Biomarker Development for Cancer Immuno-oncology/Immunotherapy: Simultaneous Digital Counting of Nucleic Acids and Proteins at 800-plex

Abstract

Abstract The ability to measure changes to the DNA, RNA, and protein is crucial to understanding both tumor and immune responses to therapy. NanoString® has pioneered 3D Biology™ – the ability to measure any combination of DNA, RNA, and protein simultaneously using a single detector from a small clinical sample – in order to characterize these complex interactions. Analytes of interest are labeled with fluorescent nucleic acid barcode (via direct hybridization for nucleic acids or antibody conjugated barcodes for protein) and detected via digital counting. This enables quantification of up to 800 analytes from a small sample (50,000 cells for DNA/RNA/protein, or 1–2 5μm FFPE slides for RNA alone). The cancer immunology profiling panel measures 770 mRNAs (unique signatures for 24 infiltrating immune cell types plus extensive immune-signaling pathways) plus 30 key IO proteins. We present two examples of 3D Biology in action. First, a targeted BRAFV600E inhibitor induces expression changes of immune genes which can be detected at both the RNA and protein levels. Second, in work from ongoing collaborations with the Cancer Immunotherapy Trials Network, clinical samples from patients treated with either mono- or combination immunotherapy can be stratified by gene expression. We also show that gene signatures predict presence of immune cell populations with a high degree of concordance to flow cytometry and immunohistochemistry measurements. Multiplexed RNA biomarkers have achieved success in predicting patient response to therapy, e.g. NanoString signatures predict response to pembrolizumab (anti-PD-1). Measuring DNA, RNA, and proteins in unison greatly expands the power of biomarker signatures to characterize responses to immunotherapy.