Nanoscale approaches to define biologic signatures and measure proteomic response to targeted therapies in hematologic and solid tumors
Authors: Alice C. Fan, Jessica L. Dermody, Christina Kong, Nancy Zhang, Liwen Xu, John P. Renschler, Mathias W. Orban, Ben Varasteh, Kunju Sridhar, Yasodha Natkunam, Stephen E. Coutre, Peter Greenberg, Dean W. Felsher
Journal: Clinical Cancer Research (2010)

Abstract The ability to quantify changes in protein activation in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. A barrier to analyzing proteomic response in patients is the large amount of specimen required to make measurements at each time point. To overcome this barrier, we have developed the use of a highly sensitive microfluidic nano-immunoassay system (NIA) to quantify unphosphorylated, single- and multiple phosphorylated isoforms of proteins in patient specimens collected by minimally invasive blood draws or fine needle aspirates. We first developed tissue collection, handling and processing protocols to produce reliable NIA results using as little as 4 nanoliters of lysate. We next developed assays to measure over 40 proteomic parameters, including changes in proteins that mediate RAS and MAP Kinase signaling, cell cycle and apoptosis. To investigate the diagnostic potential of NIA, we have collected over 300 specimens from normal controls and patients with hematopoietic and solid tumors. We found that phosphoprotein profiles in tumor cells could distinguish tumor from normal cells. Further, patient tumors could be grouped based upon different patterns of percent ERK and MEK phosphorylation. In a clinical trial setting, we have now used NIA in conjunction with multicolor intracellular flow cytometry (FACS) to monitor changes in phospho-profiles in patients with receiving novel agents for hematopoietic cancers. In a prospective study of a novel cell cycle inhibitor in myelodysplastic syndrome (MDS), we have used NIA to measure changes in MAPK and cell cycle proteins in patient leukocytes sampled throughout serial time points during the study. In a prospective study of atorvastatin in patients with non-Hodgkin's lymphoma, we have used NIA and FACS to distinguish three patterns of signaling changes in patient tumor cells, monocytes, and T cells. First, pathways that were initially activated in tumor cells decreased upon treatment with atorvastatin, with up to a 70% decrease in phospho-STAT3 and phospho-STAT5. Second, pathways that were initially suppressed in tumor cells (including phospho-PLC), normalized upon treatment. Third, expression levels of proteins in apoptotic pathways, including p38, increased upon atorvastatin treatment. Finally, changes in signaling associated with atorvastatin were found to be cell specific: effects on tumor cells were distinct from effects on non-tumor T cells and monocytes. Our studies demonstrate that NIA and FACS are complementary proteomic methods that can be used to quantify changes in cell signaling and distinguish responses in different cellular populations from very small clinical specimens sampled during therapeutic interventions. Nanoscale analysis of patient specimens is a feasible and promising approach for the development of new diagnostic markers and biomarkers of therapeutic activity. This talk is also presented as Poster A21.