Endobronchial Ultrasound-guided Transbronchial Needle Aspiration Samples Research Articles

TB PCR in BAL and EBUS-TBNA samples for the diagnosis of pulmonary and mediastinal lymph node TB: retrospective TRiBE study

IntroductionThe role of Xpert Ultra in bronchoalveolar lavage (BAL) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples for pulmonary and mediastinal lymph node tuberculosis (TB) remains unclear.MethodsThis was a retrospective...

Utility of SHOX2 and RASSF1A gene methylation detection on the residual cytology material from endobronchial ultrasound-guided transbronchial needle aspiration.

This study aims to assess the effectiveness of Short Stature Homeobox 2 (SHOX2) and RAS Association Domain Family 1 Isoform A (RASSF1A) gene methylation detection in residual liquid-based cytology (LBC) materials from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and investigate the diagnostic accuracy of a comprehensive diagnostic approach. Between June 2022 and May 2023, a total of 110 cases that underwent EBUS-TBNA were enrolled in the study. SHOX2 and RASSF1A genes methylation detection using the residual cytological material, LBC, and cell block (CB) were conducted for each EBUS-TBNA case. The sensitivity and specificity of cytology, CB histopathology, SHOX2, and RASSF1A methylation in diagnosing EBUS-TBNA samples were determined based on follow-up data. Among the 72 cases confirmed as pulmonary carcinomas, the methylation test yielded positive results in 24 adenocarcinoma cases, 10 squamous cell carcinoma cases, and 14 small cell carcinoma cases. The sensitivity of the comprehensive diagnosis (combining LBC, CB, and methylation detection) in distinguishing metastatic pulmonary epithelial malignancies in mediastinal and hilar lymph nodes or masses from benign lesions was higher (97.22%, 70/72) than that of morphological diagnosis alone (LBC and CB) (88.89%, 64/72; P < 0.05). SHOX2 and RASSF1A methylation detection demonstrates a high sensitivity and negative predictive value in the identification of pulmonary epithelial malignancies and holds promise as a valuable ancillary approach to enhance morphological diagnosis of EBUS-TBNA.

Insufficient endobronchial ultrasound-guided transbronchial needle aspiration specimens. When and why? The analysis of criteria and reasons behind the insufficient specimens.

The classification terminology systems for pulmonary cytology specimens have recently emerged. Inadequate samples, classified as "nondiagnostic," raise challenges in determining the threshold of cell numbers and the risk of malignancy (ROM). The study retrospectively reviewed 248 endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples: 46 insufficient samples, 60 low cellularity samples, and 142 adequate samples. Characteristics as cellularity, number of benign and malignant cells, and background features were assessed. Receiver operating characteristic curve analysis was performed to establish cell sufficiency thresholds for the diagnosis. Out of the 248 samples analyzed, 108 were classified as benign, 94 as malignant, and 46 as insufficient. The study found that the cellularity thresholds for diagnosis in cell blocks and cytological samples were ≥50 cells and ≥100 cells, respectively. The thresholds for tumor cell counts were ≥1 - 10 cells for both types of cells, respectively. Considerably, some low cellularity samples were initially classified as insufficient despite meeting the diagnostic thresholds upon revision. The ROM varied across sample categories, with insufficient samples having a ROM of 10.9%, benign samples 15.7%, suspicious samples 92.0%, and malignant samples 100%. Insufficient EBUS-TBNA samples raise challenges in diagnosis and management. This study identified the root cause of insufficient samples, including factors related to humans, diagnostic methods, sampling, and laboratory processing. By understanding the root causes, diagnostic recommendations can be developed to improve the diagnostic process. The findings emphasize the importance of standardized classification and terminology systems for clear communication among healthcare professionals and institutions, ultimately improving patient care and enabling quality assurance measures.

