Abstract 3621: High-plex immune marker spatial profiling quantitation by NanoString Digital Spatial Profiling technology and quantitative immunofluorescence
Authors: Toki Maria I. , Wong Pok Fai , Kluger Harriet , Liu Yuting , Merritt Chris , Ong Giang , Warren Sarah , Beechem Joseph M. , Rimm David L. 
Published: August 2018

Abstract
Background: Quantitative immunofluorescence (QIF) offers the advantage of multiple target measurement on a single slide, but is limited by the number of fluorescence channels. NanoString's Digital Spatial Profiling (DSP)* can detect and quantify immune markers at higher multiplex with spatial resolution within specific regions of interest on FFPE tissue. Here, we compare NanoString DSP to automated QIF (AQUA), for immune marker compartment specific measurement. Additionally, we assess their agreement on the prognostic value of commonly used biomarkers in Non-Small Cell Lung Cancer (NSCLC). Finally, we explore the predictive value of a 30-plex panel of immune markers on two cohorts of treated patients.
Methods: NanoString DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are UV cleaved and quantified on the nCounter platform. The comparator for this technology was the AQUA method of QIF. We retrospectively examined a NSCLC cohort of over 100 patients for prognostic markers and two treated patient cohorts for associations between immune markers expression and response to treatment, all in tissue microarray format. The treated cohorts included an Epidermal Growth Factor Receptor Tyrosine kinase inhibitor (EGFR TKI) treated NSCLC cohort and a melanoma immunotherapy (ITx) treated cohort.
Results: Multiple immune markers (CD3, CD4, CD8, CD20, PD-L1) were assessed and a high correlation was found between NanoString DSP counts and QIF scores, when the measurements were performed in the same ROIs (tumor or stroma). The prognostic value of the immune markers tested was concordant between the two assays with high expression of CD3 assessed by either assay showing a statistically significantly better overall survival (OS). For the EGFR TKI treated NSCLC cohort none of the immune markers was correlated with response to treatment or favorable outcome. However, in the ITx treated melanoma patients, PD-L1, PD-1, CD68, CD3, CD8A and b2-microglobulin tumor expression predicted response to treatment. Each marker also predicted better outcome, but only CD8A was an independent predictive marker of prolonged survival.
Conclusion: NanoString DSP offers the capacity of highly multiplexed immune marker measurements on selected compartments. It shows high concordance with AQUA, which was further validated by comparing prognostic significance. The pilot study of the Melanoma ITx cohort also illustrates the potential to simultaneously evaluate a range of markers and possibly construct new predictive signatures based on a cohort represented by very small tissue samples. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
Citation Format: Maria I. Toki, Pok Fai Wong, Harriet Kluger, Yuting Liu, Chris Merritt, Giang Ong, Sarah Warren, Joseph M. Beechem, David L. Rimm. High-plex immune marker spatial profiling quantitation by NanoString Digital Spatial Profiling technology and quantitative immunofluorescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3621.