HER2 (human epidermal growth factor receptor 2) is an important biomarker whose status plays a pivotal role in therapeutic decision making for patients with breast cancer and in determining their clinical outcomes.To ensure the accuracy and reproducibility of HER2 assays by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), a reliable standard for monitoring assay sensitivity and assessing methodological variation is needed.A workshop held at NIST in 2002 addressed this need by reaching a consensus to create cell lines as reference materials for HER2 testing.Breast carcinoma cell lines SK-BR-3 and MCF-7 were characterized quantitatively by IHC with chicken anti-HER2 IgY antibody and by FISH with biotinylated BAC DNA probes, both using quantum dots as detectors.Formalin-fixed and paraffin-embedded (FFPE) cell blocks were prepared and tested for suitability as candidate reference materials by IHC and FISH using commercially available reagents.Results from the IHC and FISH experiments were also compared with those from laser scanning cytometry (LSC)and real-time PCR, respectively.MCF-7 cells had normal gene copy number and very low protein expression for HER2; whereas, SK-BR-3 contained ~10-fold copies of the gene and exhibited~15-fold protein expression as compared with MCF-7.FFPE cells showed similar results to the SK-BR-3 cells.SK-BR-3 and MCF-7 are suitable for use as candidate reference materials in quality control of HER2 testing.Coupled with the associated assay platforms, they provide valuable controls for quantitative measurement of HER2 amplification and expression in breast cancer specimens, irrespective of the antibody/probe or detector used.