False-negative programmed death-ligand 1 immunostaining in ethanol-fixed endobronchial ultrasound-guided transbronchial needle aspiration specimens of non-small-cell lung cancer patients
Koomen, Bregje M; Vreuls, Willem; de Boer, Mirthe; de Ruiter, Emma J; Hoelters, Juergen; Vink, Aryan; Willems, Stefan M
(2021) Histopathology, volume 79, issue 4, pp. 480 - 490
(Article)
Abstract
AIMS: Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol
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prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation. METHODS AND RESULTS: FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results. CONCLUSION: Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.
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Keywords: 22C3 antibody, cytology, immunohistochemistry, immunotherapy, non-small-cell lung carcinoma, programmed cell death-ligand 1, SP263 antibody, tissue fixation, Pathology and Forensic Medicine, Histology, Journal Article
ISSN: 0309-0167
Publisher: Wiley-Blackwell
Note: Funding Information: B. M. Koomen and S. M. Willems report research grants from AstraZeneca, MSD, and Roche Diagnostics, outside the submitted work. S. M. Willems also reports research grants from Amgen, Bayer, BMS, NextCure, and Pfizer, outside the submitted work. The other authors declare that they have no conflicts of interest. Funding Information: No funding was received for this study. The graphical abstract was created by use of the following artwork from Servier Medical Art (Les Laboratoires Servier, https://smart.servier.com): analysis flask, https://smart.servier.com/smart_image/culture-flask-4/; slide, https://smart.servier.com/smart_image/slide/. Servier Medical Art is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). Publisher Copyright: © 2021 The Authors. Histopathology published by John Wiley & Sons Ltd
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