title: Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.
abstract: OBJECTIVE
  Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer.
  
  
  METHODS
  Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA.
  
  
  RESULTS
  The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status.
  
  
  CONCLUSIONS
  This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.
authors.affiliation: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27708, USA. amyebondurant@gmail.com
authors.forename: Amy E
authors.initials: AE
authors.lastname: Bondurant
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Huang
authors.forename: Regina S
authors.initials: RS
authors.lastname: Whitaker
authors.forename: Lauren R
authors.initials: LR
authors.lastname: Simel
authors.forename: Andrew
authors.initials: A
authors.lastname: Berchuck
authors.forename: Susan K
authors.initials: SK
authors.lastname: Murphy
mesh_terms: D000483:Alleles; D000077216:Carcinoma, Ovarian Epithelial; D018284:Cystadenocarcinoma, Serous / Q000097:blood / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009367:Neoplasm Staging; D009375:Neoplasms, Glandular and Epithelial / Q000097:blood / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D010051:Ovarian Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins