Measurement of Oncoproteins in Primary Hematopoietic Malignancies Pre- and Post-Therapy Using a Nano-Immunoassay System (AACR 2008)

Oncoprotein quantification in clinical specimens is important for the diagnosis of specific hematopoietic malignancies as well as the development and monitoring of effective therapies that target oncoproteins. Current protein detection methods require large samples, precluding routine serial tumor sampling to assess changes in oncoprotein levels. Here we demonstrate the use of a nano-immunoassay system (FireflyTM system) to distinguish between patient specimens of Burkitt's vs. Follicular lymphoma by characterizing patterns of MYC and BCL2 expression. Changes in the expression and activation of a variety of onco/signaling proteins including ERK, MEK, STAT and JNK in malignant hematopoietic (CML) cells and patient samples before and after treatment with therapeutic agents that impact oncogenic signaling pathways are also shown. The expression levels of the different proteins were measured with high sensitivity in samples as small as 400 cells. A key benefit of this technology is that it separates protein isoforms based on its isoelectric point. Using this assay, we were able to distinguish and quantify the phosphorylated and non-phosphorylated forms of each protein with a single antibody. Thus, we have developed a novel technique which can precisely evaluate the activity levels of signaling proteins in oncogenic pathways from very small samples.