Detecting Hypermethylated SLIT2,3-OST-2, CDH13 And TES For Early Diagnosis Of Ovarian Carcinoma

BackgroudOvarian carcinoma is the third most common gynecological malignancy and is the leading cause of death among women with gynecologic cancers. The lifetime risk of ovarian carcinoma is about 1 in 55, while the prevalence in postmenopausal women is 1 in 2,500, and the age-adjusted incidence is around 11 per 100,000. It is estimated that 82,550 new cases will be diagnosed, and 53,600 deaths from ovarian carcinoma occur, in Europe and America in 2009. About 190,000 new cases and 114,000 deaths from ovarian cancer are estimated to occur annually in the world. Unfortunately, there is no reliable data at the national level on mortality from ovarian carcinoma in China.Because there is currently no sufficiently accurate screening test proven to be effective in the early detection of ovarian carcinoma,70%～75%of patients are not diagnosed until the disease has advanced to stage III or IV. The 5-year survival rate for women with these advanced stages is less than 30%, but patients with stage I disease have 5-year survival in access of 85%if appropriately treated. Because of the association between prognosis and stage, a specific and sensitive screening test is intuitively appealing. Detection of a large fraction of ovarian carcinoma in early stage would be of clearly clinical benefit and might significantly improve the overall survival. It is estimated that if the percentage of cases diagnosed in stage I could increase to 75%by early detection, mortality could be reduced by 50%, and the 5-year survival could rise to 80-95%. Both genetic and epigenetic changes that initiate and drive tumorigenesis are promising target for early detection because they may precede clinically obvious disease. Epigenetic alterations, defined as heritable changes in gene expression but without DNA sequence changing, such as DNA methylation, histone modification, microRNAs and chromatin remodeling, are increasingly focused on recently, for their reversible nature. DNA methylation, the best studied epigenetic mechanism, is an enzymatic process to add the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3'sequence. Cytosine methylation is important for epigenetic regulation of endogenous genes, silencing of transposons and controlling of genome stability. Now, DNA methyaltion is considered a third pathway to satisfy Knudson's hypothesis. It can lead to transcriptional inactivation and gene silence by directly blocking the binding of transcriptional factors to DNA, and/or by MBP(methyl-CpG-binding protein), which recruits chromatin remodeling corepressor complexes.Numerous studies have revealed that the coexistence of gene-specific promoter hypermethylation and global genomic DNA hypomethylation is an epigenetic characteristics of cancer cells, and the methylation of CpG dinucleotides in the promoter regions of tumor suppressor genes (TSGs) can be used as a useful biomarker for early detection of cancer. However, these analyses usually are performed after surgery or biopsy, which limits their use for early screening for cancer. Fortunately, several recent studies have demonstrated the feasibility of detecting tumor-specific genetic or epigenetic alterations in body fluids, especially in serum or plasma. Tumor-associated DNA in blood originate from cell lysis, apoptosis, necrosis or active release from the primary tumor, and has almost the same characteristics as primary tumor DNA, such as oncogene expression, tumor suppressor gene mutations, as well as microsatellite and epigenetic alterations.Methylation-specific PCR(MSP), a sensitive assay that detects CpG dinucleotides in a CpG island, can identify one allele in the presence of 1,000～10,000 unmethylated alleles, and does not require prior knowledge of epigenetic alterations. Due to both its high sensitivity and specificity, MSP analysis provides a straightforward answer to cancer detection. To determine the feasibility of DNA methylation for early detection of ovarian carcinoma, we first investigated the methylation status of the human homologue of the Drosophila Slit2(SLIT2), Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2(3-OST-2), H-cadherin (CDH13) and TESTIN(TES) genes in tumor DNA from 79 ovarian carcinoma patients. Then we analysed the methylation status of plasma and ascites DNA from another 42 patients with a probable ovarian carcinoma diagnosis, in order to determine the sensitivity and specificity of DNA methylation for detection of cancer.Our research has three parts and the abstrct was as followed:Part 1:The Frequency of Hypermethylated SLIT2,3-OST-2,CDH13 and TES in Primary Ovarian CarcinomaPurpose:To determine the feasibility of DNA methylation for early diagnosis of ovarian carcinoma.Materials and Methods:Using Methylation-specific PCR, we first assessed the methylation status of SLIT2,3-OST-2, CDH13 and TES in tumor DNA from 79 patients with ovarian carcinoma, and analyzed the relationship between methylation status and clinicopathological parameters.Results:1. In the samples from primary ovarian carcinomas, the frequency of aberrant promoter hypermethylation