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The Use of Endobronchial Ultrasound–guided Transbronchial Needle Aspiration Cytology Specimens for Programmed Death Ligand 1 Immunohistochemistry Testing in Non–Small Cell Lung Cancer

Stoy, Sean MD*; Rosen, Lauren MD; Murgu, Septimiu MD, FCCP*

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Journal of Bronchology & Interventional Pulmonology: July 2017 - Volume 24 - Issue 3 - p 181-183
doi: 10.1097/LBR.0000000000000406
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The National Comprehensive Cancer Network (NCCN) 2017 Clinical Practice Guidelines for non–small cell lung cancer (NSCLC) recommend concomitant diagnosis, staging, and genetic testing.1 In addition, guidelines recommended utilizing the least invasive biopsy procedure that offers the highest yield. EBUS-guided TBNA cytology specimens have demonstrated adequacy that is at least equivalent to that from histologic specimens for the diagnosis and staging of NSCLC.2,3 The use of EBUS-TBNA has become the procedure of first choice to diagnose and stage locally metastatic lung cancer and is recommended by multiple national and international guidelines.1,4–6 As such, the majority of patients with advanced NSCLC are diagnosed by small-volume cytology or biopsy specimens, and EBUS-TBNA is the most common modality of tissue acquisition7; in fact, it may be the only sample available for testing throughout the patient’s clinical course.

The 2017 NCCN guidelines recommend testing for PD-L1 expression on tumor cells upon diagnosis with advanced NSCLC as well as for patients with progression of disease on first-line chemotherapy. There are several approved immune checkpoint inhibitors (eg, nivolumab, pembrolizumab, atezolizumab) that target programmed death receptor 1 (PD-1) or programmed death ligand 1 (PD-L1). In addition, the NCCN guidelines recommend quantification of PD-L1 tumor membrane staining expression as it is relevant to treatment decisions. For instance, pembrolizumab is approved as first-line therapy if tumor PD-L1 expression is >50% in patients without EGFR, ALK, or ROS1 aberrations and as second-line therapy for patients with disease progression on platinum-based chemotherapy. Although nivolumab does not require PD-L1 testing for second-line chemotherapy, the NCCN guidelines state that PD-L1 testing on tumor cells provides prognostic information for response and survival.1

PD-L1 expression is currently determined by immunohistochemistry (IHC) testing, which has traditionally required histologic samples.8 The ability to reliably utilize cytology specimens for PD-L1 testing would be expected to provide an advantage in terms of cost, timeliness, and patient satisfaction, especially as the majority of patients with advanced lung cancer are diagnosed on the basis of cytology specimens. There is a paucity of information regarding PD-L1 IHC testing of EBUS-TBNA samples. One recent small study compared EBUS-TBNA metastatic lymph node biopsies with transbronchial, excisional biopsies of lymph nodes and primary lung tumor with regard to PD-L1 expression. The authors found a good concordance between testing the excised lymph nodes and EBUS-TBNA samples (R-value=0.93) and between the primary tumor and EBUS-TBNA samples (R=0.75).9 It is important to also note that, although different immune checkpoint agents have specific accompanying PD-L1 IHC testing, another recent study has shown that 3 of the commercially available PD-L1 IHC assays (Ventana PD-L1 SP263; Roche Diagnostics, Indianapolis, IN), Dako PD-L1 IHC 22C3 pharmDx (Agilent Technologies, Santa Clara, CA), and Dako PD-L1 IHC 28-8 pharmDx (Agilent Technologies) have high agreement (>90%) for multiple PD-L1 tumor membrane staining cutoffs, including at 1% and 50% expression.10 Herein, we describe our institution’s workflow for PD-L1 testing of cell block formed from cytology samples obtained by EBUS-TBNA from metastatic mediastinal, hilar, or interlobar lymph nodes in patients with NSCLC.