Xpert MTB/RIF assay for the differential diagnosis between sarcoidosis and tuberculosis intrathoracic lymphadenopathy

BackgroundThe aim of this study was to evaluate the role of Xpert MTB/RIF assay in the detection of Mycobacterium tuberculosis for differentiating tuberculosis intrathoracic lymphadenopathy from sarcoidosis intrathoracic lymphadenopathy.MethodsThe patients who were suspected to having sarcoidosis or tuberculosis intrathoracic lymphadenopathy at the Shanghai Pulmonary Hospital between October 1, 2020 and June 30, 2021 were retrospectively evaluated in this study. All patients underwent endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and Xpert analysis. Differences in clinical and radiological features were recorded. The diagnostic performances of EBUS-TBNA Xpert, acid-fast bacilli, culture, and peripheral blood QuantiFERON-TB Gold (QFT) for differentiating sarcoidosis from tuberculosis intrathoracic lymphadenopathy were analyzed.ResultsA total of 119 patients were included in this analysis. Of those, 83 patients were finally diagnosed with sarcoidosis (N = 50) and tuberculosis (N = 33) intrathoracic lymphadenopathy. Young individuals were more likely to have tuberculosis versus sarcoidosis intrathoracic lymphadenopathy (P = 0.006). Markers of inflammation, including fever, leukocytes, and serum ferritin levels, were significantly higher in tuberculosis versus sarcoidosis intrathoracic lymphadenopathy (P < 0.01). Bilateral lung involvement and symmetry intrathoracic lymphadenopathy were more common in sarcoidosis intrathoracic lymphadenopathy (P < 0.01). In addition, the longest diameter of intrathoracic lymphadenopathy (in cm) was significantly larger in sarcoidosis intrathoracic lymphadenopathy (P = 0.001). However, the largest diameter of lung lesions was significantly shorter (P = 0.005). The sensitivity and specificity values of Xpert and QFT for differentiating these two diseases were 69.70% and 100%, and 96.43% and 91.84%, respectively.ConclusionXpert MTB/RIF is recommended for the diagnosis of tuberculosis intrathoracic lymphadenopathy using EBUS-TBNA samples. A negative QFT suggests the exclusion of the diagnosis of tuberculosis intrathoracic lymphadenopathy.

Diagnostic Performance and Cell Count of EBUS-TBNA Needle Gauges: A Prospective Trial.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a well-established diagnostic procedure for evaluating hilar and mediastinal lymphadenopathies and is the gold standard for lung cancer diagnosis and staging. Recent studies assessed the effectiveness of the 19-G flex needle in obtaining larger EBUS-TBNA samples, and prospective small series gave similar results in terms of diagnostic yield when testing different gauge needles. The lack of homogeneity between series and the small sample size of some prospective cohorts poses a limit to the validity of those results. This prospective controlled study compared the 19-G flex and 22-G needles in terms of diagnostic yield. An objective laboratory method was used to count cells and compare the two needles' cytologic yields. A prospective controlled study was conducted on 90 patients undergoing EBUS-TBNA for the diagnosis of hilar and mediastinal lymphadenopathies. The institutional ethic committee (IEO573) approved the study, and informed consent was obtained from all patients. A total of 90 patients were enrolled in this study, 84.4% of whom were diagnosed with malignancy and 15.6% with non-neoplastic disease. Sensitivity for malignancy was 93.4% (CI: 87.4-97.1%) for the 19-G needle and 92.6% (CI: 86.3-96.5%) for the 22-G needle (p = 0.80). The percentage of malignant cells in the cell block was 63.9% and 61.5% for the 22-G and 19-G needles, respectively. The cell count assessed by flow cytometry was 2071 cells/µL (IQR: 600,2265) with the 22-G needle and 2761 cells/µL (IQR: 505,3250) with the 19-G needle (p = 0.79). The malignant cell count was 0.05 × 103 cells/µL with the 22-G and 0.08 × 103 cells/µL with the 19-G needle (p = 0.70). There was no difference in the presence of tissue cores in the samples, and rapid on-site evaluation (ROSE) cellularity was comparable between the two needles. The 19-G flex EBUS-TBNA needle is comparable to the 22-G needle in terms of diagnostic yield for cyto-histological evaluation of hilar and mediastinal lymphadenopathies. There is no difference between the 19-G and 22-G needle cell counts evaluated by flow cytometry.

