Evaluation of the quantitative analytical methods real-time PCR for HER-2 gene quantification and ELISA of serum HER-2 protein and comparison with fluorescence in situ hybridization and immunohistochernistry for determining HER-2 status in breast cancer patients

Background: HER-2 status is generally determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Both methods are only semiquantitative, require a tumor sample, and can be difficult to reproduce. We compared these methods with 2 quantitative approaches, one measuring HER-2 gene copy number in tissue by real-time quantitative PCR (qPCR), and the other measuring shed HER-2 protein in serum by ELISA in patients with metastatic disease. Methods: We analyzed 52 cases of metastatic breast cancer for which both serum collected at the diagnosis of metastasis and stored primary breast tumor specimens were available. The within- and between-run imprecision of real-time qPCR and ELISA were evaluated according to Clinical and Laboratory Standards Institute (formerly known as NCCLS) recommendations. Concordance among the 4 methods was assessed by calculating the kappa statistic and its 95% confidence interval (95% CI). Results: The CVs for within- and between-run imprecision were both < 10% with qPCR and ELISA. There was good agreement of results between qPCR and IHC (kappa = 0.81; 95% Cl, 0.64-0.99), qPCR and FISH (kappa = 0.77; 95% Cl, 0.58-0.96), ELISA and IHC (kappa 0. 65; 95% Cl, 0.41-0.89); and ELISA and FISH (kappa 0. 69; 95% Cl, 0.46-0.92). Conclusions: Measurements of HER-2 gene expression by qPCR and of serum HER-2 protein by ELISA are highly reproducible approaches for determining HER-2 status in metastatic breast cancer. In addition, ELISA eliminates the need. for biopsy. (c) 2005 American Association for Clinical Chemistry.