BIOM-22. CLINICAL ASSESSMENT OF SIGNALING PATHWAY ACTIVITY IN GLIOMA

Abstract

Abstract Despite advances in our understanding of tumor biology, many targeted therapies for diffuse glioma have failed without an understanding of why. Improved patient stratification and assessment of biologic activity in window of opportunity clinical trials will address this failure. The clinically validated, robust biomarkers available, however, are limited. Moreover, evidence-based criteria for biospecimen acquisition are lacking. To address this need we investigated the preanalytic variables that impact the assessment of PI3K/AKT/mTOR and MAPK signaling activity at single cell resolution. To replicate the conditions in window of opportunity clinical trials, tumor tissue from a murine model for glioblastoma (GBM) was utilized and the impact of cold ischemia time (CIT), slide storage duration and temperature on detection of key phospho-proteins was determined. We then validated our findings using human glioma samples acquired in the operating room. To assess the PI3K/AKT/mTOR signaling, phosphorylation of proteins at three nodes of the pathway were investigated. Phosphorylated-PRAS40 (p-PRAS40) is upstream of mTOR and phosphorylated-RPS6 (p-RPS6) and phosphorylated-4EBP1 (p-4EBP1) are downstream of mTOR. Detection of cells expressing p-RPS6 was the most robust with no significant alterations up to 6hrs cold ischemia time (CIT). p-4EBP1 signal was unaltered up to 20min CIT, however, decreased at 2h and 6h (p< 0.001). The detection of p-PRAS40 was variable at 20min and showed a decreased tendency at 2h and 6h. For p-RPS6, p-4EBP1, and p-PRAS40 the assessment of protein intensity per cell was highly variable at all time points analyzed (p< 0.05 at 20min, 2h, and 6h). Thus, for assessment of the PI3K/AKT/mTOR pathway in diffuse glioma using clinical samples, the detection of p-RPS6 is the most stable when time elapsed between sample acquisition and downstream processing (CIT) is long, while p-4EBP1 requires short (≤20 min) delays and p-PRAS40 is more variable.