Cellular engagement and interaction in the tumor microenvironment predict non-response to PD-1/PD-L1 inhibitors in metastatic non-small cell lung cancer

Immune checkpoint inhibitors (ICI) with anti-PD-1/PD-L1 agents have improved the survival of patients with metastatic non-small cell lung cancer (mNSCLC). Tumor PD-L1 expression is an imperfect biomarker as it does not capture the complex interactions between constituents of the tumor microenvironment (TME). Using multiplex fluorescent immunohistochemistry (mfIHC), we modeled the TME to study the influence of cellular distribution and engagement on response to ICI in mNSCLC. We performed mfIHC on pretreatment tissue from patients with mNSCLC who received ICI. We used primary antibodies against CD3, CD8, CD163, PD-L1, pancytokeratin, and FOXP3; simple and complex phenotyping as well as spatial analyses was performed. We analyzed 68 distinct samples from 52 patients with mNSCLC. Patients were 39–79 years old (median 67); 44% were male and 75% had adenocarcinoma histology. The most used ICI was atezolizumab (48%). The percentage of PD-L1 positive epithelial tumor cells (EC), degree of cytotoxic T lymphocyte (CTL) engagement with EC, and degree of CTL engagement with helper T lymphocytes (HTL) were significantly lower in non-responders versus responders (p = 0.0163, p = 0.0026 and p = 0.0006, respectively). The combination of these 3 characteristics generated the best sensitivity and specificity to predict non-response to ICI and was also associated with shortened overall survival (p = 0.0271). The combination of low CTL engagement with EC and HTL along with low expression of EC PD-L1 represents a state of impaired endogenous immune reactivity. Together, they more precisely identified non-responders to ICI compared to PD-L1 alone and illustrate the importance of cellular interactions in the TME.