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.
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.
de Cremoux
P
P
Department of Pathology, Institut Curie, Paris, France. PatriciaDe-Cremou@curie.net
Martin
E C
EC
Vincent-Salomon
A
A
Dieras
V
V
Barbaroux
C
C
Liva
S
S
Pouillart
P
P
Sastre-Garau
X
X
Magdelenat
H
H
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D003520:Cyclophosphamide / Q000008:administration & dosage; D004273:DNA, Neoplasm / Q000235:genetics; D000077143:Docetaxel; D004317:Doxorubicin / Q000008:administration & dosage; D005784:Gene Amplification; D018734:Genes, erbB-2; D006801:Humans; D007150:Immunohistochemistry; D020360:Neoadjuvant Therapy; D017239:Paclitaxel / Q000008:administration & dosage / Q000031:analogs & derivatives; D016133:Polymerase Chain Reaction / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D015203:Reproducibility of Results; D043823:Taxoids
D003520:Cyclophosphamide; D004273:DNA, Neoplasm; D000077143:Docetaxel; D004317:Doxorubicin; D017239:Paclitaxel; D018719:Erb-b2 Receptor Tyrosine Kinases; D043823:Taxoids