ERBB2 GENE AMPLIFICATION DETECTED BY FLUORESCENT DIFFERENTIAL POLYMERASE CHAIN-REACTION IN PARAFFIN-EMBEDDED BREAST-CARCINOMA TISSUES

For quantificative determination of ERBBZ gene amplification in archiv al human carcinoma specimens we have developed a rapid, non-radioactiv e approach, which is based on the differential polymerase chain reacti on (FCR) and fluorescent DNA technique. Sequences from the ERBB2 gene and from a single-copy reference gene were amplified simultaneously by PCR, in which one of each primer pair was fluorescently labelled. PCR products were separated by polyacrylamide gel electrophoresis in an a utomated DNA sequencer and directly quantified after laser activation and emission scanning using appropriate software. This fluorescent dif ferential polymerase chain reaction (fd-PCR) method was used for quant ificative determination of ERBB2 gene amplification in 195 formalin-fi xed, paraffin-embedded breast carcinoma tissues. ERBB2 gene amplificat ion was found in 52 (26%) of these tumors and correlated significantly with tumor size, absence of estrogen receptor (ER) and pS2 expression , but not with absence of progesterone receptor (PR) or presence of ep idermal growth factor receptor (EGF-R) expression, lymph-node metastas es or grading. In univariate analysis, ERBB2 gene amplification showed no significant correlation with clinical outcome, either in the whole population or in the subgroup defined by positive axillary lymph-node metastases. However, within the node-negative subgroup, patients with ERBB2 gene amplification had significantly decreased relapse-free sur vival and overall survival (p < 0.05). The fd-PCR assay is a valuable tool for determination of amplification of ERBB2 gene as well as furth er oncogenes. In this way, more detailed information about individual tumor biology may be acquired by a routine assay. (C) 1995 Wiley-Liss, Inc.