Abstract 2109: Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC
Authors: Jang Anne , Feingold Daniel , Watson Mark H. , Pottiez Gwenaël , Guilbaud Rudolf , Dupuis Nicholas Francois 
Published: June 2023

Abstract
Introduction Cytokines are fundamental regulators of immune system processes and are responsible for the differentiation, proliferation, and function of immune cells. With many anti-cancer therapeutic strategies focused on targeting or harnessing the immune system, or components thereof, quantification of circulating cytokines has become important to describe the pharmacodynamic and mechanistic effects of these investigational therapies. To support these efforts, new multiplexed immunoassay panels have been developed to expand the available options for broad spectrum characterization of cytokine profiles from biospecimens. Here we characterize the analytical performance of one such panel based on proximity extension assay technology to measure 45 cytokines in plasma.
Experimental Procedures Plasma samples (n = 27) from patients with non-small cell lung cancer (NSCLC), along with age-matched plasma samples (n = 6) from healthy normal subjects were analyzed using the Olink Cytokine 48 kit. Samples were thawed and processed according to manufacturer instructions for the incubation, extension, and detection on the Olink Signature Q100 instrument. A subset of the screened samples was run in quadruplicate across multiple runs and analyzed on two separate instruments.
Results Of the 45 cytokines assayed, 41 had values above the assay limit of detection in ≥75% of samples analyzed. When compared to the normal human plasma, 26 cytokines were significantly elevated (p-value ≤ 0.05) in plasma from NSCLC subjects, including those known to be associated both with poor (e.g., IL-1β, IL-6) and good prognosis (e.g., IFN-γ). Furthermore, many of the cytokines with significant differences were of low abundance; 13 of the 26 cytokines had mean values ≤ 20.0 pg/mL. For two of the incurred samples which were run across multiple replicates and runs, with mid- to high- levels of cytokines, the median intra-assay CV across all assays was 6.2% with 90% of the assays demonstrating CVs of less than 12%. This only increased to a 7.7% median CV in inter-run comparison with 90% of the assays remaining below 12%. Additional results from the incurred sample analysis will be presented.
Conclusions These results demonstrate that multiplex cytokine analysis, based on proximity extension assays, can be effectively deployed to screen a large number of targets and can uncover clinically relevant changes in low abundant cytokines.
Citation Format: Anne Jang, Daniel Feingold, Mark H. Watson, Gwenaël Pottiez, Rudolf Guilbaud, Nicholas Francois Dupuis. Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2109.