Abstract 4691: Evaluation of the NanoString’s Digital Spatial Profiling (DSP) technology in formalin-fixed paraffin embedded (FFPE) cell line mixtures, PBMCs and non-small cell lung cancer (NSCLC) tissues
Authors: Rusbuldt Joshua James , Cash-Mason Tanesha , Tian Shaozhou , VanSchoiack Alison , Liang Yan , Rao Chandra , Smirnov Denis 
Published: August 2019

Abstract
Evaluation of the NanoString’s Digital Spatial Profiling (DSP) Technology in Formalin-Fixed Paraffin Embedded (FFPE) cell line mixtures, PBMCs and Non-Small Cell Lung Cancer (NSCLC) tissues
Purpose: The complex nature of the tumor requires multiplexed, quantitative analysis of tissues which is challenging using immunohistochemistry (IHC). Several novel approaches (including NanoString Digital Spatial Profiling (DSP) Technology) were developed to enable multiplexed analysis of tissues. Here we report on a comparative evaluation of the GeoMx™ DSP platform including a comparison to IHC.
Methods: Performance of DSP methodology was assessed using Raji and CEM/C1 cell line mixtures, as well as healthy donor PBMCs that had been evaluated using flow cytometry for expression of the targeted markers: CD4, CD8, CD68, CD14, CD3, CD45, CD45RO, CD20 and GZMB. DSP technology was compared with IHC by measuring T-cells markers (CD3, CD4, CD8), a monocytic marker (CD68) as well as PD-L1 expression in five FFPE NSCLC tumor specimens. Serial sections were stained by IHC, hematoxylin and eosin (H&E) and regions of interests (ROIs) were selected to sample areas with varied expression of each of the markers (qualified as low, medium and high). Selected ROIs were then assayed on the GeoMx DSP platform in a blinded fashion using a 39-plex immune panel that included CD3, CD4, CD8, CD68 and PD-L1 antibodies and DSP counts were compared to qualitative IHC scoring.
Results: Cell line mixture experiments demonstrated that protein counts were highly correlated to the fractions of cell lines expressing a given marker (r=0.90 - 0.94). The assay also showed good concordance with flow cytometry measurements (r=0.49-0.51 between flow cytometry MFIs and NanoString counts for PMBCs). DSP counts related well to the IHC staining patterns, as higher DSP measurements were detected in ROIs classified as high based IHC staining intensity for the markers investigated. As expected, counts were lower for markers such as CD8 and PD-L1 that were also classified as low expressing by IHC in the analyzed NSCLC tumor samples. Exploratory analysis of co-expression of all 39 markers included in the DSP panel in various areas of the tumors suggested that DSP technology can be used to perform detailed characterization of various areas of the tumor.
Conclusions: Our evaluation affirms that NanoString’s GeoMx DSP platform is a promising option for multiplexed analysis of tumor tissues. DSP technology produced measurements comparable to those obtained with flow cytometry and IHC for the evaluated markers. These data show that DSP technology can collect high-parametric proteomics data from FFPE tissue samples which can help to study the complex biology of tumor microenvironment.
Citation Format: Joshua James Rusbuldt, Tanesha Cash-Mason, Shaozhou Tian, Alison VanSchoiack, Yan Liang, Chandra Rao, Denis Smirnov. Evaluation of the NanoString’s Digital Spatial Profiling (DSP) technology in formalin-fixed paraffin embedded (FFPE) cell line mixtures, PBMCs and non-small cell lung cancer (NSCLC) tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4691.