Abstract The ability to quantify changes in protein activity in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. We have developed the use of a highly sensitive microfluidic nano-immunoassay (NIA, Nanopro1000) to quantify unphosphorylated, single- and multi- phosphorylated isoforms of proteins in clinical specimens including blood, bone marrow, and fine needle aspirates (FNAs). Using as little as 2 nanoliters of cellular lysate, we can measure over 40 proteomic parameters, and assess changes in proteins that mediate signal transduction, cell cycle progression and apoptosis. Using NIA to resolve different phosphorylated isoforms of individual proteins and directly measure percent phosphorylation has led to new hypotheses and approaches to biomarker development. To develop novel diagnostic biomarkers, we have analyzed MAP Kinase phospho-protein profiles for over 200 specimens collected from patients with hematopoietic and solid tumors. We found that quantifying even subtle differences in protein signaling between tumor calls and adjacent non-tumor tissue can distinguish various malignancies. We will also present preclinical and clinical data using NIA to develop biomarkers of therapeutic activity biologic activity of tyrosine kinase inhibitors (TKIs) in myelodysplastic syndrome and leukemia. One unanticipated finding has been that TKIs might preferentially inhibit or activate specific phospho-isoforms of proteins. Our NIA studies also suggest that dynamic changes in protein signaling may better predict therapeutic outcome than profiles from a single, pre-treatment time point. One example is that the decrease in single phospho-ERK2 (p-ERK2) is associated with response to TKIs in chronic myelogenous leukemia. Notably, measurements of the phospho-isoform at a single time point were not sufficient to distinguish the two groups, which may reflect that individual tumors may have different initial “set-points” for the pathway. In contrast, comparison of proteomic signatures serially sampled from patients before and during treatment can provide valuable information regarding degree of target kinase inhibition and also compensatory signaling that can not be gleaned from analysis of a single time point. Our studies using nanoscale proteomic analysis to profile tumor signatures and characterize biologic response to therapies have led to new potential strategies for developing biomarkers, moving us closer to individualized cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1280. doi:1538-7445.AM2012-1280