title: Use of droplet digital PCR for quantitative and automatic analysis of the HER2 status in breast cancer patients.
abstract: Digital polymerase chain reaction (dPCR) has been used to yield an absolute measure of nucleic acid concentrations. Recently, a new method referred to as droplet digital PCR (ddPCR) has gained attention as a more precise and less subjective assay to quantify DNA amplification. We demonstrated the usefulness of ddPCR to determine HER2 gene amplification of breast cancer.
  
  
  
  In this study, we used ddPCR to measure the HER2 gene copy number in clinical formalin-fixed paraffin-embedded samples of 41 primary breast cancer patients. To improve the accuracy of ddPCR analysis, we also estimated the tumor content ratio (TCR) for each sample.
  
  
  
  Our determination method for HER2 gene amplification using the ddPCR ratio (ERBB2:ch17cent copy number ratio) combined with the TCR showed high consistency with the conventionally defined HER2 gene status according to ASCO-CAP (American Society of Clinical Oncology/College of American Pathologists) guidelines (P<0.0001, Fisher's exact test). The equivocal area was established by adopting 99% confidence intervals obtained by cell line assays, which made it possible to identify all conventionally HER2-positive cases with our method. In addition, we succeeded in automating a major part of the process from DNA extraction to determination of HER2 gene status.
  
  
  
  The introduction of ddPCR to determine the HER2 gene status in breast cancer is feasible for use in clinical practice and might complement or even replace conventional methods of examination in the future.
authors.affiliation: Department of Breast and Endocrine Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Otsuji
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. takesasa@m.u-tokyo.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sasaki
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kunita
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Ikemura
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.|Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Matsusaka
authors.affiliation: Department of Breast and Endocrine Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Tada
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Fukayama
authors.affiliation: Department of Breast and Endocrine Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan.|Department of Stomach and Esophageal Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Seto
mesh_terms: D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000601:surgery; D045744:Cell Line, Tumor; D005260:Female; D005784:Gene Amplification; D018628:Gene Dosage; D018734:Genes, erbB-2; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods; D047368:Tumor Burden
keywords: Breast cancer; Digital PCR; Gene amplification; HER2; Tumor content ratio