title: Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.
abstract: BACKGROUND
  Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels.
  
  
  OBJECTIVE
  Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples.
  
  
  METHODS
  Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR.
  
  
  RESULTS
  Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22).
  
  
  CONCLUSIONS
  ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.
authors.affiliation: Guangdong Provincial Hospital of Traditional Chinese Medicine (GPHTCM), Guangzhou, China.
authors.forename: Yazhen
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Lu
authors.affiliation: Pfizer Oncology, San Diego, California, United States.
authors.forename: Maruja E
authors.initials: ME
authors.lastname: Lira
authors.affiliation: Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China.
authors.forename: Yunzhi
authors.initials: Y
authors.lastname: Du
authors.affiliation: Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China. Electronic address: mao_m@yahoo.com.
authors.forename: Mao
authors.initials: M
authors.lastname: Mao
authors.affiliation: Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, South Korea. Electronic address: UNCHUNG8@yuhs.ac.
authors.forename: Hyun Cheol
authors.initials: HC
authors.lastname: Chung
authors.affiliation: Guangdong Provincial Hospital of Traditional Chinese Medicine (GPHTCM), Guangzhou, China. Electronic address: zhengguangjuan@163.com.
authors.forename: Guangjuan
authors.initials: G
authors.lastname: Zheng
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D005784:Gene Amplification; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008875:Middle Aged; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D010232:Paraffin; D018719:Erb-b2 Receptor Tyrosine Kinases
keywords: Breast cancer; Droplet digital PCR; Gastric cancer; HER2 amplification