Abstract
432 Background: Novel agents that inhibit kinases in the hypoxia and MTOR pathways can achieve response rates of 30-60% in renal cell carcinoma (RCC); however, protein biomarkers to diagnose RCC and guide treatment have not been identified. Methods: We developed the use of two high-throughput, nanoscale immunoassays to profile hypoxia and downstream signaling in clinical specimens from patients with RCC. These automated technologies minimize errors due to manual variability, and make 96 independent measurements per overnight experiment. We used size-based protein separation (Sally Instrument, Protein Simple) to quantify proteins using antibody-specific detection, normalized to loading control. Next, we used a nano-immunoassay with charge-based separation (NIA, Nanopro1000 Instrument, Protein Simple) to distinguish multiple charged modifications of individual proteins, and measure relative ratios of individual unphosphorylated and phosphorylated isoforms. Results: We first optimized assays for proteins in MAPK, PI3K and STAT pathways, and loading controls, to analyze frozen surgical specimens and ex-vivo fine needle aspirates (FNA’s) performed immediately following nephrectomy. We analyzed more than 200 FNA’s from solid tumors, comparing RCC with paired adjacent non-tumor tissue, and other epithelial malignancies. Basal MAPK signaling of tumors varied across 3 logs of expression. Unique to NIA, we analyzed percent phosphorylation and resolved differences in single phosphorylations. Subtle differences in basal MAPK signaling between tumor calls and adjacent non-tumor tissue can distinguish RCC from other malignancies. Lastly, we prospectively collected and analyzed blood peripheral mononuclear cells from 20 RCC patients before and during treatment with standard targeted therapies. Kinase inhibitors can preferentially inhibit or activate specific protein phospho-isoforms. Conclusions: Our studies demonstrate that rapid and quantitative nanoproteomic profiling in very small amounts of clinical specimen may accelerate translational studies for novel diagnostic and predictive biomarkers.
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