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Background

Immune checkpoint inhibitors (ICIs) have transformed lung cancer treatment, yet their effectiveness appears restricted to certain patient subsets. Current clinical stratification based on PD-L1 expression offers limited predictive value. Given the mechanism of action, directly detecting spatial PD1–PD-L1 interactions might yield more precise insights into immune responses and treatment outcomes.

Methods

We applied a second-generation in situ proximity ligation assay (PLA) to detect PD1–PD-L1 interactions in diagnostic tissue samples from 16 different cancer types, a tissue microarray from 352 surgically resected early-stage non-small cell lung cancer (NSCLC) patients, and finally diagnostic biopsies from 242 advanced NSCLC patients with and without ICI treatment. RNAseq analysis was used to identify potential resistance mechanisms.

Results

In the early-stage NSCLC, only about half of the cases with detectable PD-L1 and PD1 expression showed PD1–PD-L1 interactions, with significantly lower levels in EGFR-mutated tumors. Interaction levels varied across cancer types, aligning with reported ICI response rates. In ICI-treated NSCLC patients, higher PD1–PD-L1 interactions were linked to complete responses and longer survival, outperforming standard PD-L1 expression assays. Patients who, despite high PD1–PD-L1 interactions, did not respond to ICIs, showed additional expression of stromal immune mediators (EOMES, HAVCR1/TIM-1, JAML, FCRL1).

Conclusion

Our study proposes a diagnostic shift from static biomarker quantification to assessing active immune pathways, providing more precise ICI treatment. This functional concept applies to tiny lung biopsies and can be used to further immune checkpoints. Accordingly, our results indicate concerted ICI resistance mechanisms, highlighting a need for combination diagnostics and therapies.