Formalin fixation for optimal concordance of programmed death-ligand 1 immunostaining between cytologic and histologic specimens from patients with non-small cell lung cancer

Bregje M. Koomen*, Jose van der Starre-Gaal, Judith M. Vonk, Jan H. von der Thusen, Jacqueline J. C. van der Meij, Kim Monkhorst, Stefan M. Willems, Wim Timens, Nils A. 't Hart

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

6 Citaten (Scopus)
27 Downloads (Pure)


Background Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied.

Methods Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration.

Results Agar cell blocks and tissue blocks showed substantial agreement (kappa = 0.70 and kappa = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (kappa = 0.28 and kappa = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay.

Conclusions Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.

Originele taal-2English
Pagina's (van-tot)304-317
Aantal pagina's14
TijdschriftCancer cytopathology
Nummer van het tijdschrift4
Vroegere onlinedatum27-okt.-2020
StatusPublished - apr.-2021

Citeer dit