Aberrant DNA Methylation Pattern Of Human Epithelial Ovarian Cancer And Its Applications In Molecular Classification And Clinic Diagnosis

Ovarian cancer has the highest mortality rate of the female reproductive malignancies,the majority of which(＞90%) are believed to derive from the ovarian surface epithelium. Asymptomatic and absent of efficient diagnostic approaches in its early stages,most ovarian cancers are diagnosed at the late stages ofⅢandⅣ,which makes 5-year survival for patients markedly down to less than 20%.Therefore it is very important for patients' survival to find novel tumor markers with high sensitivity and specificity in early diagnosis.Besides,patients with epithelial ovarian cancer have greatly different survival among those in divergent as well as identical clinical stages and pathologic types who reacting differently to same therapies.Distinct biomolecular characteristics of tumor cells are hypothesized underlying the heterogeneity.Therefore it is very important for improve and estimate patients' survival to establish tumor molecular classification,guiding individual treatment better.As one of the most frequent epigenetic event,DNA methylation aids in regulating genes' expression.Recent studies have shown that aberrant DNA methylation has strong association with the development and progression of tumor,including ovarian cancer.Being early events in tumorigenesis,aberrant DNA methylation could occur before the alteration of gene and its expression products.In addition,serum DNA of tumor patients would increase obviously in the level and exhibit genetic and epigenetic characteristics identical with tumor DNA.So aberrant DNA methylation including that occurred in serum DNA has intensive perspective in tumorigenesis,early diagnosis and treatment guidance.In the present study,a method called DMH(differential methylation hybridization) based on microarray technology was used to investigate genomic DNA methylation pattern in tumor cells. Molecular classification concerning this methylation profile was then interrogated for its merit in prognosis evaluation.Then large-scale ovarian cancer tissues was tested by fluorescence Taqman real-time quantitative PCR(MethyLight) to confirm the differential methylation detected using DMH and to search for novel cancer-specific tumor markers,laying the foundation for future application research of new method in early diagnosis of human epithelial ovarian cancer.Finally, the application value of serum DNA quantification and its methylation markers detection in diagnosis of ovarian cancer was discussed.The first part:Objective To profile methylation alterations of CpG islands in human epithelial ovarian cancer and interrogate its applications in molecular classification and finding new cancer related genes.Methods Cancer cells were obtained by laser microdissection from 20 frozen-preserved human epithelial ovarian tumors.Epithelial ceils secured from 5 normal ovaries were primary cultured.Differential methylation hybridization(DMH) based on microarray assay was conducted using the DNAs of the two groups of cells mentioned above to construct the aberrant DNA methylation pattern of human epithelial ovarian cancer.The correlation between the patients' survival and their molecular classification derived from the methylation pattern via hierarchical clustering and other clinical indexes was analyzed by COX regression.Sequences of the aberrant methylated DNA loci and the nearby genes were compared. Results The aberrant DNA methylation pattern of human epithelial ovarian cancer includes 182 hypermethylated loci and 64 hypomethylated loci(18 and 31 loci,individually,were positive in more than 25%arrays).The 20 patients were classified via hierarchical clustering on the basis of hyper-,hypo- and entire hyper-/hypo- methylated loci,respectively.COX regression analysis revealed that the impact factors of patients' survival were comprised of pathological type, pathological grade,operation-pathological stage and molecular type based on hyper-methylation, and the risk ratio of the latter was 13.459(P=0.022).15 loci located in CpG islands(CGI) of some genes' promoters,indicating that these genes may participate in the development and progression of ovarian cancer through aberrant promoter methylation.Conclusions Differential methylation hybridization is a high throughput method which could screen aberrant DNA methylation patterns of many diseases.The pattern of human epithelial ovarian cancer could be applied to molecular classification and finding new cancer related genes.The second part:Objective To investigate DNA methylation alterations of several promoter CpG islands in human epithelial ovarian cancer tissues,verifying the differential methylation detected by DMH in our previous study.To identify novel candidate cancer-specific epigenetic markers,laying the foundation for future application research of new method in early diagnosis of human epithelial ovarian cancer.Methods MethyLight was conducted to verify the methylation status of 7 hypomethylated promoter CGIs detected by DMH in tumor tissues of 87 patients with ovarian cancer(including the 20 primary ovarian cancer tissues used in our previous DMH assay) and 42 patients with benign ovarian diseases.Results The 7 CGIs were hypomethylated in different degrees in the 20 primary epithelial ovarian cancer tissues tested by our previous DMH assay.The methylation ratio of gene LSM2, EGFLAM and CDKN2A in tissue DNA of patients with epithelial ovarian cancer and benign ovarian diseases was 11%(10/87) versus 33%(14/42),8%(7/87) versus 21%(9/42),9%(8/87) versus 31%(13/42),respectively.The methylation degree of all the three gene was significantly decreased in patients with ovarian cancer compared to the patients with benign ovarian diseases (P＜0.05,χ~2 test).The methylation ratio of 3 CGI panel(at least one CGI methylated) was significantly decreased in all ovarian cancer patients(19/87,22%) and patients in I stage(10/33, 30%) compared to the patients with benign ovarian diseases(23/42,55%,P＜0.05,χ~2 test).Conclusions Differential methylation in tumor cells determined by DMH was well confirmed by MethyLight using large-scale tumor tissues,identifying three promoter CGIs of gene LSM2, EGFLAM and CDKN2A as novel candidate cancer-specific hypomethylated tumor markers.The total methylation status of the 3 CGIs may help in early diagnosis of ovarian cancer.The third part:Objective To quantify serum DNA of patients with epithelial ovarian cancer and to detect the methylation status of the promoter CpG islands(CGIs) of gene EGFLAM,CDKN2A and LSM2, searching for novel diagnosis methods for human epithelial ovarian cancer.Methods The preoperative serum DNA of 30 patients with primary epithelial ovarian cancer and 15 patients with benign ovarian diseases was extracted using micro-genomic DNA extraction kit and quantified by SYBR greenⅠfluorescent staining.The methylation status of the promoter CGIs of gene EGFLAM,CDKN2A and LSM2 was evaluated by Taqman fluorescent quantitative real-time PCR(MethyLight). Results The serum DNA concentration of 30 patients with primary epithelial ovarian cancer (59.69±88.43ng/ml) was significantly higher than that of 15 patients with benign ovarian diseases (21.35±10.02ng/ml,P＜0.05).In serum DNA,the methylation ratio of the promoter CGIs in gene EGFLAM,CDKN2A and LSM2 of patients with epithelial ovarian cancer(17%(5/30),10% (3/30),33%(10/30),separately) was lower,but not significantly(P＞0.05,χ~2 test),than that of patients with benign ovarian diseases(33%(5/15),20%(3/15),60%(9/15),separately).The methylation ratio of the CGI panel in serum DNA(at least one of the above three CGIs methylated) of patients with epithelial ovarian cancer(53%,16/30) was lower,but not significantly(P=0.074,χ~2 test),than that of patients with benign ovarian diseases(80%,12/15).While the CGI panel's methylation ratio of late-stage patients(40%,8/20) was significantly lower(P=0.015,χ~2 test) than that of patients with benign ovarian diseases(80%,12/15).The total diagnostic sensitivity and specificity of the serum DNA level(＞33ng/ml) and its methylation status(none of the three CGIs methylated) were 70%and 73.3%,respectively.Conclusions Quantification of serum DNA of patients with epithelial ovarian cancer and its methylation markers of the promoter CGIs in gene EGFLAM,CDKN2A and LSM2 might be novel auxiliary methods for the diagnosis of epithelial ovarian cancer.