Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185HER2/neu protein expression in breast cancer drill biopsies

✍ Scribed by Patricia de Cremoux; Emmanuel C. Martin; Anne Vincent-Salomon; Véronique Dieras; Catherine Barbaroux; Stéphane Liva; Pierre Pouillart; Xavier Sastre-Garau; Henri Magdelenat

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: French
• Weight: 373 KB
• Volume: 83
• Category: Article
• ISSN: 0020-7136
• DOI: 10.1002/(sici)1097-0215(19991008)83:2<157::aid-ijc2>3.0.co;2-l

✦ Synopsis

A PCR assay using capillary electrophoresis was designed for the detection of c-erbB-2 gene amplification in alcoholformalin-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-µm-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 G 4), 47 (58%) displayed no c-erbB-2 amplification (AGCN Յ 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 F20%. 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. Int.