Abstract A26: Elevations of intracellular kinases AKT1, GSK3β, JNK1, JNK2, and ERK2 in primary breast cancer tissue quantified with a novel multipanel of single-molecule immunoassays
Authors: Estis Joel , Hsu Jenny CY , Todd John A. , Lu Quynh Anh , Zieske Lynn , Agee Sara J. 
Published: June 2012

Abstract
Purpose: The MAPK and PI3K signaling cascades are critical pathways in malignant transformation, tumor progression and multidrug resistance (MDR) of human cancers. Elucidation of the relative contributions of distinct kinases is made difficult due to many factors, including prevalence of multiple isoforms, posttranslational modifications, synergistic activation and pathway crosstalk. Clinical investigations into the role of phosphorylation of specific kinases are further hindered by the lack of ultrasensitive assay technology with requisite isoform- and phophorylation-state specificity. Improved assays are necessary to determine clinical relevance and potential diagnostic utility with a high degree of specificity.
Methods: Adjacent normal and primary breast cancer tissue lysates were analyzed using a novel panel of ten immunoassays for quantifying the total and phosphorylated forms of AKT1, GSK3β, JNK1, JNK2 and ERK2. The ultrasensitive assay panel was developed for use with the Erenna® Immunoassay System (Singulex), which uses single molecule detection technology. Analytical sensitivity, interassay precision, linearity, and specificity were determined and compared to corresponding ELISA assays. The novel assay panel was validated in HeLa and Jurkat cell lysates, region of linearity was determined, and phospho-specificity of each assay was confirmed. All assays were validated for use in human plasma, cell culture lysates, and human tissue specimens.
Results: Testing in matched breast cancer tissue lysates showed that all total kinase concentrations (tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2) were found to be elevated in primary tumors compared to surrounding adjacent normal tissue, however concentrations of phosphorylated forms (pAKT1, pGSK3β, pERK2, pJNK1 and pJNK2) were not. All ten novel assays displayed exceptional linearity (R2=0.99) and precision (%CV 4.1 – 9.6), and were significantly more sensitive than the corresponding ELISA assay method based upon sample endpoint dilutions. The detection limits of the tAKT1, pAKT1, tGSK3β, pGSK3β, pERK2, tJNK2 and pJNK2 assays were all ≤0.3 pg/mL, while the detection limit of tERK2 was &lt; 2pg/mL, tJNK1 was 4.5 pg/mL, and pJNK1 was 7 pg/mL. The LLoQ of each assay in the panel was sufficient for quantification from a minimum of approximately 100 to 200 cells, with exception of the total JNK1 assay, which required a minimum of approximately 800 cells in order to be quantifiable.
Conclusions: We show that significant elevations in total kinase concentrations of tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2 can be quantified in primary tumors compared to surrounding adjacent tissue using a panel of isoform- and phosphorylation-specific single molecule immunoassays. We have further demonstrated that assays in this panel can quantify analytes from as little as 100 cells, and exhibit superior analytical performance over ELISA based methods. Thus, these novel assays greatly improve upon pre-existing ELISA technology that is limited by assay sensitivity, providing a new tool for clinical investigations of the MAPK and PI3K signaling cascades.
Citation Information: Clin Cancer Res 2010;16(14 Suppl):A26.