PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

SIMPLE SUMMARY: Assessing some diagnostic tests can be extremely difficult, even for highly trained clinicians. We have shown in the past that by using an advanced computer software program (QuPath), applied to high resolution images of patient tissue samples, we can assist pathologists in their assessment of a routine test that determines immunotherapy treatment. We also showed that by using a different testing method in the laboratory, called multiplexing, which detects several proteins at once rather than just one alone, we are subjectively more confident in the patient’s reported score. Here, we show that multiplexing is comparable to the traditional method, and that we can also easily apply our computer software tools to extract very specific information from the patient samples, which we are unable to do using the traditional laboratory method. We believe these tools can support pathologists to triage patient cases for this important diagnostic test. ABSTRACT: Multiplex immunofluorescence (mIF) and digital image analysis (DIA) have transformed the ability to analyse multiple biomarkers. We aimed to validate a clinical workflow for quantifying PD-L1 in non-small cell lung cancer (NSCLC). NSCLC samples were stained with a validated mIF panel. Immunohistochemistry (IHC) was conducted and mIF slides were scanned on an Akoya Vectra Polaris. Scans underwent DIA using QuPath. Single channel immunofluorescence was concordant with single-plex IHC. DIA facilitated quantification of cell types expressing single or multiple phenotypic markers. Considerations for analysis included classifier accuracy, macrophage infiltration, spurious staining, threshold sensitivity by DIA, sensitivity of cell identification in the mIF. Alternative sequential detection of biomarkers by DIA potentially impacted final score. Strong concordance was observed between 3,3’-Diaminobenzidine (DAB) IHC slides and mIF slides (R(2) = 0.7323). Comparatively, DIA on DAB IHC was seen to overestimate the PD-L1 score more frequently than on mIF slides. Overall, concordance between DIA on DAB IHC slides and mIF slides was 95%. DIA of mIF slides is rapid, highly comparable to DIA on DAB IHC slides, and enables comprehensive extraction of phenotypic data and specific microenvironmental detail intrinsic to the sample. Exploration of the clinical relevance of mIF in the context of immunotherapy treated cases is warranted.