Abstract B010: A survey of circulating biomarkers in subjects with NSCLC using library-based data independent acquisition mass spectrometry reveals host immune response mechanisms
Authors: Dupuis Nicholas , Vowinckel Jakob , Heinzmann Daniel , Escher Claudia 
Published: February 2019

Background: Identification of circulating biomarkers in cancer has proven utility in applications for early detection, differential diagnosis, predicting pre-treatment response to therapy, and treatment monitoring. More recently, circulating proteomic biomarkers have been evaluated as surrogate endpoints for early indication of benefit for immunotherapies. This last application is especially relevant during immunotherapy development where the optimal endpoint, overall survival (OS), can take longer to mature. Here, we present an unbiased survey of the circulating proteome of subjects with NSCLC to identify candidate biomarkers that may have utility in multiple stages of patient care. Methods: Unbiased, data-independent acquisition (DIA) mass spectrometry was used to analyze plasma samples from subjects with Stage III-IV non-small cell lung cancer (NSCLC, n = 15) and age matched healthy donors (n = 15), enabling simultaneous sequencing and quantification of plasma proteins. Samples were prepared for mass spectrometry and spiked with a panel of standards covering 500 plasma proteins. All samples were analyzed using 1h gradients on a C18 column coupled to a Thermo Scientific Q Exactive HF mass spectrometer. Data were extracted using Spectronaut (Biognosys) with a sample specific spectral library and statistical analysis was conducted to identify disease associated biomarker candidates. Pathway analysis highlights dysregulated biologic functions and predicts upstream regulatory pathways. Results: A protein library was created containing 771 unique proteins. In DIA acquisition, 462 proteins were quantified across all samples. Univariate statistical testing identified 26 dysregulated proteins (20 up-regulated and 6 down-regulated; q-value &gt; 0.05 and log2 fold change &gt; 0.58). Multivariate (PLS-DA) analysis identified c-reactive protein (CRP) and serum amyloid a (SAA1/SAA2), complement C9, S100A8/S100A9, and leucine rich glycoprotein 1 (LRG1) as the most significantly changed proteins across sample groups. Receiver operator analysis (ROC) identified S100A8 and complement C9 as the markers with the greatest diagnostic power at 93% sensitivity and 93% specificity. Significantly enriched pathways include acute phase response, complement system as well as IL-12 and IL-6 signaling. Similarly, upstream activated candidate pathways included STAT3, IL-6, and EZ2H. Conclusions: 26 proteins were identified as candidate biomarkers and reflect the host immune response via acute phase response signaling, innate immune response (complement system), and other proinflammatory stimuli. Several of these markers have been linked to patient outcomes and poor prognosis. Accurate monitoring of these proteins offers the possibility to define surrogate, molecular-based markers with multiple modes of utility.

Citation Format: Nicholas Dupuis, Jakob Vowinckel, Daniel Heinzmann, Claudia Escher. A survey of circulating biomarkers in subjects with NSCLC using library-based data independent acquisition mass spectrometry reveals host immune response mechanisms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B010.