title: Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.
abstract: The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in The Cancer Genome Atlas project breast cancer methylome database. For cMethDNA, a fixed physiologic level (50 copies) of artificially constructed, standard nonhuman reference DNA specific for each gene is introduced in a constant volume of serum (300 μL) before purification of the DNA, facilitating a sensitive, specific, robust, and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in training (28 normal, 24 cancer) and test (27 normal, 33 cancer) sets of recurrent stage IV patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy two to 11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer.
authors.affiliation: Authors' Affiliations: Departments of Oncology, Surgery, and Surgical Pathology, Johns Hopkins University School of Medicine; Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore Maryland; Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; Departments of Oncology and Surgery, Mayo Clinic, Rochester, Minnesota; Department of Surgical Oncology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and Department of Medicine, University of North Carolina, Chapel Hill, North Carolina.
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Fackler
authors.forename: Zoila
authors.initials: Z
authors.lastname: Lopez Bujanda
authors.forename: Christopher
authors.initials: C
authors.lastname: Umbricht
authors.forename: Wei Wen
authors.initials: WW
authors.lastname: Teo
authors.forename: Soonweng
authors.initials: S
authors.lastname: Cho
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.forename: Kala
authors.initials: K
authors.lastname: Visvanathan
authors.forename: Stacie
authors.initials: S
authors.lastname: Jeter
authors.forename: Pedram
authors.initials: P
authors.lastname: Argani
authors.forename: Chenguang
authors.initials: C
authors.lastname: Wang
authors.forename: Jaclyn P
authors.initials: JP
authors.lastname: Lyman
authors.forename: Marina
authors.initials: M
authors.lastname: de Brot
authors.forename: James N
authors.initials: JN
authors.lastname: Ingle
authors.forename: Judy
authors.initials: J
authors.lastname: Boughey
authors.forename: Kandace
authors.initials: K
authors.lastname: McGuire
authors.forename: Tari A
authors.initials: TA
authors.lastname: King
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Carey
authors.forename: Leslie
authors.initials: L
authors.lastname: Cope
authors.forename: Antonio C
authors.initials: AC
authors.lastname: Wolff
authors.forename: Saraswati
authors.initials: S
authors.lastname: Sukumar
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood*; D001943:Breast Neoplasms / Q000097:blood* / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D011446:Prospective Studies; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
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