tissues, including process of degeneration, fracture, precipitation, resuspended, hybridization, washing, extension and scanning.2.3Statistical analysis2.3.1Analysis of clinical dataSPSS17.0statistical software was used for, t-test, chi-square test, Kaplan-Meier survival analysis, Cox-regression model was used to analyze the relationship of family history, clinical pathology and survival rate between CC patients and single SCC or GCA patients.2.3.2Analysis of methylation dataChip grayscale scans were analyzed by microarray image analysis software. Same parameter values could be got, including signal intensity of the unmethylated and methylated probe, methylation level of the CpG locus in the group of samples, pvalue computed from the background model and so on.3Results3.1Analysis on family histor with CC, single SCC and GCAThe positive family history ratio of patients with CC was higher than those with single GCA (34%vs.27%, P<0.05), but similar with the single SCC patients (34%vs.31%,P<0.05).3.2Analysis on clinical pathology with CC, single SCC and GCAThe early SCC and GCA from CC was more common in those with single SCC (27%vs.15%) and GCA (18%vs.4%, P＜0.05).3.3Analysis on survival with CC, single SCC and GCAThe patients with CC had shorter survival time than those with single SCC and GCA (P<0.05), the mortality risk for CC was2folds and3folds higher than in those with single SCC (P＜0.05, HR=1.976) and GCA patients (P＜0.05, HR=2.652).3.4Clinical pathology, differentiated type and clinicopathological change analysis on the patients with CC3.4.1There were24cases (8%) of CC patients whose SCC and GCA was both on the early stage,164cases (56%) both on the middle-late stage. There were64cases (22%) of CC patients whose SCC was on the early stage and GCA was on the middle-late stage, conversely,43cases (14%) of CC patients whose SCC was on the middle-late stage and GCA was on the early stage.3.4.2The predominant type for SCC and GCA were both moderate differentiated types. Clinicopathological changes were characterized by higher rate of medullar type (37%) and ulcerative type (26%) in SCC; however the majority type of GCA were ulcerative type (48%) and infiltrating type (15%). 3.5Analysis on DNA methylation with CCData analysis of DNA methylation showed that the alone CpG loci in esophageal tissue was1276, and the alone CpG loci in cardiac tissue was1608.4Conclusions4.1The hereditary susceptibility of CC patients is higher than the single SCC/GCA patients. The patients with CC are considered as SCC and GCA from the same patient at the same time or successively, its hereditary susceptibility is higher than single tumors.4.2The early SCC and GCA in CC are more common than in those with single SCC and GCA.4.3The CC patients have shorter survival time than those with single SCC/GCA.4.4The consistent clinical stage accounts for64%in SCC and GCA from CC, the inconsistent accounts for36%; Moderate differentiation is the primary type for SCC and GCA; The predominant gross morphology for SCC are medullar and ulcerative type, ulcerative and infiltrating type are mainly for GCA. |
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