Spatial Proteome Analysis Identifies Lymphocyte CD44 as a Biomarker Associated with SBRT Resistance in Early-Stage Non-Small Cell Lung Cancer

Abstract

To discover and validate spatially-resolved protein markers associated with resistance to SBRT in early-stage NSCLC patients. We initially evaluated a discovery cohort of 44 early-stage NSCLC patients treated with SBRT as first-line treatment at the Shandong cancer hospital. Using the GeoMx DSP system, 71 proteins were measured in five molecular compartments (tumor, leukocyte, lymphocyte, macrophage, and stroma) on pre-treatment samples. Candidate biomarkers were orthogonally validated with the Gem AQUA method of quantitative immunofluorescence (QIF). For internal independent cohort validation, we assessed pre-treatment samples derived from 150 NSCLC patients who receive radiotherapy. We further analyzed 100 radiotherapy untreated patients with operable NSCLC to address prognostic significance. Using continuous log-scaled data, we identified CD44 expression in the lymphocyte compartment (CD3+) as a novel predictor of poor progression-free survival (PFS) (multivariate HR = 7.323, p = 0.0079) and overall survival (OS) (multivariate HR = 8.65, p = 0.028) in the discovery set. High CD44 expression in the tumor compartment (pan-cytokeratin, CK+) predicted significantly shorter OS (multivariate HR = 2.208, p = 0.0212), with no significant difference in PFS. We validated by QIF that lymphocyte CD44 levels were associated with resistance to SBRT therapy and prognostic for poor outcomes. Using QIF in an independent radiotherapy treated cohort, we validated that CD44 levels in the lymphocyte compartment were associated with poor PFS and OS. High lymphocyte cell CD44 was not prognostic in non-radiotherapy-treated cohort. Using DSP data, intratumoral regions with elevated lymphocyte cell CD44 expression showed prominent upregulation of CD127, ARG1 and VISTA in the discovery Cohort. In conclusion, we identified and validated lymphocyte cell CD44 as a biomarker indicative of resistance to SBRT or radiotherapy in patients with NSCLC. Further evaluation is warranted to address the predictive value of lymphocyte cell CD44 in multi-institutional studies and clinical trials.