EBUS-TBNA of the target lymph node is performed using a 25-G needle. To date, there are no published studies evaluating the differences in diagnostic yield or PD-L1 testing between the 25-G and larger-gauge needles. At least three aspirates per node are obtained during a routine EBUS-TBNA diagnostic and staging procedure. If necessary, this process continues through the remaining lymph node stations, as per standard staging procedure: N3 nodes are sampled first, followed by N2 and then N1, based on feedback from rapid on-site cytology evaluation (ROSE). Following aspiration of a sample, the needle is removed from the scope. Then a drop of material is discharged onto a glass slide first by advancing the needle stylet, and if insufficient material is visualized on the slide the needle is flushed with air. The material dispelled on the slide is then smeared with a second slide. One of the slides is stained using Diff-Quik (Medion Diagnostics AG, Düdingen, Switzerland) and undergoes ROSE. The other slide is spray-fixed with alcohol for future Pap staining. After each pass, all remaining aspirate material from the needle is ejected into Cytolyt (Hologic Inc., Marlborough, MA) solution and is subsequently processed into a cell block. On-site review of the Diff-Quik smears is performed by an attending cytopathologist. If the diagnosis is favored to be non–small cell lung cancer, the cytopathologist confirms the acquisition of sufficient material for molecular studies and requests additional material for cell block preparation used for histologic subtyping (TTF1 and P40) and for PD-L1 IHC. Cell blocks are formed by first placing aspirate material into Cytolyt solution centrifuged in the pathology laboratory into a pellet and then embedded into paraffin and submitted for histologic processing. If ROSE is not used, the specimen is deployed directly into Cytolyt or formalin, and a cell block is then prepared and tested for PD-L1.9

At our institution, PD-L1 IHC is performed using the Abcam anti-PD-L1 antibody 28-8 (Abcam, Cambridge, MA). After deparaffinizing and rehydrating the cell blocks, antigen retrieval is performed with a dilution of 1:50 on the Ventana XT platform (Roche Diagnostics). Human tonsil and placenta are used as positive controls. PD-L1 expression is quantified as the percentage of tumor membrane involved using predefined cutoff values relevant for treatment decisions (Fig. 1). Limited published evidence and our experience suggest that EBUS-TBNA cell block samples provide adequate material for PD-L1 testing. It is promising that this minimally invasive procedure using small bore needles can be used for PD-L1 IHC testing, which allows immediate treatment decisions at the time of diagnosis or disease progression without requiring additional invasive procedures for more tissue acquisition.

A, Image capture of PD-L1 IHC staining at ×400 magnification showing 0% PD-L1 tumor membrane staining. B, Image capture of PD-L1 IHC staining at ×400 magnification showing 5% PD-L1 tumor membrane staining. C, Image capture of PD-L1 IHC staining at ×400 magnification showing >50% PD-L1 tumor membrane staining. IHC indicates immunohistochemistry; PD-L1, programmed death ligand 1.

Sean Stoy*

Lauren Rosen†

Septimiu Murgu, MD, FCCP*
Departments of *Medicine, Division of Pulmonary and Critical Care
†Pathology, University of Chicago, Chicago, IL


1. Ettinger DS, Wood DE, Aisner DL, et al. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Non-small cell lung cancer. Version 3. 2017. Available at: Accessed November 16, 2016.
2. Nakajima T, Yasufuku K, Nakagawara A, et al. Multi-gene mutation analysis of metastatic lymph nodes in non-small cell lung cancer diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration. Chest. 2011;140:1319–1324.
3. Um SW, Kim HK, Jung SH, et al. Endobronchial ultrasound versus mediastinoscopy for mediastinal nodal staging of non-small-cell lung cancer. J Thorac Oncol. 2015;10:331–337.
4. Silvestri GA, Gonzalez AV, Jantz MA, et al. Methods for staging non-small cell lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2013;143:e211S–e2150.
5. De Leyn P, Dooms C, Kuzdzal J, et al. Revised ESTS guidelines for preoperative mediastinal lymph node staging for non-small-cell lung cancer. Eur J Cardiothorac Surg. 2104;45:787–798.
6. Navani N, Nankivell M, Lawrence DR, et al. Lung cancer diagnosis and staging with endobronchial ultrasound-guided transbronchial needle aspiration compared with conventional approaches: an open-label, pragmatic, randomised controlled trial. Lancet Respir Med. 2015;3:282–289.
7. Travis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;6:244–285.
8. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018–2028.
9. Sakakibara R, Inamura K, Tambo Y, et al. EBUS-TBNA as a promising method for the evaluation of tumor PD-L1 expression in lung cancer. Clin Lung Cancer. 2016; pii: S1525-7304(16)30378-3.
10. Ratcliffe MJ, Sharpe A, Midha A, et al. Agreement between programmed cell death ligand-1 diagnostic assays across multiple protein expression cut-offs in non-small cell lung cancer. Clin Cancer Res. 2017; doi: 10.1158/1078-0432.CCR-16-2375.
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