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Programmed Death Ligand 1 Testing of Endobronchial Ultrasound–guided Transbronchial Needle Aspiration Samples Acquired For the Diagnosis and Staging of Non–Small Cell Lung Cancer

Smith, Allister BSc, Eng*; Wang, Hangjun MD; Zerbo, Alix MSc*; Beaudoin, Stéphane MD; Ofiara, Linda MD; Fiset, Pierre-Olivier MD; Benedetti, Andrea PhD*; Gonzalez, Anne V. MD, MSc*,‡

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Journal of Bronchology & Interventional Pulmonology: January 2020 - Volume 27 - Issue 1 - p 50-57
doi: 10.1097/LBR.0000000000000623


Lung cancer is the leading cause of cancer-related death in both men and women worldwide,1,2 with an overall 5-year survival that remains below 20% in North America.3 Novel immune checkpoint inhibitors targeting either programmed death ligand 1 (PD-L1) or the programmed death 1 (PD-1) receptor, have changed the treatment paradigm of advanced non–small cell lung cancer (NSCLC).4 Response to the anti-PD-1 agents nivolumab and pembrolizumab correlates with PD-L1 expression on tumor cells, as assessed by immunohistochemistry performed on histologic samples.5–9

A limited number of studies to date have examined the assessment of PD-L1 expression using cytologic specimens, in particular those acquired using endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA).10,11 Minimally invasive needle techniques have become first-line diagnostic procedures in NSCLC patients with hilar and/or mediastinal node involvement.12 Sakata et al10 recently compared the results of PD-L1 testing in patients who underwent EBUS-TBNA followed by surgical resection for NSCLC over a 10-year period, and reported moderate concordance, but a significant decrease in the sensitivity of EBUS-TBNA for PD-L1 expression ≥50% of tumor cells.

The recent IASLC Atlas of PD-L1 immunohistochemistry in lung cancer warns against the use of cytology samples for determination of PD-L1 status, because none of the assays are validated for this purpose.13 Yet, EBUS-TBNA is frequently the only invasive procedure performed in patients with advanced lung cancer, as it provides the recommended, simultaneous diagnosis and nodal staging.10 We sought to examine the feasibility and results of PD-L1 testing performed on EBUS-TBNA samples acquired for the diagnosis and staging of NSCLC. These results were presented, in part, at the 2018 American Thoracic Society International Conference.


This was a retrospective study. Patients for whom PD-L1 testing was attempted on EBUS-TBNA samples, between June 2016 and December 2017, were identified from a prospectively maintained clinicoadministrative pathology database. The McGill University Health Centre Research Ethics Board approved the study (MP-37-2018-3657).

Patient demographics, procedural characteristics, and histopathologic diagnoses were extracted from electronic health records. All procedures were performed at the Montreal Chest Institute of the McGill University Health Centre, using a dedicated flexible bronchoscope with integrated ultrasound transducer (BF-UC160F; Olympus Canada Inc.). The needle gauge used, number of lymph node stations sampled, and number of needle passes were at the discretion of the operator. The occasional availability of rapid on-site evaluation (ROSE) was documented.

Specimens from each lymph node station were flushed into separate containers filled with small amounts of saline, to which CytoLyt (methanol-based fixative) was added after the procedure. The samples underwent centrifugation to prepare cell pellets. The cell pellets were fixed into 10% buffered neutral formalin for processing and then embedded into paraffin to make cell blocks. All cell blocks were cut into 4-μm thick sections and then stained with Dako PD-L1 22C3 pharmDx (Dako North America Inc., Carpinteria, CA) according to the manufacturer’s instructions.10 Sections were baked and deparaffinized, followed by antigen retrieval, and then stained on Dako Link autostainer system. Controls were prepared for each sample; positive controls were cell blocks provided by the vendor and benign tonsils. Negative controls were the cytology specimens without primary antibody.

Hematoxylin and eosin slides were reviewed by a cytopathologist (H.W.) to categorize cellularity between <100, 100 to 500, and >500 viable tumor cells. A minimum of 100 viable tumor cells are required for testing. The results of PD-L1 testing, expressed as Tumor Proportion Score (TPS), were tabulated. TPS≥50% was considered high PD-L1 expression. The concordance of PD-L1 results between EBUS-TBNA samples and additional histologic samples, when available, was examined. The time interval between the acquisitions of the different samples was recorded.

Statistical analyses were performed using SAS 9.4. Descriptive statistics were performed. The proportion of patients in whom PD-L1 testing was successfully performed on at least one of the available EBUS-TBNA samples, and the proportion of samples with <100, 100 to 500, and >500 viable tumor cells were estimated, along with associated confidence intervals. Logistic regression was used to examine potential clinical and/or procedural factors that may be associated with successful PD-L1 testing in EBUS-TBNA samples. The results of PD-L1 testing on EBUS-TBNA were compared with available histologic samples for the TPS cutoffs ≥1% and ≥50%. Concordance was calculated as the sum of concordant positive [both endobronchial ultrasound (EBUS) and tissue sample with TPS≥50%] and concordant negative (both samples with TPS<50%), divided by the total subgroup with additional, available tissue samples.


