The PI3K/AKT/mTOR pathway is an important signaling axis that is perturbed in majority of cancers. Biomarkers such as pS6RP, GLUT1, and tumor FDG uptake are being evaluated in patient stratification for mTOR pathway inhibitors. In the absence of a clear understanding of the underlying mechanisms in tumor signaling, the biomarker strategy for patient stratification is of limited use. Here, we show that no discernible correlation exists between FDG uptake and the corresponding Ki67, GLUT1, pS6RP expression in tumor biopsies from patients with head and neck cancer. Correlation between GLUT1 and pS6RP levels in tumors was observed but elevated pS6RP was noticed even in the absence of concomitant AKT activation, suggesting that other downstream molecules of PI3K/AKT and/or other pathways upstream of mTOR are active in these tumors. Using an ex vivo platform, we identified putative responders to rapamycin, an mTOR inhibitor in these tumors. However, rapamycin did not induce antitumor effect in the majority of tumors with activated mTOR, potentially attributable to the observation that rapamycin induces feedback activation of AKT. Accordingly, treatment of these tumors with an AKT inhibitor and rapamycin uniformly resulted in abrogation of mTOR inhibition-induced AKT activation in all tumors but failed to induce antitumor response in a subset. Phosphoproteomic profiling of tumors resistant to dual AKT/mTOR inhibitors revealed differential activation of multiple pathways involved in proliferation and survival. Collectively, our results suggest that, in addition to biomarker-based segregation, functional assessment of a patient's tumor before treatment with mTOR/AKT inhibitors may be useful for patient stratification. Cancer Res; 73(3); 1118-27. Ó2013 AACR.

Colorectal cancer (CRC) continues to have a dismal prognosis (overall five year survival is 11%). Majority of patients with CRC are either over-treated or under-treated as there is no technology in use for predicting treatment outcome. Currently, monoclonal antibodies that target EGFR have been approved for the treatment of metastatic CRC. However, only 10% to 20% of patients with metastatic CRC clinically benefit from anti-EGFR therapy. This indicates that molecular alterations other than EGFR are associated with their response. The aim of the present study is to use Oncoprint- our integrated platform technology that combines functional assays with molecular profiling to predict the clinical outcome to anti-cancer agents including anti-EGFR therapies. Forty tumors were obtained from patients with primary CRC and local metastasis with due informed consent. Tumors were sectioned and cultured in customized 96 well plates coated with CRC specific extracellular matrix (ECM) in the presence of their respective autologous ligands and were treated with anti-EGFR agent. Pharmacodynamic analysis of anti-EGFR agents was determined by phospho-EGFR and phospho-ERK immunohistochemistry. Tumor cell viability was assessed by WST, cellular proliferation was measured by Ki-67 and cell death was assessed by TUNEL and activated caspase-3. Tumor cell content and tumor/stroma ratio was evaluated using histopathological analysis. Mutations of KRAS/BRAF pathway and global microarray data were used to understand the molecular basis of response and non-response to anti-EGFR in these patient tumors. Functional read-out from Oncoprint platform showed that 30% of these tumors are responsive to anti-EGFR treatment. Molecular profiling of these tumors showed that they were wild type for K-RAS and B-RAF. Microarray data also suggest that RAS gene signature is a predictor of response. Mutation of either KRAS (codon 12/13/61/146) or BRAF (V600E) confers non-response to anti-EGFR treatment. However, wild type KRAS/BRAF was observed in ∼40% of non-responders. RAS gene signature was conspicuously absent in these patients. Clinical follow-up of these patients is under progress. Collectively these findings indicate that there are factors other than K-RAS mutation that may be indicative of the true response or non-response of anti-EGFR treatments under clinical setting. Oncoprint platform may be able to predict the true clinical response of anti cancer therapies including anti-EGFR treatments. Further, molecular profiling of individual tumor along with the efficacy analyses by Oncoprint would be beneficial for the identification of driver mutations and the selection of appropriate treatment options for these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3191. doi:1538-7445.AM2012-3191