Pilot Study of the Performance of 19-G Needle in Endobronchial Ultrasound-guided Transbronchial Aspiration for the Diagnosis and Testing of Molecular Markers in Lung Cancer : Journal of Bronchology & Interventional Pulmonology

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Original Investigations

Pilot Study of the Performance of 19-G Needle in Endobronchial Ultrasound-guided Transbronchial Aspiration for the Diagnosis and Testing of Molecular Markers in Lung Cancer

Wahidi, Momen M. MD, MBA*; Davidson, Kevin MD; Shofer, Scott MD, PhD*; Mahmood, Kamran MD, MPH*; Cheng, George MD, PhD*; Giovacchini, Coral MD*; Jones, Claudia MD; Jug, Rachel MB, BCh, BAO; Pavlisko, Elizabeth N. MD; Wang, Xiaofei PhD§; Gu, Lin MS§; Weimholt, Cody DO; Zhou, Zhongren MD; Chen, Alexander MD

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Journal of Bronchology & Interventional Pulmonology 28(3):p 209-214, July 2021. | DOI: 10.1097/LBR.0000000000000736
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Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become the standard for diagnosis and staging of lung cancer. Historically, 21- and 22-G needles have been paired with EBUS. We evaluated the performance of EBUS-TBNA using a larger 19-G needle in the assessment of tumor tissue obtained and success of testing for molecular markers.


We prospectively enrolled adult patients with lymphadenopathy concerning for metastatic lung cancer. Patients underwent diagnostic EBUS-TBNA utilizing 19-G needles. Cases of non–small cell lung cancer (NSCLC) were evaluated for programmed cell death receptor ligand (PD-L1) expression. Cases of adenocarcinoma or undifferentiated NSCLC were further evaluated for 3 molecular markers for driver mutations: epidermal growth factor receptor (EGFR), c-ros oncogene 1 (ROS-1), and anaplastic lymphoma kinase (ALK).


Fifty patients were enrolled and underwent EBUS-TBNA using 19-G needles. PD-L1 assay was successfully performed in 90% of NSCLC cases. In adenocarcinoma or undifferentiated NSCLC cases, the success rate in testing was 90% for EGFR and 86% for ALK. ROS-1 testing had a success rate of 67%; 24% of these specimens had adequate tumor cells but there was technical difficulty with the assay. Block quality was judged by total number of tumor cells per hematoxylin and eosin–stained slide of each cell block (58% of specimens had >500 cells and 22% had 200 to 500 cells). There were no adverse events.


EBUS-TBNA using 19-G needles can obtain a high number of tumor cells and has a high rate of success in performing assays for PD-L1, EGFR, and ALK in NSCLC patients without an increase in adverse events. The success rate of ROS-1 testing was lower.


Accurate diagnosis and staging of non–small cell lung cancer (NSCLC) is essential to determine prognosis and treatment options including surgical and medical therapies. Endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA) is gradually replacing mediastinoscopy as a less invasive, safe and cost-effective initial method to diagnose lung cancer and stage the mediastinum.1–3 Acceptance of EBUS-TBNA as a minimally invasive initial procedure is accompanied by an inherent trade off; cytologic specimens provided from needle aspirates yield smaller specimens than surgical biopsies from mediastinoscopy. In today’s management of NSCLC, it is no longer sufficient to just obtain a diagnosis and a subtype; rather a full analysis of tumor driver mutations must be obtained to guide individualized care for specific cancer biology.4 Currently, mutations with therapeutic implications in NSCLC include epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and c-ros oncogene 1 (ROS-1) with many more on the horizon. In addition to mutational analysis, there has been an interest in immunotherapy centered on inhibition of the programmed cell death receptor (PD-1) or its ligand (PD-L1) which, when expressed, can limit T-cell response to tumor cells. Clinical trials have demonstrated improved survival when using immunotherapy in patients with advanced NSCLC and PD-L1 expression.5,6

EBUS-TBNA has been shown to provide adequate cytologic samples for subclassifying NSCLC and determining molecular marker status but the demand on the tissue needed to test for additional driver mutations is threatening to exhaust the materials obtained by the needle. Success rates for EGFR, ALK, and ROS-1 testing have ranged between 80% and 94%.7–9 EBUS-TBNA has also been utilized to determine PD-L1 status and has been found to provide sufficient cytologic samples to adequately perform the assay.10,11 These studies were all performed with EBUS-TBNA needles in sizes 21- or 22-G. While one can argue that this success rate in testing is high enough with the smaller needle sizes, the new paradigm of treating NSCLC requires knowledge of molecular marker status in all patients and there is a possibility that smaller needle samples may not provide enough tissue tumor leading to additional procedures and repeat sampling of tissue.

