Preanalytical variables affect predictive immunohistochemical biomarker testing in non–small cell lung carcinoma cytology specimens

BACKGROUND: Several therapeutic targets for non-small cell lung carcinoma (NSCLC) rely on protein expression by immunohistochemistry (IHC). Most biomarkers are validated on formalin-fixed, paraffin-embedded (FFPE) tissue; however, cytologic specimens, including cell blocks (CBs), that are subject to preanalytical variables are increasingly used. The objective of this study was to evaluate predictive IHC biomarkers in NSCLC CBs and compare them with surgical pathology (SP) specimens. METHODS: The authors retrospectively reviewed NSCLC cytology specimens that underwent predictive IHC testing for hepatocyte growth factor receptor (c-MET), human epidermal growth factor receptor 2 (HER2), and programmed death-ligand-1 (PD-L1) between January 2019 and June 2025. IHC was performed on CBs that were centrifuged with equal amounts of ethanol and formalin before FFPE processing. Equal numbers of NSCLC SP specimens with available biomarker testing were included for comparison. Predictive IHC scores that were considered therapy-eligible were c-MET IHC 3+, HER2 IHC 3+, and PD-L1-positive tumor proportion scores. Statistical analyses were performed using the Fisher exact test, with p < .05 considered significant. RESULTS: The cohort included 414 cases (cytology specimens, N = 212; SP specimens, N = 212 [60% biopsy, 40% resection]). Cytology specimens demonstrated a significantly higher proportion of strong 3+ c-MET expression (40%) compared with SP specimens (10%; p < .0001). Similarly, high PD-L1 expression was more frequent in cytology specimens (32% vs. 20%; p = .0079). HER2 expression was similar between groups, with no significant difference in HER2 IHC 3+ rates (p = .34). CONCLUSIONS: Predictive IHC testing is performed on NSCLC cytology to identify patients for therapeutic targets. The current study highlights the need for optimizing preanalytical variables in cytology specimen processing, even when testing FFPE CBs, to ensure the reliability of predictive IHC results that guide therapeutic decisions.