Endobronchial ultrasound-guided transbronchial needle aspiration for the diagnosis of pulmonary sarcoidosis: A 9-year experience at a single center.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is valuable for diagnosing pulmonary sarcoidosis. We aimed to evaluate the diagnostic yield of EBUS-TBNA and cytology in sarcoidosis during the first 9 years at our institution. Patients who underwent EBUS-TBNA for suspected sarcoidosis between January 2011 and November 2019 were identified retrospectively. EBUS-TBNA was performed with rapid on-site cytological evaluation of the samples. The final diagnosis was based on the pathology and/or cytology results, radiologic features, and clinical follow-up findings. The yield rate was analyzed annually. Eighty patients underwent 83 EBUS-TBNA procedures for suspected sarcoidosis. In total, 136 lymph nodes were sampled. The mean number of lymph node stations sampled was 2.0 ± 0.6; the mean number of needle passes per lymph node was 3.5 ± 0.8. Sixty-five patients were diagnosed with sarcoidosis, with a total of 68 procedures. Nonnecrotizing granulomatous inflammation was detected in the EBUS-TBNA samples from 49/68 procedures (yield rate: 72.1%). Of 19 patients with sarcoidosis who did not obtain a pathological diagnosis with EBUS-TBNA, epithelioid cells and/or multinuclear giant cells suggestive of granulomatous inflammation were detected in five. The sensitivity, specificity, positive predictive value, and negative predictive value (NPV) for pathological diagnosis of sarcoidosis using EBUS-TBNA were 72.1%, 100%, 100%, and 24.0%, respectively. On using cytology, the sensitivity and NPV increased to 79.4% and 26.3%, respectively. The yield rate did not increase until 2016. EBUS-TBNA is useful for diagnosing sarcoidosis. Cytology resulted in an additional yield rate of 7.3%, which improved as the number of cases increased.

EP16.03-022 Endobronchial Ultrasound Guided Transbronchial Needle Aspiration Sampling Is Sufficient for Lung Cancer Molecular Profiling

Molecular testing of non-small cell lung cancer (NSCLC) specimens for somatic gene alterations is important for identification of potentially efficacious targeted therapies, as well as avoidance of therapies that are unlikely to provide clinical benefit. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique that is effective for diagnosis and staging NSCLC. Although several studies have shown two to six needle passes are sufficient for adequate molecular profiling, concern remains that EBUS-TBNA samples do not provide enough material.

EBUS-TBNA: A 2-Year Experience from a Tertiary Pathology Centre with Cyto-Histological Correlation

Introduction: Endoscopic ultrasound-guided fine-needle aspiration and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) methods are expanding due to their minimally invasive nature using real-time image guidance, a low incidence of complications, and a diagnostic rate ranging from 89% to 93.6%. The application of EBUS-TBNA was able to downgrade 40% of patients with suspicion of stage III lung cancer on PET scan. Methods: In the present study, we analysed the diagnostic outcomes of EBUS cytology at the Laboratory of Pathology, Fimlab Laboratories, Tampere, Finland, during a 2-year period. In our analysis, 1176 EBUS samples from 486 patients that underwent EBUS-TBNA in Tampere University Hospital (TAYS) from January 2017 to December 2018 were reviewed. The mean age of the patients was 64 ± 14 (SD) years with the age range from 19 to 90 years. Cytospin cytology slides stained by Papanicolaou stain and cell blocks were performed in each sample. Further immunohistochemical stains were ordered by the pathologist on demand. Results: The diagnostic yield was 89.5% and the diagnostic accuracy was 77.9% in the present study. The sensitivity for malignancy in EBUS-TBNA diagnosis was 95.69% and the negative predictive value 96.75%. Cyto-histopathological diagnosis correlation was 54.7% (95% confidence interval [CI]: 40.6%–66.4%, p < 0.001), and the histological accuracy compared to the exact original diagnosis was 43.5%. When considering the histological diagnoses that support the original diagnosis, the correlation was 61.3% (95% CI: 48.4%–71.5%, p < 0.001) and the accuracy was 72.2%. The overall inadequate rate of EBUS-TBNA samples in this study was 10.5%. Conclusions: The EBUS-TBNA diagnostic yield, diagnostic accuracy, and inadequate rate in the present study are in line with previously published studies.

Accuracy of TB-PCR using endobronchial ultrasound guided trans-bronchial needle aspiration (EBUS-TBNA) samples in mediastinal granulomatous lymphadenopathy

