Targeting of the programmed cell death protein (PD-1)/programmed death-ligand 1 (PD-L1) axis with checkpoint inhibitors has changed clinical practice in non-small cell lung cancer (NSCLC). However, clinical assessment remains complex and ambiguous. We aim to assess whether digital image analysis (DIA) and multiplex immunofluorescence can improve the accuracy of PD-L1 diagnostic testing. A clinical cohort of routine NSCLC patients reflex tested for PD-L1 (SP263) immunohistochemistry (IHC), was assessed using DIA. Samples of varying assessment difficulty were assessed by multiplex immunofluorescence. Sensitivity, specificity, and concordance was evaluated between manual diagnostic evaluation and DIA for chromogenic and multiplex IHC. PD-L1 expression by DIA showed significant concordance (R² = 0.8248) to manual assessment. Sensitivity and specificity was 86.8% and 91.4%, respectively. Evaluation of DIA scores revealed 96.8% concordance to manual assessment. Multiplexing enabled PD-L1+/CD68+ macrophages to be readily identified within PD-L1+/cytokeratin+ or PD-L1-/cytokeratin+ tumor nests. Assessment of multiplex vs. chromogenic IHC had a sensitivity and specificity of 97.8% and 91.8%, respectively. Deployment of DIA for PD-L1 diagnostic assessment is an accurate process of case triage. Multiplex immunofluorescence provided higher confidence in PD-L1 assessment and could be offered for challenging cases by centers with appropriate expertise and specialist equipment.
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