Abstract The MAP Kinase pathway is commonly dysregulated in cancer. We have developed the use of a highly sensitive microfluidic nano-immunoassay system (NIA) to perform detailed analysis of ERK and MEK activation in hematopoietic and solid tumors. We recently reported use of NIA for measurement of proteins in as little as 4 nanoliters of lysate from preclinical and clinical lymphoma and leukemia specimens (Fan et al, Nature Medicine 2009). Now, we present results measuring specific isoforms of MAP Kinase proteins in cells sampled from patients with solid tumors and Myelodysplastic Syndrome (MDS). First, we developed tissue collection, handling and processing protocols to produce reliable NIA results in a small number of cells obtained by minimally invasive techniques. We collected over 100 blood buffy coat and fine needle aspirate (FNA) specimens from normal controls and patients with MDS or solid tumors. Using a single antibody that recognizes both the phosphorylated and unphosphorylated isoforms of ERK, we can determine not only relative levels of phosphorylated isoforms of ERK protein, but also calculate the percent phosphorylation of ERK in each clinical specimen. We have developed a similar method to quantify MEK isoforms. NIA revealed that adenocarcinoma, squamous cell carcinoma, renal cell carcinoma, and MDS samples could be distinguished from non-tumor specimens based upon different patterns of phosphorylated ERK isoforms. To determine if signaling changes can be detected by NIA upon treatment of cells with novel targeted therapies, human leukemic TF1 cells were treated with a polo-1 kinase (PLK-1) inhibitor, ON01910 (Onconova, Inc.). We found the surprising result that only 5% of ERK1 and ERK2 were found to be phosphorylated in the TF1 cell line, whereas 45% and 90% of MEK1 and MEK2 were phosphorylated, respectively. Additionally, NIA analysis revealed a 20% decrease in the percent of phosphorylated MEK2 after treatment with ON01910. Taken together, our studies demonstrate that NIA can be used to identify specific changes in MAPK signaling pathway that distinguish normal from malignant cells as well as quantitatively measure the response of malignant cells to a targeted therapeutic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B178.