PD-L1 testing in advanced stage lung cancer using cytology samples: Suitability and reporting issues. Comparison between two tertiary referral centers

Background: Lung cancer is the most common cause of cancer-related death worldwide and unfortunately up to 80% of patients amongst newly diagnosed are inoperable therefore the cytological sample is often the only material available for diagnosis and assessment of molecular characteristics driving the treatment. Recently immunotherapy has shown promising results in tumors expressing Program Death Ligand 1 (PD-L1). The expression of PDL1 can routinely be detected by immunohistochemistry. However, the presence of several antibodies with different cut-off and the expression of this marker by normal immune cells are generating confusion in interpretation and the need for harmonization amongst pathologists. Materials and methods: We assessed the suitability of 74 consecutive cell blocks from cytology samples for PDL1 testing and evaluate the concordance between two different antibodies (Ventana assay SP263 and Dako 223C pharmDx assay) and amongst different pathologists from two different tertiary referral center for thoracic pathology. The degree of agreement was measured by Fleiss K statistic (FKS) for categorical scores after dichotomization based on specifi ed cutoffs. A review of discordant cases was also performed. Results: Review of the slides stained with both antibodies showed substantial agreement within our department and moderate agreement with results from the other institution. Overall less than 10% of cases were deemed inadequate. Discordant cases showed a decreased amount of tumor cells, therefore, tumor heterogeneity could be responsible for the variation in the reading. Conclusions: Our results show overall concordance between the two antibodies and the suitability of cytology material for PDL-1 testing. Research Article PD-L1 testing in advanced stage lung cancer using cytology samples: Suitability and reporting issues. Comparison between two tertiary referral centers Elshiekh Mohamed1, Spinelli Manuela2, Iles Sandra1, Hopcroft Danielle1, Trivedi Pritesh1, Gupta Nandita1, Bhagwat Priya1 and Viola Patrizia1* 1Department of Cellular Pathology, NWLP Hosted by Imperial College Healthcare. NHS Trust, Charing Cross Hospital, London, UK 2Department of Pathology, Worcestershire Royal Hospital, Charles Hastings Way, Worcester, UK Received: 24 December, 2020 Accepted: 22 January, 2021 Published: 25 January, 2021 *Corresponding author: Viola Patrizia, Department of Cellular Pathology, NWLP Hosted by Imperial College Healthcare. NHS Trust, Charing Cross Hospital, London, UK, E-mail:


