Heterogeneity of PD-L1 Expression in Lung Mixed Adenocarcinomas and Adenosquamous Carcinomas Federica Zito Marino, BD,* Giulio Rossi, MD,† Marco Montella, MD,* Gerardo Botti, MD,‡ Rossella De Cecio, MD,‡ Alessandro Morabito, MD,§ Carmine La Manna, MD,§ Andrea Ronchi, MD,* Mariacarolina Micheli, BD,∥ Giuseppe Salatiello, BSc,∥ Pietro Micheli, MD,∥ Danilo Rocco, MD,¶ Marina Accardo, MD,* and Renato Franco, MD, PhD* Abstract: Immune checkpoint inhibitors against programmed cell death protein 1/programmed death-ligand 1 (PD-L1) have proven to be remarkably effective in non–small cell lung cancer. PD-L1 represents a predictive biomarker in lung cancer, although its heterogenous expression represents an emerging challenge for accurate biomarker-based patient selection. Lung adenocarcinomas (ADCs) show a high rate of intratumor morphologic heterogeneity that may reflect a heterogenous molecular and immunophenotypic profile. The aim of our study was to analyze the expression of PD-L1 in different intratumor subtypes and/or growth patterns in a series of mixed ad- enocarcinomas (mADCs) and adenosquamous lung carcinomas (AdSqLCs). As many as 73 mADCs and 6 AdSqLCs were selected. Comprehensive histologic subtyping was performed, and PD-L1 expression was assessed by immunohistochemistry assay using different primary antibodies and automated im- munostainers. Overall, PD-L1 expression was observed in 37 of 79 cases (39.2%) (31 mADCs and all AdSqLCs). PD-L1 ex- pression was heterogenous in 22 of 37 PD-L1-positive cases (23.2% mADC and 83% AdSqLC). PD-L1 expression was ob- served more frequently in ADC with solid pattern. Hetero- geneity of PD-L1 expression was significantly related to the presence of micropapillary (P = 0.028) and solid (P = 0.017) patterns. All PD-L1-positive cases were epidermal growth fac- tor receptor wild-type, 2 cases harbored concomitantly PD-L1 expression and ALK rearrangement. Our data suggest that PD- L1 expression is quite heterogenous in mADCs and AdSqLCs, partly contributing to explaining the discrepant results between biopsy and surgical resections and discordant clinical effec- tiveness in regard to PD-L1-positive or negative ADC diag- nosed on cytology/small biopsy. Key Words: PD-1, PD-L1, immunotherapy, lung adenocarcinoma, lung adenosquamous carcinoma, immunohistochemistry (Am J Surg Pathol 2019;00:000–000) R ecent advances in immune-targeted therapies have significantly improved the treatment of various types of cancer, by virtue of the development of immune checkpoint inhibitors, which are involved in mechanisms of immune surveillance evasion. The programmed death 1 immune checkpoint receptor (PD-1) and its ligand—the programmed death-ligand 1 (PD-L1)—are key targets for immunotherapeutic approaches in several tumors. 1 PD-1 is an inhibitory receptor activated by at least 2 ligands, PD-L1 and PD-L2. It was originally identified in T lymphocytes, wherein it was found to be involved in the inhibitory signals that downregulate T-cell functions, in the mechanism of T-cell tolerance, and in the down- regulation of physiological immune response. The ex- pression of PD-L1 by cancer cells impairs T-cell–mediated antitumor cytotoxicity. 2 Several studies have shown that PD-L1 is commonly upregulated in non–small cell lung cancer (NSCLC) patients, regardless of the histology; in particular, the prevalence of PD- L1 positivity has been shown to vary from 19.6% to 75%. 3–8 In lung cancer patients, PD-1/PD-L1 pathway inhibitors represent a recent paradigm shift in the first-line therapy of NSCLCs lacking EGFR, ALK, and ROS1 gene alterations. 9 PD-1/PD-L1 pathway inhibitors have shown to induce a therapeutic response in patients with NSCLC, including squamous and nonsquamous histology. 3,6,10,11 Although patients have, at the beginning, been effectively treated with inhibitors targeting PD-1/PD-L1 axis blockade regardless of their PD-L1 immunohistochemical (IHC) expression, 3,12,13 subsequent clinical trials have recognized high PD-L1 expression (positive membrane staining ≥ 50% of tumor cells with any intensity level) as a predictive marker. 14,15 PD-L1 expression is currently a prerequisite parameter to select lung cancer patients eligible for treatment with PD-1/PD-L1 inhibitors, particularly in the first line of treatment. In unselected lung cancer patients, the response rate was ∼20%, increasing up to 50% in patients with PD-L1 expression. 3,10,11,14,16–18 These findings suggest that an accurate quantitative assessment of PD-L1-positive tumor From the *Pathology Unit, Department of Mental and Physical Health and Preventive Medicine, University of Campania “L. Vanvitelli”; ‡Pathology Unit; §Thoracic Department, National Cancer Institute, IRCCS—Fondazione G. Pascale; ∥Pathology Unit; ¶Pneumo-Oncology Unit, Monaldi Hospital Naples; and †Pathology Unit, S. Maria delle Croci Hospital, Ravenna, Italy. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Renato Franco, MD, PhD, Pathology Unit, Università degli Studi della Campania “Luigi Vanvitelli,” Via Luciano Armanni, 5, Napoli 80138, Italia (e-mail: renato.franco@unicampania.it). Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. ORIGINAL ARTICLE Am J Surg Pathol  Volume 00, Number 00, ’’ 2019 www.ajsp.com | 1 Copyright r 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.