MORPHEUS-lung: Biomarkers and clinical response to atezolizumab + bevacizumab + stereotactic body radiotherapy in patients with metastatic non-small cell lung cancer

Purpose: Results from the MORPHEUS-Lung (NCT03337698) study suggest that atezolizumab+bevacizumab+stereotactic body radiotherapy (SBRT) was associated with numerically improved efficacy outcomes vs docetaxel (control) in immune checkpoint inhibitor-exposed patients with metastatic non-small cell lung cancer. We report exploratory analyses to identify potential biomarkers associated with this clinical response. Experimental Design: Tumor samples were analyzed for differential gene expression and pathway/immune subset gene signatures by single cell RNA-sequencing and further validated with bulk RNA-sequencing. Results: A total of 58 patients were included in the biomarker-evaluable population (atezolizumab + bevacizumab + SBRT (Atezo+Bev+Radio), n = 26; docetaxel control, n = 32). In the discovery analysis comparing patients with clinical benefit (CB) versus non-clinical benefit (non-CB) at baseline, tumor-resident memory CD8⁺ T cells (CD8 T RM ) were significantly enriched in the CB group (P = 0.027) accompanied by an increased T-cell receptor clonality, indicating clonal expansion of tumor-reactive T cells (Gini index P = 0.018). PDCD1, the target molecule of immune checkpoint therapy, was highly expressed in CD8 T EX , supporting their responsiveness to atezolizumab treatment. Furthermore, the CB group showed increased abundance of CXCL10⁺ and FOLR2⁺ macrophages, suggesting enhanced myeloid-T cell crosstalk. CD8 T RM with high tumor-recognition potential exhibited strengthened antitumor activity through CXCL-mediated interactions with CXCL10⁺ macrophages, which expressed PD-L1 at the highest level. Following Atezo+Bev+Radio treatment, CD8 T RM -identified as predictive biomarkers-maintained a persistent tumor-reactive state, whereas CD8 TEMRA cells showed increased cytotoxic function likely driven by radiation exposure. Atezo+Bev+Radio treatment also induced a marked increase in FOLR2⁺ and FABP4⁺ macrophages in the post-treatment group. FOLR2⁺ macrophages interacted with CD8 T cells expressing ICOS via the ICOS-ICOSL signaling pathway, promoting T cell-mediated antitumor immunity. In contrast, FABP4⁺ macrophages engaged Treg cells expressing TIGIT through the PVR-TIGIT signaling axis, thereby contributing to immune suppression. Integration of the data revealed T cell- and macrophage-related gene signatures validated in an independent cohort, correlating with survival benefit after atezolizumab + bevacizumab + SBRT treatment. Conclusions: These results suggest that activation of T cells and macrophages are associated with a favorable clinical response Atezo+Bev+Radio. Moreover, CD8 T RM , CD8 T EX , and CXCL10 + macrophages may serve as potential biomarkers of response to Atezo+Bev+Radio treatment and potentially aid in tailored, precision medicine for individual patients.