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// Erika Maria Parasido 1,2 , Alessandra Silvestri 1 , Vincenzo Canzonieri 3 , Claudio Belluco 4 , Maria Grazia Diodoro 5 , Massimo Milione 6 , Flavia Melotti 6 , Ruggero De Maria 7 , Lance Liotta 1 , Emanuel F. Petricoin 1,* and Mariaelena Pierobon 1,* 1 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA 2 Department of Experimental Oncology, CRO-National Cancer Institute, Aviano, Italy 3 Department of Pathology, CRO-National Cancer Institute, Aviano, Italy 4 Department of Surgical Oncology, CRO-National Cancer Institute, Aviano, Italy 5 Department of Pathology, Istituto Nazionale Tumori Regina Elena, Roma, Italy 6 Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy 7 Department of Pathology, Sacred Heart Catholic University of Rome, Roma, Italy * These authors have contributed equally to this work Correspondence to: Mariaelena Pierobon, email: // Keywords : intra-tumor heterogeneity, personalized therapy, kinase signaling, laser capture microdissection, reverse phase protein microarray Received : August 16, 2016 Accepted : December 09, 2016 Published : December 15, 2016 Abstract Introduction: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates. Material and methods: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array. Results: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were

This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of c-Kit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT-mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms.

Background: The evaluation of intratumor heterogeneity has become a major topic in the oncological field and it has major implications in the delivery of individualized therapy. While a number of genomic studies have reported significant intratumoral heterogeneity the analysis of the activation of protein kinase-driven signaling networks, which constitute the targets of most of the targeted compounds, has been sparse, or did not focus specifically on the tumor cells. This study evaluated the activation/phosphorylation of the protein signaling networks and a number of major precision medicine-oriented drug target to assess whether the intratumoral heterogeneity could impact patient stratification for targeted treatment in the context of metastatic disease. Methods: 3 independent study sets were used for this analysis: 1) metastatic colorectal cancer (mCRC) liver biopsies were collected from 6 patients; for each biopsy, 2 different regions were microdissected, 2) 18 needle biopsies were collected from 2 different regions of the same lesion of 9 mCRC liver metastases, 3) 7 serial liver biopsies were collected from 3 mCRC before and after treatment. Specimens were frozen upon collection, stored at -80°C and subjected to laser capture microdissection to isolate pure tumor epithelium. Protein pathway activation mapping was performed using reverse phase protein microarray. To assess changes in the activation level of the members of the MAPK and the AKT-mTOR pathway, unsupervised hierarchical clustering analysis and fold change between the different areas of the tumors were evaluated. Results: Hierarchical clustering analysis of the activation of 47 key signaling protein drug targets revealed that the signaling activation of any given protein or pathway was largely the same within a patient's tumor, no matter where the biopsy was obtained, but large interpatient variation was observed. When we evaluated activation of the MAPK and AKT-mTOR pathways, we found that the relative levels of pathway activation were found to be similar in different regions of the tumor, while each patient looked distinct. Conclusions: This first of its kind protein drug target activation analysis of microdissected tumor cells from human metastatic tumors revealed that the activated signaling architecture is largely maintained within the same tumor while each patient's tumor has a distinct patient-specific signaling portrait. The data suggest that interpatient protein drug target activation heterogeneity is much larger than intratumoral heterogeneity and point to the need to determine precision medicine based profiling on a patient-by-patient basis. Citation Format: Erika M. Parasido, Alessandra Silvestri, Claudio Belluco, Vincenzo Canzonieri, Maria Grazia Diodoro, Massimo Milione, Lance A. Liotta, Emanuel F. Petricoin, Mariaelena Pierobon. Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2014-3465