Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185(HER2/neu) protein expression in breast cancer drill biopsies

Int J Cancer. 1999 Oct 8;83(2):157-61. doi: 10.1002/(sici)1097-0215(19991008)83:2<157::aid-ijc2>3.0.co;2-l.


A PCR assay using capillary electrophoresis was designed for the detection of c-erbB-2 gene amplification in alcohol-formalin-acetic acid (AFA)-fixed, paraffin-embedded biopsies from 81 consecutive breast tumors. c-erbB-2 expression was analyzed in the same samples using immuno-histochemistry (IHC). In the competitive PCR assay, a single pTag plasmid containing a 4-nucleotide (nt)-deleted copy of a 124-nt sequence of c-erbB-2 and a 4-nt-deleted copy of a 120-nt sequence of GAPDH was co-amplified with genomic DNA extracted from 3 10-micrometer-thick tissue sections of the tumor biopsy. The percentage of tumor cells in the biopsy specimen and the percentage of tumor cells stained with the membrane anti-c-erbB-2 monoclonal antibody CB11 were recorded by a single pathologist on 2 consecutive sections. Among 81 consecutive tumor biopsies assayed by PCR, 21 (26%) displayed unequivocal c-erbB-2 amplification (actual gene copy number, AGCN > 4), 47 (58%) displayed no c-erbB-2 amplification (AGCN </= 2) and 7 (9%) could not be analyzed due to an insufficient amount of DNA. Six samples (7%) were considered inconclusive since the percentage of tumor cells was <20%. Analysis of c-erbB-2 expression by IHC showed that among the 21 amplified specimens 15 displayed strong staining, while all non-amplified samples (47) displayed no or only weak staining. The concordance of the 2 techniques was 91%. We conclude that c-erbB-2 gene amplification can be accurately quantitated by competitive PCR performed on small, fixed and embedded tumor samples.

Publication types

  • Clinical Trial
  • Comparative Study
  • Randomized Controlled Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / drug therapy
  • Adenocarcinoma / genetics*
  • Adenocarcinoma / pathology
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / pathology
  • Cyclophosphamide / administration & dosage
  • DNA, Neoplasm / genetics
  • Docetaxel
  • Doxorubicin / administration & dosage
  • Gene Amplification*
  • Genes, erbB-2*
  • Humans
  • Immunohistochemistry
  • Neoadjuvant Therapy
  • Paclitaxel / administration & dosage
  • Paclitaxel / analogs & derivatives
  • Polymerase Chain Reaction / methods
  • Receptor, ErbB-2 / biosynthesis*
  • Receptor, ErbB-2 / genetics
  • Reproducibility of Results
  • Taxoids*


  • DNA, Neoplasm
  • Taxoids
  • Docetaxel
  • Doxorubicin
  • Cyclophosphamide
  • Receptor, ErbB-2
  • Paclitaxel