PD-L1 testing was attempted on a total of 120 EBUS-TBNA samples between June 2016 and December 2017. Baseline patient and procedural characteristics are detailed in Table 1. The mean age was 68.1 years (SD: 10.5 y), and 53% of patients were women. The most common NSCLC subtype was adenocarcinoma (78%), while squamous cell carcinoma accounted for 14% of cases. Over 90% of patients had locally advanced or metastatic disease. The vast majority of samples were acquired using a 22 G needle, and the median number of needle passes was 4 (range, 2 to 8). The majority of procedures (95%) were performed in the absence of ROSE by a cytopathologist. The EBUS samples selected for PD-L1 testing were acquired from a variety of nodal stations: subcarinal (24%), paratracheal (33%), and hilar (43%).

Patient and Procedural Characteristics

Successful PD-L1 testing was performed on 110/120 samples (92%), with testing canceled in 8 samples due to insufficient tumor cells (specimen cellularity as evaluated on hematoxylin and eosin slides is displayed in Fig. 1). The majority of samples consisted of >100 tumor cells: 76 specimens (63%) had a cellularity >500 tumor cells, while 34 (28%) had between 100 and 500 tumor cells (Table 2). Among patients with an adequate EBUS sample, 53/110 (48%) had high PD-L1 expression (TPS≥50%). TPS categorization of PD-L1 staining is shown in Figure 2.

Specimen cellularity assessed on hematoxylin and eosin slides. A, Less than 100 viable tumor cells. B, 100 to 500 viable tumor cells. C, More than 500 viable tumor cells. Arrow indicates viable tumor cells (×100 magnification).
PD-L1 Expression According to EBUS-TBNA Specimen Cellularity
Examples of programmed death ligand 1 immunocytochemical staining on endobronchial ultrasound–guided transbronchial needle aspiration cell blocks using 22C3 pharmDx kit. A, Tumor Proportion Score (TPS) <1%. B, TPS 1% to 49%. C, TPS≥50%. Arrow indicates viable tumor cells (×200 magnification).

No predictors of an adequate EBUS sample for PD-L1 testing were identified using logistic regression; these results are presented in a data supplement (Table E1, Supplemental Digital Content 1,

Eighteen patients had additional histologic samples that were tested for PD-L1, which allowed comparison with PD-L1 expression on EBUS-TBNA samples. These paired results are displayed in Table 3, with the time interval between acquisition of the various samples indicated. The median time between acquisition of histologic samples and needle aspirates was 47.5 days (interquartile range: 15 to 120 d). EBUS PD-L1 results were concordant with histologic samples in 14/18 patients (concordance 78%). Table 4 displays the data according to PD-L1 TPS (<1%, 1% to 49%, and ≥50%). With the cutoff of ≥50%, the sensitivity of EBUS-TBNA samples was 100%, with a specificity of 60%. There were no false-negative results.

Results of PD-L1 Testing in EBUS-TBNA Versus Additional Histology Samples


Immunotherapy has rapidly become an integral part of the management of patients with advanced NSCLC, with its use guided by levels of PD-L1 expression.4,9,14 The present study suggests that PD-L1 testing is feasible in the majority of EBUS-TBNA samples acquired for the diagnosis and staging of NSCLC. There was moderate concordance between EBUS results and histologic samples, with no false-negative results.

In the current study, EBUS-TBNA samples were adequate for evaluating PD-L1 expression in 92% of patients in whom it was attempted. Specimens acquired using EBUS-TBNA were the only samples available in the majority of patients. More than half the EBUS samples consisted of >500 tumor cells. The high success rate of PD-L1 testing on EBUS-TBNA samples is consistent with that reported by other authors,15–17 and the results of these recently published series are detailed in Table 5. Among the 110 patients with an adequate EBUS sample, 53/110 (48.2%) had positive PD-L1 expression defined as TPS≥50%; this is generally higher than has been previously reported.15–17

Comparison of EBUS PD-L1 Results Versus Additional Histology Samples

Recent series using cytologic samples and the 22C3 antibody have reported PD-L1 expression ≥50% in 33%, 10%, and 13% of patients.15–17 However, in the study by Lerner et al,18 using formalin-fixed cytology samples, high PD-L1 expression (≥50%) was reported in a comparable 47.3% of tumors. A combination of CytoLyt and 10% buffered neutral formalin fixation was used in the current study, and results do not support the recent suggestion that CytoLyt fixation may be responsible for lowered PD-L1 expression.19 CytoLyt-fixed samples are not specifically approved for PD-L1 testing. However, PD-L1 testing on CytoLyt-fixed samples has been previously reported,16 and a recent study by Wang et al20 showed no significant difference in PD-L1 expression between Cytolyt and formalin-fixed cytologic samples. Over 90% of patients in this series had advanced or metastatic disease, and high PD-L1 expression has been previously correlated with more advanced disease.20,21 Pembrolizumab has been approved as a first-line monotherapy for patients with metastatic lung cancer with PD-L1 expression ≥50% of tumor cells,9,22 and it is also conceivable that pathologists reviewing cytology samples may preferably classify borderline cases as PD-L1-positive, to ensure patients are not denied the potential benefits of immunotherapy.

