title: Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.
abstract: Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Jackson
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Choi
authors.affiliation: Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: James D
authors.initials: JD
authors.lastname: Luketich
authors.affiliation: Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Arjun
authors.initials: A
authors.lastname: Pennathur
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts; Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenberg, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts. Electronic address: godfreyt@bu.edu.
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D005787:Gene Frequency; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012680:Sensitivity and Specificity; D051901:Smad4 Protein / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D004259:DNA-Directed DNA Polymerase; D016283:Proto-Oncogene Proteins p21(ras)