title: Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.
abstract: BACKGROUND
  Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods.
  
  
  METHODS
  In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples.
  
  
  RESULTS
  The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001).
  
  
  CONCLUSIONS
  Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression.
authors.affiliation: Optipharm M&amp;D, Inc., Wonju Eco Environmental Technology Center, Wonju, Republic of Korea.
authors.forename: Hye-Young
authors.initials: HY
authors.lastname: Wang
authors.forename: Sungwoo
authors.initials: S
authors.lastname: Ahn
authors.forename: Sunyoung
authors.initials: S
authors.lastname: Park
authors.forename: SeungIl
authors.initials: S
authors.lastname: Kim
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Lee
mesh_terms: D014408:Biomarkers, Tumor; D001940:Breast / Q000378:metabolism* / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human