Use of Nano-ImmunoAssay to Generate Rapid, Quantitative Nanoscale Proteomic Profiling of the Hypoxia Pathway in Renal Cell Carcinoma Clinical Specimens (Stanford University, ASCO 2012)

Novel inhibitors of the hypoxia pathway [VEGF, PDGF] achieve response rates of 30-57% in renal cell carcinoma (RCC); yet threshold levels of targets and downstream signaling proteins have not been identified as biomarkers to guide treatment. Methods: To profile hypoxia proteins in RCC clinical specimens, we have developed the use of automated nanoscale immunoassays for charge-based protein separation (NIA, NanoPro 1000) and charge-based protein separation (Simple Western, Sally). To decrease the amount of tissue and invasive procedures required to obtain cells for analysis, we optimized assays to profile specimens acquired by fine needle aspiration (FNA). Results: We used Simple Western to quantify proteins of the MAPK (ERK1, ERK2, pERK1, pERK2, MEK2), PI3K (S6, GSK3b, AKT2, pan-AKT) and STAT pathways (p-STAT5) and loading controls (tubulin, HSP-70) in more than 200 FNA's from solid tumors including RCC. Profiles can be completed overnight after receiving the specimen. Unique to NIA, we also analyzed percent phosphorylation and resolved differences in even a single phosphorylation in FNA specimens, allowing us to group tumors based upon different patterns of phosphorylation and percent phosphorylation. Conclusions: Rapid and quantitative nanoproteomic profiling in very small amounts of clinical specimen is enabling translational studies for novel diagnostic and predictive biomarkers.