Abstract 2239: Bone metastasis protein signaling pathway mapping in seven primary tumor types via Reverse Phase Protein Microarray
Authors: Chiechi Antonella , Espina Virginia A. , Novello Chiara , Magagnoli Giovanna , Benassi Mariaserena , Picci Piero , Petricoin Emanuel F. , Liotta Lance A. 
Published: February 2011

Abstract
We have developed a workflow using protein kinase signal pathway mapping technology to identify key cell signaling signatures in bone metastasis samples dependent and independent of primary tumor histotype. There is little known about molecular alterations during the metastatic process, especially to the bone, as well-preserved frozen tissue sources are limiting. The activation state of kinase-driven signal networks contains important information about disease pathogenesis and the state of kinase-associated therapeutic targets. Proteomic molecular profiling can provide a means to identify activated pathways or specific protein endpoints that may be potential drug targets or molecules useful for diagnosis/prognosis in bone metastasis pathology.
The data set consists of metastatic bone tissue (n=166) and matched normal tissue (n=115) with corresponding clinical data. 142 bone metastasis were from different types of primary carcinomas: 13 head & neck, 45 urogenital, 16 gastroenteric, 14 breast, 25 lung, 7 melanoma and 22 unknown carcinomas. 24 bone metastasis were from primary sarcomas.
The objectives are a) identify activated kinase pathway interconnections in each bone metastasis from different primary tumors to identify predictive/prognostic biomarkers, and b) correlate this pathway information with the matched normal tissue.
Our workflow overcomes 3 major limitations to analyzing bone tissue: 1) the presence of contaminating blood in the tissue, 2) the need to completely homogenize the bone, and 3) the need to verify tissue histomorphology for each sample. Many bone metastasis tissue samples are lytic and embody a large amount of blood. In order to overcome the blood contamination problem, we developed an original method using the DNA concentration in each sample to normalize protein expression data, since red blood cells are devoid of a nucleus. Data normalization by DNA staining was very stable, consistent with sample cell number, and did not cross-react with RNA or proteins.
Samples were completely homogenized using CryoPrep™ and Adaptive Focus Acoustic™ (AFA) technology by Covaris, with very good protein extraction efficiency. A portion of every sample was decalcified, paraffin embedded and Hematoxylin/Eosin stained to verify the tumor cell percentage in the tissue (&gt;90%). Reverse phase protein arrays were used to quantitatively map 150 key proteins involved in bone metabolism, tumor-host interactions, hormone response, growth-proliferation, stress-inflammation, and adhesive-cytoskeletal proteins.
Our workflow overcomes the difficulties in processing and analyzing bone metastasis samples. We obtained a proteomic profile that enables elucidation of molecular mechanisms leading to bone metastasis formation and its clinical manifestations. This is a fundamental step for developing new effective therapeutic strategies to treat bone metastasis pathology.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2239.