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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
Author(s) -
de Cremoux Patricia,
Martin Emmanuel C.,
VincentSalomon Anne,
Dieras Véronique,
Barbaroux Catherine,
Liva Stéphane,
Pouillart Pierre,
SastreGarau Xavier,
Magdelenat Henri
Publication year - 1999
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19991008)83:2<157::aid-ijc2>3.0.co;2-l
Subject(s) - microbiology and biotechnology , biology , polymerase chain reaction , immunohistochemistry , staining , concordance , gene duplication , pathology , gene , medicine , genetics , immunology
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‐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 > 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. Int. J. Cancer 83:157–161, 1999. © 1999 Wiley‐Liss, Inc.