A larger more flexible EBUS-TBNA needle (19-G) became available in the recent past and has been shown to provide larger sample sizes without an increase in complication rate.12,13 We hypothesized that the 19-G needle would provide a large number of tumor cells and may afford a high performance of testing for molecular markers and PD-L1 assays.


Study Design

We conducted a prospective pilot and feasibility study at 2 academic medical centers: Duke University and Washington University in Saint Louis.

Candidates for the study were consecutive adult patients with suspected lung cancer identified on chest imaging with associated hilar or mediastinal lymphadenopathy who were referred to undergo EBUS-TBNA sampling of the lymph nodes (LN). Patients were enrolled between August of 2017 and January of 2019 after they signed an informed consent as approved by the Institutional Review Board.


EBUS-TBNA was performed by experienced operators and followed the recommendation of national guidelines.14 All samplings were preformed using a ViziShot Flex 19-G needle (Olympus Respiratory America, Redmond, WA). Using standard sampling technique, the LN was penetrated with the needle as confirmed by ultrasound and the needle was passed back and forth through the node 10 times to obtain each sample. Suction was used during aspiration; the operator had the option of not using suction on subsequent aspiration attempts if the first sample obtained with the use of suction was deemed too bloody. Rapid on-site evaluation (ROSE) was available at each procedure to perform immediate assessment of diagnostic adequacy of obtained specimens. In cases with multiple enlarged LN, only the first LN sampled was included in the analysis. The order of sampled LN followed practice guidelines (highest stage LN sampled first). In cases where a target LN was negative for malignancy by ROSE or there were no other suspicious LN for malignancy (eg, <5 mm by EBUS sizing) then the primary lung lesion was chosen as a target for sampling if accessible through EBUS.

Cytology Specimens Preparation

A droplet of each needle pass was placed on a slide and smeared. Two slides were prepared for each pass: one slide was immediately placed in alcohol fixative for Papanicolaou stain, the other was air dried and stained with Diff Quik for ROSE evaluation to determine the presence of tumor. After the droplet was placed on the slide as above, the remaining sample was rinsed from the needle with either air or normal saline into the cell block solution.


The primary outcome of this study is to determine the success rate of testing for molecular markers (EGFR, ALK, and ROS-1) and PD-L1. EGFR, ALK, and ROS-1 were tested on every sample with adenocarcinoma or NSCLC using the following assays, respectively: rtPCR cobas EGFR Mutation Test v2 (Roche Diagnostics, North America), ALK Vysis ALK Break Apart FISH Probe Kit (Abbott, Chicago IL) and Vysis ROS-1 FISH probes (Abbott).

PD-L1 was tested on every sample with NSCLC. PD-L1 protein expression was determined from cell block specimens using the Dako PD-L1 22C3 pharmDx TM assay (Agilent, Santa Clara, CA). We recorded patient demographics including age, sex, race, and procedural details including number of needle passes and adverse events. Adverse events included any intraprocedural or postbronchoscopy complications. Bronchoscopists were asked to note any bleeding in the airways following needle puncture that required more than usual amount of suctioning.

Secondary outcomes included the following:

  • Tumor quantity: number of tumor cells per hematoxylin and eosin–stained section of each cell block using the following scoring scale: 0=no tumor cell (TC), 1=<200 TC, 2=200 to 500 TC, 3=>500 TC.
  • Average number of tumor cells in 10 high power fields of the area containing the highest number of tumor cells (tumor hot spot) using the following scoring scale: 0=no TC, 1=1 to 10 TC, 2=11 to 30 TC, 3=>30 TC.
  • Percent of tumor DNA in area selected for molecular analysis using the following scoring scale: 1=<10%, 2=10% to 20%, 3=21% to 40%, 4=>40%.

Statistical Analysis

Descriptive statistics were performed using SAS 9.4 (SAS, Cary, NC) to characterize study subjects and procedure, which include the mean for continuous variables and percentages for categorical variables, such as subject’s age at procedure performed, race, sex, the number of biopsy passes, location of LN, and adverse events. Descriptive statistics were also used to calculate the success rate of the procedure in successful performance of molecular marker and PDL-1 testing.


A total of 50 patients were enrolled. Basic demographic characteristics of patients included an average age of 65, male sex of 56% and a race of White in 78%, Black in 20%, and Asian in 2%. All patients completed the EBUS-TBNA procedure and underwent aspiration of the planned targets.

