Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray

Abstract

Programmed Death 1 Ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut-points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index TMA to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index Tissue Microarray (TMA) containing a panel of 10 isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142-FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other 4 assays. The assays for 22C3-FDA, 28-8-FDA, SP263-FDA and E1L3N-LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.

Multiomic analysis and immunoprofiling reveal distinct subtypes of human angiosarcoma

“…mIHC/IF, which allows for staining of up to 6 markers on a single slide, allows a broader range of analysis compared with IHC. The reproducibility of this technique and correlation with conventional IHC have been reported by our group and others for various markers, including some of those used in this study, such as CD8, CD68, FOXP3, and PD-L1 (34)(35)(36)(37). Moreover, mIHC/IF workflow has been partially automated from staining to analysis (38), reducing hands-on time.…”

Multiplex immunohistochemistry/immunofluorescence (mIHC/IF) for PD-L1 testing in triple-negative breast cancer: a translational assay compared with conventional IHC

“…Thus, we embarked on the present study, which used mIHC/IF as an objective, standardised staining and analytic pipeline to overcome interobserver variability. Reproducibility of such a technique has been reported by our group and others for various markers including PD-L1 25 42–44. This approach may also be applicable in other types of cancer treated with immunotherapy that also require PD-L1 scoring on immune cells specifically, such as urothelial carcinoma45 and gastric cancer 46.…”

Transcriptional Spatial Profiling of Cancer Tissues in the Era of Immunotherapy: The Potential and Promise

“…FISH and RNAscope are useful clinical tools for the diagnosis and prognostication of solid and haematological cancers [ 130 , 131 , 132 ]. Newer techniques, such as MERFISH and Visium, have overcome some of the limitations to their multiplexing capacity by enabling bulk transcriptome analysis with an unprecedented level of resolution and sensitivity [ 79 , 133 ]. The increasing accessibility of such techniques is exciting for the field of cancer immunology, as they enable the discovery of novel biomarkers that serve to predict responses to immunotherapy and permit personalized treatment approaches based on the heterogeneity of their unique TME [ 108 , 109 ].…”