PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry

Ju-Yoon Yoon; Rakesh Nayyar; Graeme Quest; Dana Pabedinskas; Prodipto Pal; Ming-Sound Tsao; Joerg Schwock; Hyang-Mi Ko

doi:10.1016/j.lungcan.2020.07.013

PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry

Lung Cancer. 2020 Oct:148:55-61. doi: 10.1016/j.lungcan.2020.07.013. Epub 2020 Aug 1.

Authors

Ju-Yoon Yoon¹, Rakesh Nayyar², Graeme Quest³, Dana Pabedinskas⁴, Prodipto Pal⁵, Ming-Sound Tsao⁵, Joerg Schwock⁵, Hyang-Mi Ko⁶

Affiliations

¹ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² CytoQuest, Toronto, Ontario, Canada.
³ Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.
⁴ Division of Pathology, University Health Network, Toronto, Ontario, Canada.
⁵ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Division of Pathology, University Health Network, Toronto, Ontario, Canada.
⁶ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Division of Pathology, University Health Network, Toronto, Ontario, Canada. Electronic address: hyangmi.ko@uhn.ca.

PMID: 32799091
DOI: 10.1016/j.lungcan.2020.07.013

Abstract

Objectives: Pathologists encounter several challenges with programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) tests in malignant effusions, including lineage specification (distinction between carcinoma vs. immune and mesothelial cells), background staining, sample fixation issues and inter-observer variability. We explored flow cytometric (FC) quantification of PD-L1 expression in malignant pleural effusions of lung adenocarcinoma patients as an alternative, automated, and objective quantification method compared to PD-L1 IHC.

Materials and methods: We examined 23 malignant pleural effusions of TTF-1-positive adenocarcinoma were subjected to FC with a panel of antibodies against CD45, CD3, CD200, EpCAM, D2-40 (podoplanin), and PD-L1 (clone MIH1). The PD-L1 gate was established using fluorescence-minus-one (FMO) isotype controls. Lineage-specific PD-L1 surface expression was quantified and the FC tumor proportion score (TPS) was assessed. PD-L1 IHC was performed on cell block sections using Dako PD-L1 IHC 22C3 pharmDx assay and assessed by two cytopathologists blinded to the FC PD-L1 TPS.

Results: FC analysis allowed for the distinction between carcinoma cells (CD45^-/EpCAM⁺/D2-40-), leukocytes (CD45⁺/EpCAM-/D2-40-) and mesothelial cells (CD45-/EpCAM-/D2-40⁺). FC PD-L1 TPS ranged from 0% to 77 %, while the 22C3 IHC PD-L1 TPS ranged from 0% to 97 %. The FC and IHC TPS values correlated positively (R = 0.8). Best concordance was observed when FC was performed and cell blocks were generated in parallel (R = 0.99). FC also allowed for simultaneous PD-L1 quantification in mesothelial and T-cells. PD-L1 expression on mesothelial cells ranged from 0% to 90.9 %, which also correlated positively with IHC TPS (R = 0.54). PD-L1 expression on T-cells was limited (0.1-2.9 %).

Conclusion: FC permits rapid, objective and lineage-specific PD-L1 surface expression quantification with limited specimen manipulation. The FC and IHC concordance was impacted by different antibody clones being used, but the positive correlation suggests potential clinical utility, especially in malignant effusion specimens.

Keywords: Adenocarcinoma; Effusion; Flow cytometry; Immunohistochemistry; Lung; PD-L1.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenocarcinoma of Lung* / diagnosis
B7-H1 Antigen
Flow Cytometry
Humans
Lung Neoplasms* / diagnosis
Pleural Effusion, Malignant* / diagnosis

Substances

B7-H1 Antigen
CD274 protein, human