An urgent clinical goal is to identify molecular networks associated with subpopulations of cancer patients who may respond individually to molecular targeted inhibitors. Current molecular targeted therapeutics is directed at protein kinases and/or their phosphorylated substrates. Therefore, measurement of this new class of phosphoprotein signal pathway epitopes in tumor biopsy samples is crucial for individualizing molecular targeted therapies. Phosphoprotein antigen epitopes are not adequately preserved by formalin fixation and paraffin embedding, and freezing of tissue is very expensive and compromises diagnostic accuracy. We propose the advanced development and clinical validation of an innovative and transformative technology for preserving tissue phosphoproteins and diagnostic histomorphology for clinical cancer molecular profiling. Applying knowledge gained under an NIH R21 funded study, we created a novel tissue preservation chemistry that stabilizes all classes of phosphoproteins, is compatible with paraffin embedding, while maintaining complete diagnostic histomorphology, and fully preserving critical diagnostic immunohistology (IHC) antigens including Estrogen Receptor, Progesterone Receptor, HER2, and Ki-67. These IHC antigens are not preserved by special research fixatives used for tissue RNA preservation. Our new non-formalin tissue preservative, termed Biomarker and Histology Preservative (BHP) can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment. At the time of procurement, tissue can be immersed directly in the new fixative and processed into a paraffin block for routine diagnosis, obviating the need for costly freezing during shipping or storage. BHP offers the potential for substantial improvements over conventional formalin fixation. In the present application we propose the blinded clinical validation of our novel preservation chemistry in community hospital settings, utilizing a team of international pathologists for validation. The goal of the project is one-step paraffin block stabilization of all classes of cellular phosphoproteins, diagnostic histomorphology, and diagnostic immunohistochemistry antigens, while at the same time maintaining full diagnostic morphology equivalent or superior to standard formalin fixation. We will collect fresh surgical tissue, under informed consent, covering a broad variety of organs and cancer histology to develop an archive of 150 cases of matched paraffin and frozen specimens. We will measure 100 validated phosphoprotein epitopes spanning membrane, cytoplasmic and nuclear compartments from extracted paraffin sections using Reverse Phase Protein Microarray (RPMA) and Laser Capture Microdissection (LCM) technology. Following objective independent validation by diagnostic pathologists, this transformative technology will be ready for widespread clinical and research use. Adoption of the technology would mean that only one diagnostic paraffin block could be used for all classes of molecular profiling rather than the current requirement for multiple blocks. This would increase diagnostic accuracy while substantially reducing costs.

Public Health Relevance

Our technology for preserving proteins, at the time of collection, will enable analysis of molecular targeted kinase inhibitors for individualized therapy, while considerably reducing healthcare costs, and increasing diagnostic accuracy. Our new non-formalin tissue preservative can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1-SRLB-5 (J1))
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Chuaqui, Rodrigo F
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George Mason University
Other Basic Sciences
Schools of Arts and Sciences
United States
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Pierobon, Mariaelena; Ramos, Corinne; Wong, Shukmei et al. (2017) Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer. Clin Cancer Res 23:4919-4928
Staunton, Lisa; Tonry, Claire; Lis, Rosina et al. (2017) Pathology-Driven Comprehensive Proteomic Profiling of the Prostate Cancer Tumor Microenvironment. Mol Cancer Res 15:281-293
Spreafico, Filippo; Bongarzone, Italia; Pizzamiglio, Sara et al. (2017) Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread. Oncotarget 8:46177-46190
Pin, Elisa; Stratton, Steven; Belluco, Claudio et al. (2016) A pilot study exploring the molecular architecture of the tumor microenvironment in human prostate cancer using laser capture microdissection and reverse phase protein microarray. Mol Oncol 10:1585-1594
Gallagher, Rosa I; Espina, Virginia (2014) Reverse phase protein arrays: mapping the path towards personalized medicine. Mol Diagn Ther 18:619-30
Holmes, Frankie Ann; Espina, Virginia; Liotta, Lance A et al. (2013) Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling. BMC Res Notes 6:507
Chiechi, Antonella; Novello, Chiara; Magagnoli, Giovanna et al. (2013) Elevated TNFR1 and serotonin in bone metastasis are correlated with poor survival following bone metastasis diagnosis for both carcinoma and sarcoma primary tumors. Clin Cancer Res 19:2473-85
Federici, Giulia; Gao, Xi; Slawek, Janusz et al. (2013) Systems analysis of the NCI-60 cancer cell lines by alignment of protein pathway activation modules with ""-OMIC"" data fields and therapeutic response signatures. Mol Cancer Res 11:676-85
Silvestri, Alessandra; Calvert, Valerie; Belluco, Claudio et al. (2013) Protein pathway activation mapping of colorectal metastatic progression reveals metastasis-specific network alterations. Clin Exp Metastasis 30:309-16
Golubeva, Yelena; Salcedo, Rosalba; Mueller, Claudius et al. (2013) Laser capture microdissection for protein and NanoString RNA analysis. Methods Mol Biol 931:213-57

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