Impact of Prolonged Ischemia and Fixation on the Immunohistochemical Expression of PD-L1 in Non-small Cell Lung Cancer Specimens

Abstract

Humanized antibodies targeting PD-1 or PD-L1 are established standards of care for non–small cell lung cancer (NSCLC), and there seems to be a correlation between tissue expression of PD-L1 and response rate in patients.1,2 Most of the analytical challenges in the immunohistochemical evaluation of PD-L1 expression have been extensively analyzed.3 However, preanalytical issues have been scarcely explored. The impact of prolonged ischemia and tissue fixation on PD-L1 false-negative cases has been established, and the fixation time window to obtain optimal results in control tissue,4 most commonly tonsil, has also been determined.5,6 Extrapolation of such results to specific tumor tissues should be made with caution, as tonsil tissue is less affected by variability than tumor samples. Heterogeneity of expression may preclude the adequate interpretation of suboptimally processed samples.7 Moreover, small-size samples, including bronchoscopic biopsy specimens or cytology blocks, may be even more sensitive to prolonged prefixation periods.8 The main objective of this study was to determine the proportion of routine samples processed within acceptable preanalytical times and to evaluate the extrapolatability to NSCLC samples of ischemia and fixation time limits established for the assessment of PD-L1 expression in control tissue.

We retrospectively reviewed consecutive and unselected samples received at a referral pathology unit, selected from the unit database over a one-year period. Samples were eligible if they included an NSCLC diagnosis and PD-L1 determination. Sample processing was uniform across all the evaluated period (see File S1). Age, gender, biopsy site and method, processing method, histological subtype, and PD-L1 results, including proportion and intensity of positive cells, were recorded for each patient (Table 1, Table S1). Samples were considered PD-L1 positive if at least 1% of tumoral cells were positive, although other cut points were also explored. Based on previous studies in control tissue, an optimal preanalytical time of up to 72 h was defined. Time from surgical procedure to laboratory admission and time from laboratory arrival to end of fixation were obtained from the traceability system, and their addition was considered the full preanalytical time. Bivariate analysis was performed to assess significant relationships between the duration of the preanalytical process and PD-L1 results. The R-package version 3.0.1 was used for all statistical analyses (R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ ). The study was approved by the the Comitè d’Etica de la Investigació amb medicaments, Hospital Universitari Germans Trias i Pujol (PI-18-072 approval number) and performed in adherence to the STROBE guidelines.