Evaluation Of Plasma Cell-free DNA Quantification And Aberrant Promoter Hypermethylation Of COL14A1Gene For The Early Detection Of ESCC

Part one:Evaluation of plasma cell-free DNA quantification for the early detection of ESCCBackground and Objective:Esophageal cancer is one of the most common malignancies. Linzhou, a town in north of Henan province, is the world’s highest-prone areas. In China, esophageal squamous cell carcinoma is most common histological type, accounting for more than90%. Endoscope is the primary means of diagnosis and treatment monitoring of esophageal cancer currently. Compared with other common cancers,such as liver cancer, breast cancer, colon cancer, ovarian cancer, esophageal cancer lack of effective tumor marker. Circulating free DNA (circulating cell-free DNA, cfDNA), is the serum or plasma separated from the fragments of extracellular DNA. Studies have shown, quantitative detection of circulating free DNA has the potential effect of tumor markers. But in these studies, esophageal squamous cell carcinoma is rare reffered. The purpose of this study was to evaluate quantitative detection of plasma free DNA in the diagnosis of esophageal squamous cell. Methods:Plasma free DNA was extracted with a TIANamp Micro DNA Kit and quantified by real-time quantitative PCR(SYBR Green Ⅰ)in60healthy individuals,49ESCC patients. Difference of plasma free DNA levels between healthy individuals and ESCC patients was analysised.Discrimination power was evaluated through the receiver operating characteristic (ROC) curve. Difference between different ESCC patients subgroup was also analysised.Results:The60healthy individuals plasma free DNA levels median17.91ng/ml (12.99～29.90ng/ml). Male plasma free DNA levels median19.10ng/ml and the female group17.17ng/ml,no significant difference was not found(P=0.382); there was no significant difference between<60years of age group (18.17ng/ml) and>60years of age group (15.46ng/ml)(P=0.374).49ESCC patients plasma free DNA levels median49.72ng/ml(28.17～68.421ng/ml). No significant difference was not found between gender group and age group.The value of plasma free DNA quantification in ESCC patients(49.72ng/ml) were significantly increased, compared with that in the healthy individuals(17.91ng/ml)(p<0.001). The area under the curve ROC was0.956(95%CI,0.922to0.990) for plasma free DNA quantification for the healthy individuals versus ESCC patients.29.43ng/ml was the best cut-off point for the detection of ESCC with sensitivity75.5%, specificity93.3%,6.7%false positive rate, false negative rate24.5%, Youden index of0.708. According to tumor location, regional lymph node status, histopathological grade, TNM clinical stage grouping, the49ESCC patients were devided into different subgroups,of which:upper throax10cases, middle throax29cases, lower throax10cases; regional lymph node invasion (+)26cases, local lymph node invasion (--)23cases; well differentiated10cases, moderately differentiated in29cases, poorly differentiated10cases; Ⅰ/Ⅱ stage,30cases,Ⅲ stage19cases. Statistical results showed that:The median level of plasma free DNA Ⅲ patients (67.92ng/ ml) was significantly higher than Ⅰ/Ⅱ patients (33.42ng/ml)(P=0.000); plasma free DNA levels in patients of regional lymph node invasion (+)(62.82ng/ml) was significantly higher than those with regional lymph node invasion (-) patients (P=0.007); No significant difference was observed among different ages,sex, tumor positions and histopathological grade(p>0.05).Conclusion:Our results suggest that:(1) quantitative determination of plasma free DNA has significant implications for the identification of esophageal squamous carcinoma and healthy people, it may provide some supplementary information for the diagnosis of esophageal squamous cell carcinoma, but doesn’t enough sensitivity and specificity to became an independent marker for the diagnosis of esophageal squamous cell carcinoma;(2) plasma cell-free DNA level is related to clinical staging and lymph node metastasis status of esophageal squamous cell carcinoma,it may provide some clues to prognosis of patients with ESCC. Part two:Evaluation of aberrant promoter hypermethylation of COL14A1gene for the early detection of ESCCBackground:Epigeneticsis a case study of the nucleotide sequence of the gene does not change, and a branch of genetics heritable gene expression changes. Epigenetic phenomena including DNA methylation, genomic imprinting, histone modification and so on. DNA methylation is one of the most important, is currently the most in-depth study of an epigenetic form. DNA methylation refers to the DNA double helix, DNA methyltransferase catalytic activity of the process-specific methylation of bases. In mammals, DNA methylation occurs mainly in the two nucleotide cytosine (CpG) bit5carbon atoms, forming a5’methylcytosine (5-MethylCytosine). Many studies show, DNA methylation plays an important role in the development of tumorigenesis, is one of the early events in tumorigenesis, tumor markers with potential significance. COL14A1is present in the extracellular matrix associated with mature collagen glycoprotein molecules. Change of extracellular matrix components are considered to tumor progression and metastasis. COL14A1has close relationship with decorin, a small molecule which is a leucine-rich proteoglycans have been shown to strongly inhibit the progressive growth of tumor cell, such a core protein and its possible inhibition of epidermal growth factor ErbB family of receptors and other proteins mediate, In renal cancer cell lines and primary renal cell carcinoma was detected COL14A1gene promoter region methylation status, and high methylation and gene silencing and associated mRNA down. In our previous chip screening test, it was found that in ESCC COL14A1gene promoter region is hypermethylation.In this study, based on results of preliminary high-throughput screening, we detected status of the promoter region of the gene COL14A1in ESCC tumor tissue and plasma free DNA of ESCC patients and healthy individuals to evaluate aberrant promoter hypermethylation of COL14A1gene for the detection of ESCC.Methods:Aberrant promoter hypermethylation of the p16gene was detected by methylation specific PCR in50healthy human’s plasma free DNA,42ESCC patients’plasma free DNA and its matched ESCC biospy DNA and paired adjacent tissues DNA to the analysis of the difference between healthy individuals and ESCC patients,the difference between tumor tissue and its paired adjacent tissues. In addition, the correlation of COL14A1gene promoter methylation status between plasma free DNA and ESCC biopsy was also analysised.Results:Compared to healthy individuals (0%0/50), COL14A1gene promoter methylation positive rate was significantly higher in plasma of ESCC patients(30.95%13/42)(χ2=8.02, P=0.000).Also, in ESCC biospy(45.24%19/42), COL14A1gene promoter methylation positive rate was higher than in its paired adjacent tissues(11.9%5/42)(χ2==11.43,P=0.001).No significant difference in COL14A1gene promoter methylation positive rate was observed in ESCC patients with different ages, sex, histopathological grades and TNM stages. Only patients whose ESCC biopsy harboredhypermethylated p16gene exhibited aberrant methylation in theirplasma DNA, Spearman correlation coefficients is0.737(P<0.001), indicating that COL14A1hypermethylation in plasma was generally representative of that in its matched biopsy.Conclusion:(1)In ESCC patient, tumor tissue and plasma free DNA in the presence of high COL14A1gene promoter hypermethylation has potential clinical value for the diagnosis of ESCC;(2) plasma free DNA can accurately reflect the tumor tissue COL14A1promoter methylation status, which provides basic and clinical research specimens source for esophageal squamous cell carcinoma.