of SLIT2,3-OST-2, CDH13 and TES genes was 86.1% (68/79),77.2%(61/79),43.0%(34/79) and 30.4%(24/79), respectively. Overall, in 77 of the 79 tumors, at least one of the four gene promoters was hypermethylated, which provides 97.5%diagnostic coverage. In contrast, none of the 40 benign ovarian tissues exhibited abnormal promoter hypermethylation of any gene.2. Hypermethylation was detected in patients of all ages, in all histological cell types, in all pathological grades, in all stages of ovarian carcinoma, and even in borderline tumors of low malignant potential. The methylation status had no correlation with tumor stage, histological classifications, grade or patient age. There was no association between the mean number of methylated genes and the tumor stage (2.590.91 in stageⅠ-Ⅱvs 2.28±0.96 in stageⅢ-Ⅳ, P= 0.195).3. Hypermethylation of CDH13 was associated with CA 125 level (P= 0.034) and SLIT2 was associated with the lymph node metastasis (P= 0.027).4. The combination of SLIT2,3-OST-2, CDH13 and TES provided 97.5% diagnostic coverage, which improved the sensitivity of the gene panel for cancer detection.Conclusions:Ovarian carcinoma harbored CpG island hypermethylation of SLIT2,3-OST-2, CDH13 and TES genes. Promoter hypermethylation showed tumor-specific features and was a relatively early event in ovarian tumorigenesis. The combination of multiple genes could improve the sensitivity of the gene panel for cancer detection.Part 2:Detecting Methylated SLIT2,3-OST-2, CDH13 and TES in Plasma for Early Diagnosis of Ovarian CarcinomaPurpose:To determine the feasibility of circulating DNA for early diagnosis of ovarian carcinoma.Materials and Methods:Using Methylation-specific PCR, we investigated the methylation status of SLIT2,3-OST-2, CDH13 and TES in plasma DNA from 42 patients with a probable ovarian carcinoma diagnosis, and compared the methylation status of DNA from tissues to that from plasma.Results:1. In the 42 patients with suspected ovarian carcinoma, the frequency of promoter hypermethylation detected in plasma was 38.1%(16/42) for SLIT2, 28.6%(12/42) for 3-OST-2,19.0%(8/42) for CDH13 and 19.0%(8/42) for TES. Overall,54.8%(23/42) of the plasma DNA samples had at least one gene promoter hypermethylated. But no methylation of any gene was detected in the 30 plasma DNA samples from healthy age-matched women. 2. After surgery, we examined the hypermethylation status of the panel genes in the matched tumor tissues.Comparing the methylation results from the plasma samples to the tumor samples revealed that the sensitivity of the gene panel was 88.5%(23/26) in plasma and 96.2%(25/26) in tissues, and the specificity both were 100%.3. The methylation concordance between plasma and paired tumor DNA was 95.2%(40/42).4. If plasma DNA was substituted for tumor DNA for diaagnosis, the sensitivity, specificity, accuracy, positive prognostic value and negative prognostic value were 92.0%(23/25),100%(17/17),95.2%(40/42),100%(23/23) and 89.5%(17/19), respectively.5. The methylation frequency of at least one gene in plasma DNA form stage I-Ⅱpatients was 70%(7/10).6. The frequency of CDH13 hypermethylation was still associated with CA 125 level (P=0.021) in tumor DNA.Conclusions:The aberrant promoter hypermethylation of tumor suppressor genes can be detected in the plasma of ovarian carcinoma patients. Circulating hypermethylated DNA detection is a promising strategy for early detection of ovarian carcinoma.Part 3:Evaluation of Methylated SLIT2,3-OST-2, CDH13 and TES in Ascites for Diagnosis of Ovarian CarcinomaPurpose:To determine the feasibility of ascites DNA for diagnosis of ovarian carcinoma.Materials and Methods:Using Methylation-specific PCR, we analysed the methylation status of SLIT2,3-OST-2, CDH13 and TES in ascites DNA from 42 patients with a probable ovarian carcinoma diagnosis, and compared the results to that from tissues and from plasma.Results: 1.In the 42 patients with suspected ovarian carcinoma, the frequency of promoter hypermethylation detected in ascites was 47.6%(20/42) for SLIT2, 38.1%(16/42) for 3-OST-2,21.4%(9/42) for CDH13 and 23.8%(10/42) for TES. Overall,52.4%(22/42) of the ascites DNA samples had at least one gene promoter hypermethylated.2. The sensitivity of the gene panel for ovarian carcinoma diagnosis was 84.6% (22/26) in ascites. If ascites DNA was substituted for tumor DNA for diaagnosis, the sensitivity, specificity, accuracy, positive prognostic value, negative prognostic value were 88.0%(22/25),100%(17/17),92.9%(39/42),100%(22/22) and 85.0% (17/20), respectively.3. In the abnormal cytological patients,19 showed aberrant promoter hypermethylation, the sensitivity and specificity was 95.0%(19/20) and 100%(22/22), respectively.4. In the benign tumor patients, no methylation of any gene was detected in their ascites DNA, which was coincident with the results of plasma.5. The methylation frequency of panel genes in ascites DNA from stage I-II patients was 60%(6/10).Conclusions:The aberrant promoter hypermethylation of tumor suppressor genes can be detected in the ascites of ovarian carcinoma patients. Ascites DNA is a surrogate for methylation analysis of tumor DNA or plasma DNA.