Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model.

Expression of Met, the Hepatocyte Growth Factor receptor, has been shown to have prognostic value in numerous types of cancer including breast, gastric, cervical and head and neck carcinomas. However, traditional analyses of expression have shown variable results and a lack of reproducibility. The AQUA system is a method of quantitative in situ analysis of protein expression that allows the assessment of reproducibility of both antibodies and assay conditions. Here, we illustrate the necessity for antibody validation when assaying the prognostic value of a potential biomarker. Using five antibodies to the intracellular domain of the Met receptor and 10 cell line controls, we quantitatively assess reproducibility of protein expression. We show that many antibodies are not reproducible at a quantitative level from lot to lot or assay to assay, suggesting new criteria for antibody validation. We also build upon past literature addressing the prognostic value of Met in a cohort of 640 cases of invasive breast cancer on a tissue microarray. We show that high levels of expression of nuclear Met, as determined by antibodies to the intracellular domain and defined as nuclear by subcellular compartmental analysis, is associated with shorter disease-specific survival in breast cancer.