LAUSR

Background: Protein expression in formalin-fixed, paraffin-embedded (FFPE) tissue is routinely measured by Immunohistochemistry (IHC) on only one protein, or with quantitative fluorescence (QIF) on a handful of proteins on a single section. NanoString’s Digital Spatial Profiling (DSP)* technology can detect and quantify proteins at significantly higher multiplex (currently 30 plex, potential for up to 800 plex) with spatial resolution using molecular digital color-coded “barcodes”, within specific regions of interest on FFPE tissue. Here, we compare Nanostring DSP technology to automated QIF (AQUA), for accurate and reproducible measurement of protein expression on a continuous scale. Additionally, using the multiplexing potential of Nanostring technology, we did a pilot study to assess cold ischemic time as a variable to monitor tissue quality by assessment of epitope degradation in Non-Small Cell Lung Cancer (NSCLC). Methods: The DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos with a UV Photocleavable linker that can be counted on the nCounter™ platform. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are released via UV mediated linker cleavage. Free oligos are captured via microcapillary fluidics into a microtiter plate and then quantitated on the nCounter. The comparator for this technology was the AQUA method of QIF. We examined a breast cancer tissue microarray with a range of HER2 expression, and a NSCLC time to fixation standardization array with timepoints from 20 min to 48 hrs. Results: Multiple markers were assessed and a high correlation was found between Nanostring DSP counts and QIF scores (R²>0.7), when the measurements were performed in the same region of interest (defined by cytokeratin expression). The dynamic range of DSP exceeded the quantification range of QIF (nearly 4 logs vs about 2 logs). When the 28 protein markers’ expression was compared at different fixation timepoints, most were found to be stable over different cold ischemic timepoints. Two markers, including phospho-ERK (Extracellular Signal-Regulated Kinases) and phospho-GSK (Glycogen Synthase Kinase) showed epitope loss as a function of delay to fixation (R²=0.0064 and R²=0.05 respectively). Conclusion: The Nanostring DSP is a reproducible method with the capability of highly multiplexed measurements of protein expression on a field of view averaged basis. It shows high concordance with the AQUA method of QIF, an extensively validated technique for protein quantification. For the first time, the high-plex capacity of DSP allows inclusion of markers that are sensitive to time to fixation as an intrinsic control for tissue quality. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Maria I. Toki, Chris Merritt, Giang Ong, Joseph M. Beechem, Daniel E. Carvajal-Hausdorf, Yalai Bai, David Rimm. Validation of novel high-plex protein spatial profiling quantitation based on NanoString9s Digital Spatial Profiling (DSP) technology with quantitative fluorescence (QIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3810. doi:10.1158/1538-7445.AM2017-3810