Assessment of Programmed Death–ligand 1 (PD-L1) Immunohistochemical Expression on Cytology Specimens in Non–Small Cell Lung Carcinoma

A Comparative Study With Paired Surgical Specimens

Andrea Hernandez, DO; Tamar C. Brandler, MD; Fang Zhou, MD; Andre L. Moreira, MD, PhD; Nina Schatz-Siemers, DO; Aylin Simsir, MD


Am J Clin Pathol. 2019;151(4):403-415. 

In This Article

Materials and Methods

Case Selection

The current study protocol was approved by the New York University Institutional Review Board. A retrospective review of the pathology database (Powerpath; Sunquest) was performed between January 1, 2016, and December 30, 2017, to identify NSCLC surgical biopsy and resection cases that had PD-L1 testing performed and reported. The concurrent or subsequent cytology preparations for each surgical pathology case yielded by our search were evaluated for (1) the presence of a formalin-fixed, paraffin-embedded (FFPE) cell block and (2) adequate tumor cellularity. Cases with fewer than 50 tumor cells were excluded from this study.

Cytologic Preparation and Study Samples

Cell blocks were prepared using fine-needle aspiration (FNA) material placed in RPMI solution (Corning Cellgro). Effusion specimens were decanted into a conical tube and centrifuged to obtain a sufficient cell pellet. The supernatant fluid was removed. Cell blocks were further prepared by the plasma-thrombin method, fixed in 10% neutral buffered formalin for at least 6 hours, and embedded in paraffin, and sections were stained with H&E. Both surgical and cytologic specimens were diagnosed using the 2015 World Health Organization classification for lung tumors.[10] Immunohistochemical expression of protein markers including, but not limited to, thyroid transcription factor 1 (TTF-1), p40, p63, napsin-A, MOC-31, or BER-EP4 were performed, when necessary, to aid in the diagnosis of NSCLC.

In total, 52 cytology cases from 50 patients (two patients had two cytology specimens) with paired surgical specimens were selected for this study. Cytology samples were formalin-fixed cell blocks from pleural fluid (n = 20), lung FNA (n = 16), lymph node FNA (n = 14), bronchial brush (n = 1), and mediastinal FNA (n = 1).

PD-L1 Immunohistochemical Staining and Evaluation

Cell blocks of the cases selected for this study were retrieved and 4-μm serial sections were obtained. Unstained slides were used within the same week of serial sectioning for PD-L1 immunostaining. PD-L1 IHC expression was quantified using the PD-L1 clone 22C3 pharmDx kit (Dako/Agilent Technologies) and a Ventana BenchMark automated stainer (Ventana Medical Systems) on FFPE surgical and cell block sections. The Ventana platform was previously validated for surgical specimens in our laboratory by comparing the gold-standard (Keytruda test) scores with the same in-house NSCLC cases run on the Ventana platform with the OptiView detection kit, resulting in a correlation that was positive and significant (r = 0.88, P < 0.001). The sensitivity, specificity, positive predictive value, and negative predictive values were 92.3%, 100%, 100%, and 86.7%, respectively.[11] Each PD-L1 run included positive and negative cell line controls provided by Dako. In addition, an in-house positive control (tonsil tissue) was included on every PD-L1 immunostained slide.

PD-L1 expression was evaluated using the tumor proportion score (TPS), defined as the percentage of viable tumor cells with partial/complete membranous staining at any intensity with respect to all viable tumor cells within the slide.[12] The TPS results were then categorized as negative (TPS <1%), low positive (TPS ≥1%-49%), or high positive (TPS ≥50%). The following were not included in the TPS: cytoplasmic staining without any membranous staining, any staining in background cells (such as nonneoplastic cells including macrophages, hematopoietic cells, bronchial cells, and mesothelial cells), and any staining in necrotic cells.

PD-L1 expression in cytology samples was scored by two board-certified pathologists at a multiheaded microscope (A.S. and A.H.; a cytopathologist and a cytopathology fellow). The pathologists were blinded to the corresponding surgical specimen PD-L1 scores and were previously trained in PD-L1 evaluation by an in-house experienced pulmonary pathologist/cytopathologist (A.L.M.). All cases of PD-L1 score discordance between surgical and cytology samples were subsequently blindly reviewed by A.L.M.

All surgical specimens used for paired comparison had prior PD-L1 IHC performed with Dako PD-L1 pharmDx (clone 22C3), with TPS reported between January 2016 and December 2017 by a group of three pulmonary surgical pathologists. As part of the current study, these scores were also categorized as negative (<1%), low positive (≥1%-49%), and high positive (≥50%).


Cohen's κ statistic was calculated for the matched cytology and surgical pathology cases using IBM SPSS Statistics Version 23 (SPSS). The strength of association (agreement) was categorized as follows: 1.00, perfect agreement; 0.81 to 0.99, almost perfect agreement; 0.61 to 0.80, substantial agreement; 0.41 to 0.60, moderate agreement; 0.21 to 0.40, fair agreement; 0.00 to 0.20, slight agreement; and less than 0, poor agreement.