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Clinical Chemistry 49: 219-229, 2003; 10.1373/49.2.219
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(Clinical Chemistry. 2003;49:219-229.)
© 2003 American Association for Clinical Chemistry, Inc.

HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue

Melanie Königshoff1,2, Jochen Wilhelm1, Rainer M. Bohle2, Alfred Pingoud1 and Meinhard Hahn1a

1 Institut für Biochemie, FB 08, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.

2 Institut für Pathologie, FB 11, Justus-Liebig-Universität Giessen, Langhansstrasse 10, D-35392 Giessen, Germany.

aAddress correspondence to this author at: Abteilung Molekulare Genetik, H0700, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Fax 49-6221-424639; e-mail m.hahn{at}dkfz.de.

Background: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods.

Methods: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer.

Results: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies.

Conclusions: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.




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