Research Papers:

Determinants of variability of five programmed death ligand-1 immunohistochemistry assays in non-small cell lung cancer samples

Ross A Soo _, Joey Sze Yun Lim, Bernadette Reyna Asuncion, Zul Fazreen, Maria Cynthia Herrera, Mohd Feroz Mohd Omar, Nguyen Hoang Diem Phuong, Ju Ee Seet, Benhur Amanuel, Barry Iacopetta, David Byrne, Shona Hendry, Stephen Fox and Richie Soong

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Oncotarget. 2018; 9:6841-6851. https://doi.org/10.18632/oncotarget.23827

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Ross A. Soo1,2,3, Joey Sze Yun Lim2, Bernadette Reyna Asuncion2, Zul Fazreen2, Maria Cynthia Herrera2, Mohd Feroz Mohd Omar2, Nguyen Hoang Diem Phuong2, Ju Ee Seet4, Benhur Amanuel5,6, Barry Iacopetta3, David Byrne7, Shona Hendry7, Stephen Fox7,8 and Richie Soong2,9

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

2Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore, Singapore

3School of Surgery, University of Western Australia, Perth, Australia

4Department of Pathology, National University Hospital, Singapore, Singapore

5Department of Anatomical Pathology, Queen Elizabeth II Medical Centre, Perth, Australia

6School of Pathology and Laboratory Medicine, The University of Western Australia, Perth, Australia

7Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia

8Department of Pathology, The University of Melbourne, Melbourne, Australia

9Department of Pathology, National University of Singapore, Singapore, Singapore

Correspondence to:

Ross A. Soo, email: [email protected]

Keywords: programmed death ligand-1; immunohistochemistry; non-small cell lung cancer; immunotherapy

Received: June 29, 2017     Accepted: December 23, 2017     Published: January 02, 2018


Programmed death ligand-1 (PD-L1) expression as determined by immunohistochemistry (IHC) is potentially predictive of clinical outcome. The aim of this study was to assess the concordance of reported PD-L1 IHC assays and investigate factors influencing variability. Consecutive sections from 20 non-small cell lung cancers (NSCLCs) comprising resection, core biopsy, cytology and pleural fluid samples underwent IHC with 5 different antibody/autostainer combinations: 22C3/Link48, 28-8/BOND-MAX, E1L3N/BOND-MAX, SP142/BenchMark and SP263/BenchMark. PD-L1 RNA levels were assessed using RNAscope. The frequency of positive cases using scoring thresholds from clinical trials was 72%, 33%, 61%, 56%, and 33% for the 5 IHC protocols respectively, and 33% for RNAscope. Pairwise agreement on the classification of cases as positive or negative for PD-L1 expression ranged from 61%-94%. On a continuous scale, the lowest correlation was between 28-8/BOND-MAX and SP142/BenchMark (R2=0.25) and highest was between 22C3/Link48 and E1L3N/BOND-MAX (R2=0.71). When cases were ordered according to tumor cell (TC)%, a similar ranking of cases across IHC protocols could be observed, albeit with different quanta and limits of detection. Single-slide OPAL 7-color fluorescence IHC analysis revealed a high degree of co-localization of staining from the 5 PD-L1 antibodies. Using SP142 antibody in a BOND-MAX protocol led to increased TC% quanta, while retaining a similar ranking of samples according to TC%. The results of this study highlight tumor PD-L1 status can vary significantly according to IHC protocol. Protocol-dependent staining intensities and nominated thresholds for positivity contribute to this variability, while the antibody used appears to be less of a factor.

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