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 immunohistochemistry 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% CI, 0.64-0.99), qPCR and FISH (kappa = 0.77; 95% CI, 0.58-0.96), ELISA and IHC (kappa = 0.65; 95% CI, 0.41-0.89); and ELISA and FISH (kappa = 0.69; 95% CI, 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.