Abstract 415: A single molecule detection system for comprehensive analysis of cancer related cellular pathways

The purpose of this study was to evaluate the system capabilities of a new single-molecule detection platform capable of both genomic and proteomic analysis of cellular pathways using very small amounts of tumor material. The system has 4-color optics, single-fluorophore detection capability, localization of molecules to within a 20nm area, a flow cell with an area of 940 mm2 and the ability to detect > 109 molecules on the surface. NSCLC cell lines were cultured and untreated or treated with the EGFR tyrosine kinase inhibitor erlotinib. Cells were harvested and used to generate protein lysates, isolate mRNA and gDNA. Cellular molecules were covalently attached to an epoxysilane-coated surface on the flow cell and then repeatedly probed 10-15 times with labeled antibodies and oligonucleotides to detect, count and quantitate proteins, protein phosphorylation levels, mRNAs, fusion transcripts, and DNA mutations. We detected protein phosphorylation changes for EGFR, ERK, MET and MEK comparable to that observed by traditional western blots; however, the system required only 0.5 to 2 cell equivalents of protein lysate, containing sub pico-Molar levels of protein. We demonstrated robust and statistically significant detection of 2-fold changes in protein levels across 3 slides, in triplicate lanes and performed over 3 non-consecutive days by 3 operators (Tukey-Kramer tests). We also detected 8 mRNAs, with no PCR amplification required, in a multiplexed format with relative levels similar to that observed with TaqMan® qPCR tests. We demonstrated protein detection at ~ 100% recovery compared to an ELISA test and nucleic acid recovery at ~ 30% with no amplification. We also detected exon 19 deletions in EGFR, point mutations at L858R, T790M in EGFR and V600E in BRAF, at 0.5% minor allele levels, by using an oligonucleotide ligation assay off-chip and then attaching the ligated product to the surface. mRNA levels were detected using 10-20 cell equivalents of RNA. We believe the system will enable comprehensive analysis of cancer-related pathways, requiring only a few cells, to help decipher cellular pathway activity induced by driver mutations and consequently help with selection of efficacious drugs tailored for specific individuals based on comprehensive molecular analysis. Citation Format: Manohar R. Furtado, Rixun Fang, Norman Burns, Rebekah Burich, Catherine Le, Soumya Ivaturi, Rob Hartlage, Ed Zizminskas, Jim Jahncke, Philip C. Mack, Bryan P. Staker. A single