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BackgroundMost patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC.MethodsWe initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively.ResultsUsing continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK–/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted pConclusionsThis work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC.

Introduction: Most patients with advanced NSCLC fail to develop durable responses from PD-1 axis blockade, and more robust predictive biomarkers are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition across multiple NSCLC cohorts. Methods: We first used the GeoMx Digital Spatial Profiling (DSP) system in a discovery cohort of 56 NSCLC patients treated with PD-1 axis inhibitors at Yale (USA). Pre-treatment tumors were represented in a tissue microarray (YTMA471) and analyzed using a 71-plex primary antibody panel. Proteins were measured from three molecularly defined tissue compartments: tumor (CK+), leukocytes (CD45+/CD68-), and macrophages (CD68+). We used quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external validation of identified predictors, we assessed pre-treatment whole tissue sections from a cohort of 128 NSCLC patients treated with single-agent PD-1 axis inhibitors at the Hospital 12 de Octubre (Madrid, Spain) using DSP (39-plex protein panel, measured from CK+ and CD45+ compartments). In addition, we analyzed two immunotherapy untreated cohorts to address prognostic significance: YTMA423 (Yale, USA; n = 252) and CIMA-CUN (UNAV, Spain; n = 124), using QIF and DSP respectively. Results: Using continuous log-scaled data, we found 5 markers independently associated with progression-free survival (PFS) in the tumor compartment (including PD-L1, p = 0.002). Among the novel candidate predictors, tumor cell CD44, a marker of pluripotency and stemness, was associated with longer PFS (multivariate HR = 0.63, p= 0.002). Using QIF, we orthogonally validated that CD44 expression in the tumor compartment was associated with longer PFS (p 1.5, p Conclusion: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in NSCLC. NSCLCs with high CD44 expression in epithelial cells may be associated with an immune contexture primed for higher response to immune checkpoint blockade. Further studies are needed to understand the interplay between CD44+ cancer stem cell phenotype and mechanisms of immune evasion. Citation Format: Myrto Moutafi, Magdalena Molero, Sandra Martinez-Morilla, Javier Baena, Ioannis Vathiotis, Niki Gavrielatou, Laura Castro, Gorka Ruiz de Garibay, Vera Adradas, Daniel Orive, Karmele Valencia, Alfonso Galvo, Luis M. Montuenga, Santiago Ponce, Kurt Schalper, Luis Paz-Ares, David L. Rimm, Jon Zugazagoitia. Spatially resolved proteomic profiling identifies tumor cell CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2028.