A list of targets and diagnoses are summarized in Table 1. The average number of passes per target was 5. The average size of the sampled LN or masses was 22.8 mm. Cytologic samples had full concordance between ROSE intraprocedural evaluation and final histologic result.

TABLE 1 - List of Sampled Targets and the Associated Diagnosis
Variables Targets (N=50) [N (%)]
Target location
 LN 4R 20 (40)
 LN 4L 9 (18)
 LN 7 5 (10)
 LN 11R 5 (10)
 LN 10L 2 (4)
 LN 2L 1 (2)
 LN 10R 1 (2)
 LN 11L 1 (2)
 LN 2R 1 (2)
 LN 12R 1 (2)
 LUL mass 1 (2)
 RUL mass 1 (2)
 RLL mass 1 (2)
 Right hilar mass 1 (2)
Cytologic diagnosis of target
 Adenocarcinoma 16 (32.0)
 Small cell carcinoma 15 (30.0)
 Squamous cell carcinoma 13 (26.0)
 Non–small cell carcinoma, not otherwise specified 3 (6.0)
 Other-pulmonary carcinosarcoma 1 (2.0)
 Other-undifferentiated carcinoma 1 (2.0)
 Other-carcinoma of unknown primary 1 (2.0)
LN indicates lymph node; LUL, left upper lobe; RLL, right lower lobe; RUL, right upper lobe.

Clinical Outcomes

The success rate of testing for EGFR was 90% (18/20) with 15% of patients testing positive for the mutation. The success rate of testing for ALK was 86% (18/21); no ALK rearrangements were detected.

For ROS-1, the success rate for testing was 67% (14/21); 24% of these specimens (5/21) were deemed to have adequate tumor tissue but experienced technical difficulty with the assay. Only 1 patient (7%) had a ROS-1 rearrangement detected.

PD-L1 assay was successfully performed in 28 of 31 NSCLC cases (90%), there was 1 case of NSCLC where PD-L1 was not performed on the EBUS specimen as the pathologist performed it on a biopsy sample obtained during the same procedure. Of those cases, 68% demonstrated positive expression (defined as >1% expression). Table 2 summarizes the results of testing success. Figure 1 shows an example of PD-L1 staining of a squamous cell carcinoma.

A, Endobronchial ultrasound-guided transbronchial needle aspiration specimen of squamous cell carcinoma using 19-G needle. B, Programmed cell death receptor ligand staining on the same specimen (75% positive).
TABLE 2 - Success of Testing for Molecular Markers and PD-L1
Test Outcome EGFR [n (%)] ALK [n (%)] ROS-1 [n (%)] PDL-1 [n (%)]
Inadequate tissue 2 (4.0) 2 (4.0) 2 (4.0) 3 (6.0)
Adequate tissue, technical difficulty 0 1 (2.0) 5 (10.0) 0
Negative 15 (30.0) 18 (36.0) 12 (24.0) 9 (18.0)
Not ordered 30 (60.0) 29 (58.0) 29 (58.0) 19 (38.0)
Positive 3 (6.0) 0 1 (2.0) 19 (38.0)
Atypical 0 0 1 (2.0) 0
Overall testing success rate (%) 90 86 67 90
ALK indicates anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; PD-L1, programmed cell death receptor ligand; ROS-1, c-ros oncogene 1.

Histologic Outcomes

Block quality was judged by total number of tumor cells per hematoxylin and eosin–stained slide of each cell block: 58% of specimens had >500 cells and 22% had 200 to 500 cells. Average number of tumor cells in 10 high power fields of the area containing the highest number of tumor cells showed that 84% of specimens had >30 tumor cells. The percentage of tumor DNA in areas selected for molecular analysis showed that 62% of specimens had >40% and 10% had 21% to 40%. Table 3 summarizes block quality test results.

TABLE 3 - Quality of Tumor Cells Slides
Tumor cells per H&E slide, n (%)
 0=no tumor 5 (10.0)
 1=<200 5 (10.0)
 2=200 to 500 11 (22.0)
 3=>500 29 (58.0)
Tumor cells per 10 HPF, n (%)
 0=No tumor 4 (8.0)
 1=1 to 10 3 (6.0)
 2=11 to 30 1 (2.0)
 3=>30 42 (84.0)
Percent of tumor DNA, n (%)
 1=<10% 9 (18.0)
 2=10 to 20% 5 (10.0)
 3=21 to 40% 5 (10.0)
 4=>40% 31 (62.0)
H&E indicates hematoxylin and eosin; HPF, high power fields.

