Nationwide differences in cytology fixation and processing methods and their impact on interlaboratory variation in PD-L1 positivity

Abstract

Programmed death ligand-1 (PD-L1) immunostaining, which aids clinicians in decision-making on immunotherapy for non-small cell lung cancer (NSCLC) patients, is sometimes performed on cytological specimens. In this study, differences in cytology fixation and cell block (CB) processing between pathology laboratories were assessed, and the influence of these differences on interlaboratory variation in PD-L1 positivity was investigated. Questionnaires on cytology processing were sent to all Dutch laboratories. Information gathered from the responses was added to data on all Dutch NSCLC patients with a mention of PD-L1 testing in their cytopathology report from July 2017 to December 2018, retrieved from PALGA (the nationwide network and registry of histo- and cytopathology in the Netherlands). Case mix-adjusted PD-L1 positivity rates were determined for laboratories with known fixation and CB method. The influence of differences in cytology processing on interlaboratory variation in PD-L1 positivity was assessed by comparing positivity rates adjusted for differences in the variables fixative and CB method with positivity rates not adjusted for differences in these variables. Twenty-eight laboratories responded to the survey and reported 19 different combinations of fixation and CB method. Interlaboratory variation in PD-L1 positivity was assessed in 19 laboratories. Correcting for differences in the fixative and CB method resulted in a reduction (from eight (42.1%) to five (26.3%)) in the number of laboratories that differed significantly from the mean in PD-L1 positivity. Substantial variation in cytology fixation and CB processing methods was observed between Dutch pathology laboratories, which partially explains the existing considerable interlaboratory variation in PD-L1 positivity.

Keywords: Cytological techniques; Fixatives; Immunocytochemistry; Interlaboratory variation; Non-small cell lung cancer; Programmed death ligand-1.

Fig. 1

Overview of the various combinations of fixation and cell block methods for cytology samples as described by the survey respondents. The final column displays the number of times each combination is used. Colors depict the overall fixation method for each combination (see legend). ^#Fluids were not always received in a collection medium, but rather as fresh fluids. Abbreviations: FFPE, formalin-fixed paraffin-embedded; N/A. not applicable

Fig. 2

Flowchart of the patient selection process from the PALGA data set. CB, cell block; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand-1; TPS, tumor proportion score

Fig. 3

Funnel plots showing interlaboratory variation in programmed death ligand-1 (PD-L1) positivity. PD-L1 positivity was determined using either a 1% cutoff (a) or a 50% cutoff (b). For each laboratory, case mix-adjusted positivity rates are displayed against the total number of patients tested for PD-L1 (dots). The variables age, sex, histological subtype, and source of material used for PD-L1 testing were included in the case mix adjustment analysis. Colors are used to indicate the fixative that was used the most in each laboratory (see legend). The black line shows the overall mean proportion of PD-L1 positive patients, surrounded by its 95% confidence limits (black dotted lines). The colored dotted lines display the mean PD-L1 positivity rate for each fixative category

Fig. 4

Funnel plots showing interlaboratory variation in programmed death ligand-1 (PD-L1) positivity, with PD-L1 positivity rates adjusted for case mix including the variables fixative and cell block method. PD-L1 positivity was determined using either a 1% cutoff (a) or a 50% cutoff (b). For each laboratory, case mix-adjusted positivity rates are displayed against the total number of patients tested for PD-L1 (diamonds). The color of the diamonds indicates the fixative that was used the most in each laboratory (see legend). The black line shows the overall mean proportion of PD-L1 positive patients, surrounded by its 95% confidence limits (dotted lines)