Abstract LB-275: Measuring concordance of CD8 and PD-L1 expression in non-small cell lung cancer between a novel multiplex immunofluorescence assay and a brightfield laboratory developed test
Authors: Shitole Shravani , Johnson Monique , Hebert Courtney , Buell Jamie , Ly Amy , Downing Sean R. 
Published: September 2020

Abstract
The quantitative assessment of PD-L1 protein expression has been widely used in the clinic to predict response to immune checkpoint therapy in non-small cell lung cancer (NSCLC) patients. The conventional method of assessment is the manual quantification (scoring) of PD-L1 positive tumor cells by a certified pathologist on samples stained by immunohistochemistry (IHC). To improve the accuracy of the PD-L1 scoring, we assessed the use of digital image analysis tools to accurately quantify the positive tumor cells and correlate the data with the conventional manual scoring method used in the clinical setting by both brightfield and multiplex immunofluorescence assays. Thus, the aim of this study was to evaluate the digital analysis scoring in NSCLC samples stained by IHC and by the ImmunoVUE™ PD-L1 multiplex assay and to correlate the results with manual scoring as well as assess the concordance in the digital scoring using the two assays.
The ImmunoVUE PD-L1 multiplex assay is currently being validated for clinical use by showing concordant results in manual scoring of PD-L1 and CD8 in NSCLC samples with the multiplex assay compared to the IHC laboratory developed test staining in terms of precision, accuracy, and reproducibility. Digital analysis was performed using the HALO software on the manually scored images stained with IHC and the ImmunoVUE PD-L1 multiplex assay. For the digital analysis, the samples were segmented into tumor and non-tumor regions using random forest classification. Positive cells were quantified using the CytoNuclear algorithm for IHC images and the HighPlex FL algorithm was used to quantify images stained with the ImmunoVUE PD-L1 multiplex assay. Total tumor cells and percentage of PD-L1+ tumor cells and CD8+ T cells associated with the tumor were obtained and compared to the manual PD-L1 and CD8 scoring.
Digital analysis of the individual samples stained by IHC or by ImmunoVUE PD-L1 multiplex showed no significant differences compared to the manual scoring in terms of total tumor cell count or the percent of PD-L1+ tumor cells and CD8+ T cells associated with tumor. Coefficients of correlation were greater than 0.9 when comparing manual scoring to digital analysis. Additionally, the digital analysis results obtained from IHC stained slide was concordant with an ImmunoVUE PD-L1 multiplex stained slide from the same sample showing correlation in terms of positive cell counts. The correlation for PD-L1 between brightfield LDT and mIF was measured at 0.8 while that for CD8 was 0.99.
This study shows that digital image analysis can provide an accurate method of PD-L1 and CD8 analysis and is comparable with the manual scoring method using IHC staining. Digital image analysis can be used on samples stained with IHC or with the ImmunoVUE PD-L1 multiplex assay and can be used for assessment of PD-L1 and CD8 positivity in NSCLC patient samples. Lastly, the ImmunoVue assay appears to be concordant with an established LDT.
Citation Format: Shravani Shitole, Monique Johnson, Courtney Hebert, Jamie Buell, Amy Ly, Sean R. Downing. Measuring concordance of CD8 and PD-L1 expression in non-small cell lung cancer between a novel multiplex immunofluorescence assay and a brightfield laboratory developed test [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-275.