Adverse Events

EBUS-TBNA procedures performed with 19-G needle demonstrated no adverse events. No excessive airway bleeding noted following puncture with the 19-G needle.


Despite advancements in lung cancer screening, the majority of patients continue to present with advanced stage disease at the time of diagnosis. With the development of targeted therapy for these advanced stages of lung cancer, obtaining adequate amounts of tissue and testing for molecular markers has become a priority for patients.

Immunotherapy has quickly become standard of care for subtypes of advanced stage NSCLC, and agents directed against PD-1 and PD-L1 are recommended based on improved clinical outcomes. NSCLC treatment guidelines now recommend testing for PD-L1 to identify patients who might benefit from immune checkpoint inhibitors.15,16 Original studies of PD-L1 were based on tissue expression, not on cytologic specimens using standard gauge needles; however, not all patients are suitable candidates for invasive biopsy because of comorbid conditions or specific tumor anatomy. While some studies have demonstrated the ability to perform PD-L1 and molecular marker assays on cytologic specimens, resource availability varies tremendously between institutions and there has been increasing interest in the ability to obtain larger amounts of tissue through minimally invasive, nonsurgical approaches.

The results of this pilot and feasibility study suggest that performing EBUS-TBNA with a 19-G needle results in a high success rate in testing for molecular markers and immune checkpoint inhibitor targets.

The success rate of testing EGFR, ALK, and PD-L1 was high in this study but testing for ROS-1 was less successful. Following discussions with pathology, this was attributed to difficulty with the assay as opposed to adequacy of the number of tumor cells. The ROS-1 assay was found to be challenging for 2 reasons: the ROS-1 FISH probe covers a small range of base pairs in comparison to other probes making them less visible under the fluorescent microscope. The second factor deals with tissue integrity. As cytology specimens tend to have less tissue architecture, there are more cells lost from the slide during the process of digestion and application of the probe. It is important to note that this is not an issue related to the needle size or the number of malignant cells obtained but rather an assay and probe issue. Newer assays are now overcoming this issue.

Not every patient in this study had testing for molecular markers and PD-L1 as we encountered cancer diagnoses that do not require such testing (small cell lung cancer and carcinosarcoma); in our sample of 50 patients, EGFR, ALK, ROS-1, and PD-L1 were performed in 40%, 42%, 42%, and 62% of patients, respectively. This made our sample smaller and weakened our study.

While this study was designed as a pilot and feasibility study assessing the 19-G needle’s ability to provide adequate material for molecular analysis, an added benefit was establishing a method for objectively analyzing biopsy specimens for quantity of tumor cells and specimen quality. Given the variability in institutional practice regarding the process of analyzing molecular marker specimens, we feel strongly that reporting an objective quantification of tumor burden in any study on specimen acquisition is as important as reporting the ability to perform tests of individual markers. We do acknowledge that these cytologic metrics are novel and future studies with different quantitative techniques should be conducted to support our findings.

As clinical practice shifts from single gene testing (as performed in this study) to next-generation sequence testing to investigate multiple genes or even entire exomes,17 it is more important than ever to provide adequate tissue material to stratify testing needed for the lung cancer patient.

The primary limitations of this study are the relatively small sample size as well as the utilization of 2 tertiary care, academic institutions with dedicated cytopathology programs as study sites. Both of these aspects may limit generalizability of the results of this study. In addition, this was designed as a pilot and feasibility study, and therefore contained no control arm. There also could have been some selection bias leading the investigators to pick patients who might have fared better with EBUS-TBNA sampling.

Our study did not prove that the 19-G needle is better than the 22- or 21-G needles; it merely showed that the 19-G needle can provide ample tissue and facilitate a high success rate in molecular marker and PD-L1 testing. In addition, the 19-G could have theoretically performed worse for tissue analysis because of blood contamination and additional trauma compared with the smaller needles but our findings refute these concerns.

In conclusion, this pilot study demonstrated a favorable performance of EBUS-TBNA using a 19-G needle in obtaining a large number of tumor cells and achieving a high success rate in performing assays for PD-L1, EGFR, and ALK in NSCLC patients without an increase in adverse events. The success rate of ROS-1 testing was lower because of difficulty in the assay and not because of the adequacy of tumor cells. Additional investigation regarding specimen adequacy for advanced molecular analysis should be comparative to smaller needle sizes to establish whether large gauge needles are superior to small gauge needles. Future studies should utilize an objective metric of specimen quantity and quality in a multidisciplinary manner inclusive of bronchoscopists and pathologists.


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lung cancer; EBUS; molecular markers; PD-L1

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