Feasibility of PD-L1 Testing on EBUS Samples

The majority of EBUS samples in this series were acquired using the 22 G needle, with a median of 4 needle passes, and in the absence of ROSE. The recent Chest guidelines on technical aspects of EBUS-TBNA recommend that additional samples, beyond the ≥3 needed to establish a diagnosis, be obtained for molecular analysis. Either the 21 or 22 G needles are considered acceptable options.23 No data are currently available on the role of the 19 G needle in PD-L1 assessment. A recent study concluded to the benefits of ROSE in ensuring sufficient EBUS-TBNA specimens are acquired for PD-L1 testing (n=53).24 However, access to ROSE is variable across institutions, and the current results are reassuring in that mostly adequate samples were acquired in the absence of ROSE.

The results of PD-L1 testing in EBUS-TBNA were compared with those from additional histologic samples in a subset of patients and revealed moderate concordance (78%). Recent studies comparing the results of PD-L1 testing between histology and either EBUS-TBNA samples, or cytology specimens that included EBUS samples, are reviewed in more detail in Table 6. Sakakibara et al11 reported moderate correlation of PD-L1 results between EBUS-TBNA samples and either transbronchial biopsies (r=0.75, n=16) or resected specimens (r=0.75, n=6 for primary tumor and r=0.93, n=5 for lymph node metastasis). Similarly, moderate concordance was reported by Sakata et al,10 in 61 paired EBUS-TBNA samples and surgical resection specimens (82%, at PD-L1 cutoff≥50%).

Concordance of PD-L1 Results Between Cytology Samples That Include EBUS-TBNA, and Histology Samples

There were no false-negative PD-L1 EBUS results in the current series, whether at ≥1% or ≥50% cutoff. This is in contrast to the results of Sakata et al,10 where at ≥50% PD-L1 cutoff, EBUS misclassified the status of 8/15 PD-L1-positive tumors, leading the authors to warn of the limitations of EBUS-TBNA for assessment of PD-L1 status. Both studies used the 22C3 PD-L1 assay. In the current study, PD-L1 expression was evaluated using the Food and Drug Administration–approved Dako platform, rather than a laboratory-developed test. All patients in the Sakata series underwent surgical resection of their tumor, with 26% demonstrating high PD-L1 expression on resected specimen tumor cells. Stage differences may account for varying levels of PD-L1 expression, and could partly explain the limitations of EBUS in this setting. Finally, samples dating back to 2006 were tested for PD-L1, and fading of PD-L1 immunohistochemistry expression with storage time has been previously reported in NSCLC tissues.29

There are limitations to this study. This retrospective series is based on a clinicoadministrative database of pathologic samples for which PD-L1 testing was attempted. Samples are sent for molecular testing in the presence of a seemingly adequate cell block. As such, there may be patients who underwent EBUS-TBNA for diagnosis and staging of NSCLC in whom the EBUS sample was diagnostic of malignancy, but the cell block was insufficient to proceed with molecular testing. The institution’s tumor registry data were reviewed for the study period and suggests that 5% of all EBUS-TBNA samples that were diagnostic of malignancy would have been insufficient for molecular analysis, had it been indeed requested. To our knowledge, this is the largest series to date looking at PD-L1 testing in EBUS-TBNA samples exclusively. Additional histologic samples were available in only a fraction of patients who underwent EBUS-TBNA, but nevertheless these results contribute to the existing data on concordance between EBUS samples and histologic specimens.

Assessment of PD-L1 status has well-established technical and biological pitfalls as a biomarker.30 Both spatial and temporal heterogeneity of PD-L1 expression has been described,31 and may account for variable positivity levels across studies. There is currently an important disconnect between the specimens required in clinical trials and those acquired in daily practice, as recently reviewed by Beattie et al.30 As recommended by current guidelines,12 EBUS is frequently the first and only invasive diagnostic procedure performed in patients with advanced NSCLC, hence the importance of ensuring EBUS can provide an adequate assessment of PD-L1 status. It is hoped that tissue requirements for future clinical trials will more closely reflect tissue samples that are being acquired routinely in clinical practice.30 In addition, PD-L1 expression is likely to be supplemented by assessment of tumor mutation burden.31

In conclusion, PD-L1 testing was feasible in the majority of EBUS-TBNA samples acquired for the diagnosis and staging of NSCLC. High PD-L1 expression (TPS≥50%) was noted in a higher proportion of patients than previously reported in histologic samples, and this finding warrants further investigation. Comparison of EBUS results with histologic samples revealed moderate concordance, and no false-negative results. Future work will seek to examine the response to immunotherapy based on PD-L1 expression, as assessed in EBUS-TBNA samples.


The authors thank Luke Jeagal for his contribution to data collection and Pei-Zhi Li for assistance with statistical analyses.


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carcinoma; non–small cell lung; PD-L1; EBUS-TBNA

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