Contents lists available at ScienceDirect Acta Histochemica journal homepage: www.elsevier.com/locate/acthis Pd-ligand 1 is expressed in inflammatory cells but not in neoplastic cells in hepatocellular carcinoma: An immunohistochemical study Francesco Vasuri a , Azzurra Nerpiti a , Stefano Zagnoni a , Matteo Ravaioli b , Antonia D’Errico a , Michelangelo Fiorentino a, * a Pathology Unit, S.Orsola Malpighi University Hospital, Bologna, Italy b Surgery Unit, S.Orsola Malpighi University Hospital, Bologna, Italy ARTICLE INFO Keywords: Hepatocellular carcinoma Immunohistochemistry PD-L1 Tissue micro-array ABSTRACT Nowadays, the major limit to the immunohistochemical (IHC) analysis of tissue PD-L1 is the high variability of the monoclonal antibodies commercially available. Aims of the present paper are to assess the best clone and the most suitable scoring for PD-L1 IHC determination on human hepatocellular carcinoma (HCC) among three commercially available clones, and to evaluate which PD-L1 clone is the best in predicting HCC aggressiveness in vivo. We built a tissue microarray (TMA) with 60 retrospective HCC cases, including the correspondent non- tumoral tissue. IHC was automatically performed using the following anti-PD-L1 clones: 28.8, SP142, and SP263. As results, we did not find any immunoreactivity for PD-L1 in both neoplastic and normal hepatocytes in- cluded in the TMA using the three antibodies. Positivity for PD-L1 was exclusively seen in inflammatory cells within the HCC tissue and in cirrhotic parenchyma. When a gold standard was assessed, the sensitivity of SP142, 28.8 and SP263 was 46 %, 54 % and 85 % respectively. Using the SP263 clone, the absolute number of PD-L1- positive inflammatory cells in the HCC cores was paired with the number of PD-L1-positive inflammatory cells in the corresponding non-tumoral tissue (P = 0.001). Finally, using SP263, the mean number of PD-L1-positive cells was 11.3 ± 12.6 in HCC from deceased patients, versus 4.7 ± 5.2 in alive patients (p = 0.039). SP263 is the most sensitive clone for PD-L1 IHC tissue determination in HCC, as well as the best antibody for the as- sessment of its biological behavior. 1. Introduction Hepatocellular carcinoma (HCC) is the most frequent malignant primary liver tumor, sixth for incidence among malignant tumors worldwide (WHO, 2018). The high mortality of HCC is due to the morphological and biological heterogeneity of this tumor, that makes difficult the application of non-surgical therapies. The recent in- troduction of the immune check-points inhibitors (ICPI) has represented a milestone in the therapy of several human tumors. By the production and regulation of different cytokines, interleukins and growth factors, cancer cells are able to induce a status of immunosuppression, hiding themselves from the host immune system. This process, called “cancer immunoediting” requires a fine interaction with the extratumoral mi- croenvironment, and it is carried out by immune check-points (ICP), among which the axis PD-1/PD-L1 plays a fundamental role (Sprinzl and Galle, 2017; Xu et al., 2018). In the liver, PD-L1 is mainly expressed by Kupffer cells and, in a minor amount, by antigen presenting cells (APC) (Wu et al., 2009; Calderaro et al., 2016). The link between PD-1 (expressed by CD8 + T lymphocytes) and PD-L1 (expressed by APC) is responsible for the im- munetolerance in HCC. The role of PD-L1 expression in HCC is con- troversial: some papers did not find any correlation between PD-L1 expression and HCC biological behavior 5 , while others found a higher risk of metastatization and cancer-related death in HCCs with PD-L1 expression in peritumoral hepatocytes (Dai et al., 2017; Semaan et al., 2017). In particular, the concomitant presence of a dense CD8+ lym- phocytic infiltrate (tumor-infiltrating lymphocytes, TILs) and PD-L1 expression seems promising for proposing the immunotherapy with ICPI in at least a subset of HCCs. The major limit to the analysis of tissue PD-L1 is represented by the high variability of the monoclonal antibodies commercially available for the immunohistochemical (IHC) determination on tumors. Different reagents have been validated on different human cancers for clinical application, such as lung (Scheel et al., 2016; Hirsch et al., 2017; Giunchi et al., 2018), bladder (de Jong et al., 2018; Wang et al., 2019), kidney (Shin et al., 2016), melanoma (Sunshine et al., 2017) and https://doi.org/10.1016/j.acthis.2019.151468 Received 27 August 2019; Received in revised form 24 October 2019; Accepted 11 November 2019 ⁎ Corresponding author at: Pathology Unit, S.Orsola Malpighi University Hospital, v.le Ercolani 4/2, 40138, Bologna, Italy. E-mail address: michelangelo.fiorentino@unibo.it (M. Fiorentino). Acta Histochemica xxx (xxxx) xxxx 0065-1281/ © 2019 Published by Elsevier GmbH. Please cite this article as: Francesco Vasuri, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.151468