Phosphoprotein Quantitation Via Quantum Dot Immunofluorescence and Multispectral imaging Using Liquid-Based Cytology System: Adapting a Clinical Laboratory Platform for Biomarker Studies
J Bodo, L Durkin, ED Hsi. Cleveland Clinic, Cleveland, OH
Background: Recently we have assessed feasibility of quantitative analysis of phosphoproteins in paraffin embedded fixed tissues. In this study we evaluated conditions required in a liquid based cytology (LBC) system that will preserve phosphoproteins (PPs) for IF quantitation.
Design: Lymphoma cell lines treated with hydrogen peroxide to modulate PP levels of pERK1/2, pS6, and pSTAT3 served as a model system. An FDA-approved LBC system (ThinPrep™) with CytoLyt™ (wash/transfer buffer) and PreservCyt™ (preservative/fixative) served as the test clinical laboratory model platform. Imaging was performed using a multispectral imaging system (Nuance™). In order develop QD IF absolute quantitation, we applied an ELISA assay to determine absolute concentrations of pERK.
Results: First, we evaluated the effect of exposure of cells to PreservCyt™ on phospho ERK level and demonstrated good correlation (R= 0.98, P= 0.01) compared to Western Blot quantitation. Next, we tested the effects of pre-treatment of cells with CytoLyt™ (15 min) prior to exposure to PreservCyt™. This resulted in a marked loss (71% on average) of QD IF signal in all analyzed phosphoproteins. Time course studies for exposure to PreservCyt™ indicate that overnight (18 hrs) exposure is optimal for QD IF staining with 80% decrease of pERK signal after 48 hours compared to 18 hrs incubation. To optimize signal we subsequently studied the effect of adding 2% paraformaldehyde to PreservCyt™ and found an approximately 1.4 fold increase in pERK signal at 18 hrs. Interestingly we found optimal QD IF signal with best morphologic preservation by sequencing 10% buffered formalin fixation (30 min) followed by 18 hrs incubation in PreservCyt™ and storage of prepared slides in 96% EtOH. As a proof or principle for an absolute quantitation assay, we performed pERK Elisa and QD IF on paired samples and demonstrate that values of QD IF correlate with ELISA-based measurements. (R= 0.98, P= 0.02).
Conclusions: These preliminary results indicate feasibility of adapting a clinical LBC platform for cytology based in situ quantitative phosphoprotein assays. These studies have implications for 1) monitoring of targeted therapies (such as novel kinase inhibitors) using a minimally invasive FNA technique during early phase clinical trials and 2) predictive theranostic assays by ex vivo demonstration of active pathways that are the target of specific therapies. Further proofs of concept studies are ongoing.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 71, Wednesday Afternoon