IntroductionMediastinal granulomatous lymphadenopathies, such as tuberculous lymphadenitis, sarcoidosis, are frequently encountered by respiratory physicians, and their diagnosis is based on histological and microbiological tests. Endobronchial ultrasound-guided Trans bronchial needle aspiration (EBUS-TBNA) is widely used to perform mediastinal lymph node sampling. However, very limited data is available on the yield of polymerase chain reaction for Mycobacterium tuberculosis (TB-PCR) using EBUS-TBNA samples in patients with mediastinal granulomatous lymphadenopathy. Materials and methodsA retrospective study using a prospectively collected database was performed from January 1, 2018 to December 31, 2018, to evaluate the efficacy of the TB-PCR test using EBUS-TBNA samples in patients with benign mediastinal lymphadenopathy which included both granulomatous lymphadenopathy and reactive lymphadenopathy. The cohort with reactive lymphadenopathy acted as the control group of the study population. The patients with mediastinal lymphadenopathy who were awaiting EBUS-TBNA either for diagnostic evaluation of primary disease or for staging of a known malignancy were included in the study. The patients were then followed up for 1 year post procedure with clinical and radiological evaluation. ResultsOf the 310 patients with mediastinal lymphadenopathy who underwent EBUS-TBNA, 190 cases had a benign pathology with granulomatous lymphadenopathy in 120 and reactive lymphadenopathy in 70 patients. The sensitivity, specificity, the positive predictive value and the negative predictive value of TB-PCR was at 90%, 97.14%, 98.18%, and 85% respectively. The accuracy of TB-PCR is 92.63%. ConclusionTB-PCR using EBUS-TBNA samples is an effective tool for diagnosing mediastinal granulomatous lymphadenopathy. This technique can prevent further invasive interventions like mediastinoscopy in patients whose histological and microbiological tests are non-diagnostic. It should always be performed when tuberculosis is in the differential diagnosis of a patient with mediastinal lymphadenopathy.

Randomized Cross-over Trial of Endobronchial Ultrasound Transbronchial Needle Aspiration With or Without Suction in Suspected Malignant Lymphadenopathy.

The benefit from applying suction during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is unclear. The purpose of this study was to evaluate the impact of applying suction during EBUS-TBNA of lymph nodes (LN) on the diagnostic yield and specimen quality of EBUS-TBNA smears and cellblocks. We conducted a randomized blinded cross-over trial comparing the diagnostic yield and quality of EBUS-TBNA samples obtained with suction (S+) and without suction (S-) from suspected malignant mediastinal LN. Each LN was aspirated 4 times in a randomly assigned alternating order: S+/S-/S+/S- or S-/S+/S-/S+, resulting in 2 matched S+/S- smears pairs and 1 matched S+/S- cellblocks pairs. Specimen were assessed by a blinded cytopathologist. The primary outcome was the diagnostic yield of EBUS-TBNA smears and cellblocks. Secondary analyses accounted for LN size (≤2 vs. >2 cm) and station (mediastinal vs. hilar). Of 234 EBUS-TBNA smears and 114 cellblocks were obtained from 59 LN. The concordance of S+ and S- cellblocks diagnostic yield was low (43.8%; 95% confidence interval: 30.7-57.6) with a significantly higher diagnostic yield among S+ compared with S- cellblocks (56.1% vs. 10.5%, respectively, P<0.001), regardless of LN station and size. However, S+ and S- smears were moderately concordant (66.7%; 95% confidence interval: 57.4-75.1) with no overall difference in their respective diagnostic yield. Numerically higher diagnostic yield among S+ smears in LN≤2 cm or hilar LN was not statistically significant. Results were similar for specimen adequacy, sensitivity and diagnosis of malignancy. Applying suction during EBUS-TBNA of suspected malignant LN enhanced the diagnostic yield of cellblocks regardless of mediastinal LN station or size, but not of smears.

Adequacy of small biopsy and cytology specimens for comprehensive genomic profiling of patients with non-small-cell lung cancer to determine eligibility for immune checkpoint inhibitor and targeted therapy

AimsIn advanced-stage non-small-cell lung cancer (NSCLC), incomplete genotyping for guideline-recommended genomic biomarkers poses a significant challenge to making informed and timely clinical decisions. We report our institution’s experience in assessing...

Comparison of adequacy between transbronchial lung cryobiopsy samples and endobronchial ultrasound-guided transbronchial needle aspiration samples for next-generation sequencing analysis.

