Abstract 644: Validation of PD-L1 staining by IHC in solid tumors: A journey from xenograft model to ex vivo culture of clinical samples
Authors: lin Xiaoqian , Carlson Jennifer , Quast Megan , Sun Yuliang , Jepperson Tyler , Williams Casey , RojasEspaillat Luis , Solomon Benjamin , Starks David , Krie Amy , De Pradip , Sulaiman Raed , Dey Nandini , Leyland-Jones Brian 
Published: August 2018

Abstract
Background: Blockade of tumor immune evasion by immune checkpoints inhibitors (ICI) which target PD-L1/2 and PD-1 signaling axis ensured durable clinical benefit in solid tumors including melanoma, Non-Small Cell Lung Cancer, and colorectal cancers which led to the approval of Atezolizumab, Nivolumab, and Pembrolizumab. Studies revealed that response to PD-1/PD-L1 blockade might correlate with PD-L1 expression in tumor cells (Herbst et al., Nature 2014; Robert et al., NEJM 2015a, 2015b; KEYNOTE-001; -012; -040 Trials). Currently, assays for the prognostic/predictive role of tumor PD-L1 expression are “not standardized with respect to either quantity or distribution of expression” (McLaughlin et al., JAMA Oncol. 2016).
Objective: We aimed to standardize, validate and set up criteria for the evaluation of PD-L1 protein expression using conventional immunohistochemistry (IHC) staining to demonstrate PD-L1 protein distribution in cultured ex vivo tumors obtained from surgically resected tumor samples.
Methods: We used cell lines, their xenografts tumors, and Formalin-Fixed Paraffin-Embedded tumor samples from patients with different solid tumors to compare results from two different PD-L1 antibodies (Rb Anti-Human PD-L1/CD274 Mono-Ab Clone SP142 from SPRING BIOSCIENCE CATALOG# M4420; Mono-Mouse Anti-Human PD-L1 [Concentrate] Clone 22C3 from DAKO Code: M3653).TNBC (MDA-MB231, MDA-MB468, SUM149) and NSCLC (H1975 and H2228) cell lines, human term placenta and tonsil whole tissue sections were used for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using diaminobenzidine chromogenic IHC in FFPE sections/ fixed cells. Tumor-infiltrating lymphocytes (TILs) in tissue and ex vivo samples were scored in hematoxylin-eosin slides using current consensus guidelines. Finally, we validated PD-L1 expression in ex vivo cultured live tumor samples from patients with lung cancers.
Results: With both PD-L1 antibodies, we find that PD-L1 IHC expression was heterogeneous. When validated using human tonsil sections, a focal and sporadic predominantly membranous positivity was observed in xenograft tissues and tumor cells. Both antibodies showed limited concordance, significant intra-assay heterogeneity, and significant interassay discordance. We also observed a markedly high non-specific staining in both necrotic tumor areas and scars.
Conclusion: We had standardized and validated IHC method for the PD-L1 protein expression in (1) xenografts of several breast cancer cell lines, (2) FFPE from different solid tumors and (3) finally in ex vivo cultured tumors from lung cancer patients. Expression of PD-L1 is currently being correlated with TILs / inflammatory response in tumors the result of which will be presented at the meeting. This evaluation of PD-L1 expression based on ex vivo model may help to stratify patients for the treatment with ICI.
Citation Format: Xiaoqian lin, Jennifer Carlson, Megan Quast, Yuliang Sun, Tyler Jepperson, Casey Williams, Luis RojasEspaillat, Benjamin Solomon, David Starks, Amy Krie, Pradip De, Raed Sulaiman, Nandini Dey, Brian Leyland-Jones. Validation of PD-L1 staining by IHC in solid tumors: A journey from xenograft model to ex vivo culture of clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 644.