title: Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer.
abstract: If detected early, breast cancer is eminently curable. To detect breast cancer in samples with little cellularity, a high level of sensitivity is needed. Tumor-specific promoter hypermethylation has provided such a valuable tool for detection of cancer cells in biological samples. To accurately assess promoter hypermethylation for many genes simultaneously in small samples, we developed a novel method, quantitative multiplex-methylation-specific PCR (QM-MSP). QM-MSP is highly sensitive (1 in 10(4)-10(5) copies of DNA) and linear over 5 orders of magnitude. For RASSF1A, TWIST, Cyclin D2, and HIN1, we observed significant differences in both the degree (P < 0.003) and incidence (P < 0.02) of hypermethylation between normal and malignant breast tissues. Evaluation of the cumulative hypermethylation of the four genes within each sample revealed a high level of sensitivity (84%) and specificity (89%) of detection of methylation. We demonstrate the application of this technique for detecting hypermethylated RASSF1A, TWIST, Cyclin D2, HIN1, and RARB in 50-1000 epithelial cells collected from breast ducts during endoscopy or by lavage. Such an approach could be used in a variety of small samples derived from different tissues, with these or different biomarkers to enhance detection of malignancy.
authors.affiliation: Breast Cancer Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231-1000, USA.
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Fackler
authors.forename: Megan
authors.initials: M
authors.lastname: McVeigh
authors.forename: Jyoti
authors.initials: J
authors.lastname: Mehrotra
authors.forename: Marissa A
authors.initials: MA
authors.lastname: Blum
authors.forename: Julie
authors.initials: J
authors.lastname: Lange
authors.forename: Amanda
authors.initials: A
authors.lastname: Lapides
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Garrett
authors.forename: Pedram
authors.initials: P
authors.lastname: Argani
authors.forename: Saraswati
authors.initials: S
authors.lastname: Sukumar
mesh_terms: D000199:Actins / Q000235:genetics; D001940:Breast / Q000378:metabolism / Q000502:physiology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D044584:Carcinoma, Ductal / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013431:Sulfates / Q000737:chemistry
chemical_list: D000199:Actins; D017931:DNA Primers; D004273:DNA, Neoplasm; D013431:Sulfates; C012036:sodium sulfate