BackgroundMost lung cancer patients present with lesions in both lung fields and lymphadenopathy. Thus, transbronchial lung cryobiopsy (TBLC) and endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) are commonly performed for diagnosing lung cancer. However, the adequacy of these samples for next‐generation sequencing (NGS) analysis remains unclear. This study aimed to compare the adequacy between TBLC and EBUS‐TBNA samples for NGS analysis.MethodsThis retrospective cohort study included patients whose lung samples were collected via TBLC or EBUS‐TBNA and analyzed using NGS. Out of 46 genes, the number of genes in TBNA and TBLC samples that could not be assessed via NGS analysis was mainly evaluated.ResultsA total of 37 patients were included and classified into two groups (TBLC group, n = 18 and TBNA group, n = 19). The mean number of genes that could not be evaluated via NGS analysis was significantly lower in the TBLC group than in the TBNA group (0.9 vs. 10.3, P = 0.024). The median total area of tumor cells in TBLC samples was significantly greater than that in TBNA samples (6.3 [1.6–4.2] vs. 2.6 [0.2–17.3] mm2, P < 0.01). In the TBNA group, there were two fully inadequate samples for NGS analysis with a high degree of cell crush or low tumor content, while there was no fully inadequate sample in the TBLC group.ConclusionsTBLC is more effective in obtaining adequate samples for NGS analysis than EBUS‐TBNA. TBLC should be performed to obtain adequate samples for NGS analysis in lung cancer patients with target lesions in lung fields, even if they have lymphadenopathy.Key points Significant findings of the study The mean number of genes that could not be evaluated was significantly lower in TBLC samples than in EBUS‐TBNA samples (0.9 vs. 10.3, P = 0.024). TBLC could obtain adequate samples with a high concentration of uncrushed tumor cells for NGS. What this study adds To obtain samples for NGS analysis, the use of TBLC should be aggressively considered in lung‐cancer patients with target lesions located in lung fields, even if they have lymphadenopathy.

EBUS in optimizing non-small cell lung cancer diagnosis and treatment

BackgroundEndobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a commonly used minimally invasive method for the diagnosis and staging of lung cancer. In order to improve its diagnostic accuracy, rapid on-site cytologic evaluation (ROSE) is being utilized in some institutions. ROSE, performed by a cytopathologist in the examination room, allows the assessment of the adequacy of the collected samples, identifies malignant cells and sometimes establishes diagnosis on the spot, thus improving diagnostic sensitivity. As non-small cell lung carcinomas (NSCLC) require not only pathological subtyping, but also molecular characterization, obtaining the adequate amount of tissue is crucial. Only a limited number of studies have analyzed the suitability of EBUS-TBNA samples for assessment of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) and programmed death-ligand 1 (PD-L1) status.AimWe intended to examine the diagnostic yield of ROSE in NSCLC and the results and feasibility of molecular analysis performed on EBUS-TBNA small samples.Methods100 patients with lung tumors and hilar and/or mediastinal lymphadenopathy on CT or PET/CT scans were retrospectively identified over a 3-year period, from a prospectively maintained EBUS-TBNA database. All examinations were accompanied by on-site cytological exam - ROSE, histopathological exam (HPE) and, in the case of NSCLC, molecular testing. After the sampling of the lymph nodes, specimens were Diff-Quik stained and a rapid preliminary diagnosis was established. Immunohistochemistry and mutational testing were performed using cell blocks.ResultsAdenocarcinoma was the most frequent diagnosis in both ROSE (34%) and histopathology (53%). Overall sensitivity and positive predictive value of ROSE in NSCLC, considering HPE the gold standard, were 92.18% and 93.65%, respectively, with a specificity and negative predictive value of 75% and 70.58%, respectively. All samples that were tested for EGFR mutation and ALK rearrangement were adequate for analysis. The adequacy ratio for PD-L1 was 91.66%; 37.5% of patients showed a high PD-L1 expression level, with a tumor proportion score TPS ≥50%.ConclusionEBUS-TBNA is a valuable method for lung cancer diagnosis. ROSE proved to have a moderate prediction of the final diagnosis in NSCLC. Molecular analysis of EGFR, ALK and PD-L1 can be successfully accomplished on EBUS-TBNA small tissue samples.

Methylation Assessment for the Prediction of Malignancy in Mediastinal Adenopathies Obtained by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration in Patients with Lung Cancer.

