A Quantitative Multiplex Protein Assay for Analysis of Intracellular Signaling Pathways in Sarcoma Samples
M Bui, W Brazelle, J Gemmer, S Altiok. Moffitt Cancer Center, Tampa, FL
Background: Patients with advanced metastatic sarcomas have poor prognosis with a disease-free survival at 5 years less than 10%. New and better agents are warranted for the treatment of these diseases. The modern revolution in molecular biology has led to characterization of biologically important signaling pathways in cancer, the elements of which have focused drug development efforts towards novel, targeted therapies, and to identify specific molecular biological profiles for each tumor. The greatest challenge in clinical studies is to identify subgroups of patients who will benefit from different types of targeted treatments. The purpose of this study is to develop quantitative pharmacodynamic assays to predict and assess tumor response to targeted therapeutics in sarcoma patients.
Design: Tumor tissues corresponding to approximately four core biopsy samples were collected from resection samples in accordance with IRB requirements. Cellular extracts were prepared and the expression levels and phosphorylation status of intracellular signaling proteins as well as markers of apoptotic cell death were analyzed in by using the Luminex xMAP system. The levels of phospho proteins were normalized to their total expression levels.
Results: The quantitative multiplex assays enabled simultaneous analysis of activation status of the extracellular signal regulated MAP kinase (Raf/Mek/Erk), PI3K/Akt and mTOR/p70S6 pathways and of apoptotic protein caspase 3 in as small as 3 micrograms of cellular proteins. Western blot methods corroborated the results of multiplex analysis. The sarcoma cells obtained from patients with spindle cell sarcoma, myxoid chondrosarcoma, synovial sarcoma, myxoid spindle cell sarcoma, osteosarcoma and myxoid liposarcoma showed significant variation in their signaling profiles, suggesting predictor of patient response to targeted therapeutics before the initiation of treatment.
Conclusions: Collectively, these results demonstrate that the components of the signaling pathways can be quantitatively analyzed in sarcoma samples to assess the efficacy of the therapy in vivo. This approach may also open the avenues to explore the molecular mechanisms leading to drug resistance in individual patients, which may lead to design of combination therapies to block aberrantly activated pathways in individual patient's tumor. We will apply the methods described here to clinical samples collected from patients with extremity and retroperitoneal sarcomas treated with an mTOR inhibitor in an upcoming clinical trial.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 248, Tuesday Morning