Introduction
Lung cancer is the most common cause of cancer-related death worldwide with an incidence of 14% per year [1]. Nonsmall cell lung carcinoma accounts for more than 85% of cases leading to an overall 5 years survival rate around 18% [2,3]. About 80% of Non-Small Cell Lung Carcinomas (NSCLC) are diagnosed in an advanced stage and are therefore usually inoperable for locally advanced and/or metastatic disease. In these cases, surgical excision provides no or minimal benefi t, thus diagnosis is often made with minimally invasive procedures such as transthoracic, TC-guided core biopsies, bronchoscopic biopsies, Endo-Bronchial Ultrasound-Guided Fine-Needle Aspiration (EBUS-FNA) or thoracentesis [4,5]. Therefore, in many patients, cytology is increasingly playing an essential role either diagnostic or prognostic role [6][7][8][9][10]. The premises seem to be good but, in contrast with Immunohistochemistry (IHC), which has already highly standardized and validated protocols, Immunocytochemistry (ICC) in clinical practice is still limited by lack of standardization and pre-analytical and analytical variability. Studies in the past few years have been compared ICC procedures versus the well-established IHC protocols, focusing on the selection of material (direct smears, monolayer preparations, cell blocks), use of different fi xative agents, use of different pre-analytic platforms. [11,12] One of the larger comparison studies by Fowler in 2008 and Fisher in 2014 [7,10], confi rmed that ICC on cytology can be as safe and reliable as IHC on histology, providing the use of appropriate techniques and strict adherence to quality protocols.
In the past few years a protein called Programmed Death receptor-1 (PD-1) and its ligands PD-L1 and PD-L2 have been identifi ed as potential new markers, to guide the treatment of NSCLC.
PD-1 is a membrane immunoglobulin with a crucial role in regulating immune-mediated tissue damage. PD-1 acts as a membrane receptor with an immunomodulatory role in B and T cells, natural killer cells and macrophages, and as an essential regulatory factor in activated T cells [8]. Some studies demonstrated that the binding of PD-1 with specifi c ligands can block the T cell response to a tumor in different sites including the lung. Disruption of the PD-1/PD-L1 interaction results in activation of T cell immune response and this may play a crucial role in infl uencing tumor microenvironment and leading to downregulation and apoptosis of tumorreactive T cells. Therefore, the block of PD-1/PD-L1 interaction leads to an increased response of T cells against tumor cells [3,13,14]. Several ligands to PD-1 have been identifi ed and amongst them, PD-L1 is the most studied in lung cancer for its emerging role in tailoring the treatment of NSCLC. Monoclonal antibodies have been developed to target either PD-1 and PD-L1 and block the PD-1 receptor activity to allow T cells to attack tumor cells. Clinical trials with antagonists of PD-1 showed increased survival rates in patients with advanced, metastatic tumors including melanoma and NSCLC [8]. The study from Garon et al is of central importance for pathologists as it proved that tumors which expression of PD-L1 in 50% or more of malignant cells have a signifi cantly increased response to molecular therapy with Pembrolizumab [15], which has been also approved by US Food and Drugs Administration (FDA) for patients with at least 1% expression of PD-L1 [4].
For the last 30 years, the only therapeutic option for advanced/metastatic NSCLC was based on cytotoxic chemotherapy. Immunotherapy is used increasingly since studies proved the effectiveness of inhibitors that target PD-1 receptors such as Pembrolizumab, Atezolizumab, Nivolumab [13,15,16]. The expression of PD-L1 is evaluated with Immunohistochemical assay therefore it can be routinely detected. However, several clones are commercially available, each featuring different platforms and cut-offs; which can generate confusion and is urging the need for standardization, quality control, and harmonization amongst different laboratories and pathologists.
In this study, we tried to assess the suitability of cytology as material for PD-L1 testing and evaluated the concordance between two different antibodies and amongst pathologists in two different referring centers in reporting PD-L1 in cytology specimens.

Materials and methods
We retrieved 74 consecutive cell blocks from cytology FKS scores of 0.81 or higher were considered near perfect, scores of 0.61 to 0.80 were considered substantial agreement, scores of 0.41 to 0.60 were considered moderate agreement, scores of 0.21 to 0.40 were considered fair agreement, scores of 0.01 to 0.20 were considered slight agreement whilst less than 0 was considered poor agreement [21].

Results
The majority of our samples were obtained through  and strong positive at the other, however, the patient was also bearing EGFR mutation and was treated with Erlotinib.

Discussion
The introduction of IHC for PD-L1 assessment put the pathologist in a crucial role in the diagnosis and management of lung cancer [3].
Quantifi cation of PD-L1 is essential to assess patient eligibility for immunotherapy and to determine their possible response to PD-1 and PD-L1 inhibitors in advanced NSCLC. As    Our results confi rm those previously published showing good suitability of cytology material for this test and an overall good intradepartmental concordance of PD-L1 evaluation when a specialist or trained pathologists report PD-L1 at different cut-offs. Of note in our study, two pathologists were fully trained to report PD-L1 whilst the third pathologist is an expert cytopathologist who did not take any training. In our study, we showed we also compared the staining of two different antibodies on the same cytological material and the comparison showed only moderate agreement. This lower degree of agreement can be easily explained with loss of cellularity in the samples and can be therefore still be considered acceptable.
Our work confi rmed the suitability of cytology material for PD-L1 testing, with results that are in line with previously published studies. Our results also demonstrated overall concordance between the two antibodies used (Ventana clone SP263 and Dako clone 22C3). We also highlight the importance of the cellularity present in the sample as well as the need for fully trained pathologists or at least highly specialized cytopathologists in reporting these cases as staining of nonneoplastic cells can affect the fi nal score.