Rational design cancer drugs that target specific molecular lesions have shown great promise when appropriate patient subsets are identified. Because of the therapeutic costs and specificity of patient subsets in which such drug may be effective, universally available physical standards and reference materials become increasingly important when the drug, its molecular target and molecular stratification of patients are well-defined. Approximately one-third of breast cancers have over expression of HER2/ERBB2/neu gene and cognate HER2 receptor. For such patients, trastuzumab (a humanized monoclonal therapeutic, or trade name Herceptin) may be effective. Because of the cost of drug and the risk of side effects, accurate diagnostic measurements of HER2 gene (by FISH) and receptor (by IHC) are crucial. To address benchmarking of FISH, IHC and other diagnostic modalities, NIST has developed background materials and methods for HER2 metrology. We created the HER2 BAC clone as FISH probe1, and designed anti-HER2 chicken antibody for IHC study. To improve signal quantitation1, 2, we have incorporated quantum dot fluorophores into measurement of HER2 analytes at both the FISH and IHC levels of analysis in batch model reference material preparations with DNA and antibody probes and methods developed at NIST. To further improve HER2 metrology, we have applied z-plane corrections to fluorescent signals with 3D quantitative analysis of quantum dot fluorescence supplemented by deconvolution algorithms. These methods and materials will go into interlaboratory validations soon. This model system for rational design cancer drugs illustrates a general approach to improving metrology of complex molecular genetic diagnostic tests through quantitative technology development in the process of generating stable, highly-certified fixed biological substrates coordinated for measurements at the gene, mRNA, protein and receptor levels of analysis. (Supported in part by NIST HER2 SRM and the Office of Women’s Health, NIH). References 1. Yan Xiao and Peter E. Barker, (2004) Nucleic Acids Research, 32 (3), e28 2. Yan Xiao, William G. Telford, J. Christopher Ball, Laurie E. Locascio and Peter E. Barker, (2005) Nature Methods, 2(10), 723.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]