The evaluation of mediastinal lymph nodes is critical for the correct staging of patients with lung cancer (LC). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for mediastinal staging, though unfortunately lymph node micrometastasis is often missed by cytological analysis. The aim of this study was to evaluate the predictive capacity of methylation biomarkers and provide a classification rule for predicting malignancy in false negative EBUS-TBNA samples. The study included 112 patients with a new or suspected diagnosis of LC that were referred to EBUS-TBNA. Methylation of p16/INK4a, MGMT, SHOX2, E-cadherin, DLEC1, and RASSF1A was quantified by nested methylation-specific qPCR in 218 EBUS-TBNA lymph node samples. Cross-validated linear regression models were evaluated to predict malignancy. According to EBUS-TBNA and final diagnosis, 90 samples were true positives for malignancy, 110 were true negatives, and 18 were false negatives. MGMT, SHOX2, and E-cadherin were the methylation markers that better predicted malignancy. The model including sex, age, short axis diameter and standard uptake value of adenopathy, and SHOX2 showed 82.7% cross-validated sensitivity and 82.4% specificity for the detection of malignant lymphadenopathies among negative cytology samples. Our results suggest that the predictive model approach proposed can complement EBUS-TBNA for mediastinal staging.

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.

Immunotherapy has become an integral part of management in patients with advanced non-small cell lung cancer (NSCLC). Programmed death ligand 1 (PD-L1) expression in at least 50% of tumor cells on histologic samples has been correlated with improved efficacy of the immune checkpoint inhibitor pembrolizumab. A limited number of studies have examined the suitability of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimens for assessment of PD-L1 status. 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. Patients were identified from a prospectively maintained pathology database. Baseline characteristics were tabulated. Hematoxylin and eosin slides were reviewed to categorize cellularity between <100, 100 to 500, and >500 viable tumor cells. Samples were tested using Dako's PD-L1 IHC 22C3 pharmDx kit, with a minimum of 100 viable tumor cells. For patients in whom additional tissue samples were available, the results of PD-L1 testing were compared. PD-L1 testing was attempted on 120 EBUS-TBNA samples. The most common NSCLC subtype was adenocarcinoma (78%). Seventy-six specimens (63%) had a cellularity >500 tumor cells. Among 110 of 120 (92%) patients with an adequate endobronchial ultrasound (EBUS) sample, 53 of 110 (48.2%) had high PD-L1 expression, defined as a Tumor Proportion Score ≥50%. EBUS PD-L1 results were concordant with an available histologic sample in 14 of 18 patients (78%), with no false-negative results. PD-L1 testing was feasible in the majority of EBUS-TBNA samples acquired for the diagnosis and staging of NSCLC. Comparison of EBUS results with histologic samples revealed moderate concordance, with no false-negative results.

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing.

Next‐generation sequencing (NGS) has been implemented in clinical oncology to analyze multiple genes and to guide therapy. In patients with advanced lung cancer, small biopsies such as computed tomography‐guided needle biopsy (CTNB), endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) and transbronchial biopsy (TBB) are less invasive and are preferable to resection to make a pathological diagnosis. However, the quality of DNA/RNA and NGS from small lung tumor biopsy samples is unknown. Between April 2017 and March 2018, 107 consecutive samples were obtained from thoracic tumors or metastatic sites for targeted NGS analysis. Fifteen samples were obtained through CTNB, 11 through EBUS‐TBNA, 11 through TBB and 70 through surgical resection. All samples were formalin‐fixed and paraffin‐embedded. DNA and RNA quality was measured using the ddCq method and the percentage of RNA fragments above 200 nucleotides (DV200), respectively. Our custommade probes were designed to capture exon sequences of 464 cancer‐related genes and transcripts of 463 genes. DNA and RNA yield from the 3 biopsy methods were similar, and less than the yield obtained from resected samples. The quality of DNA and RNA was similar across all methods. Overall, 12 of 15 CTNB samples (80%), all 11 EBUS‐TBNA samples, and 9 of 11 TBB samples (82%) underwent successful NGS assays from DNA. NGS analysis from RNA was successful in all 12 CTNB samples, 9 of 11 EBUS‐TBNA samples (82%), and 8 of 11 TBB samples (73%). CTNB, EBUS‐TBNA and TBB mostly resulted in adequate DNA and RNA quality and enabled high‐quality targeted NGS analysis.

Effect of Preparation Technique on Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Sample Adequacy: 3 Years of Experience from a Single Center.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used to sample mediastinal lymph nodes and lesions. However, the methodological details of an optimal preparation technique for aspirated material have not yet been clearly determined. This study was an evaluation of the effect of 2 preparation techniques on the adequacy of aspirated specimens. A retrospective analysis was performed of EBUS-TBNA samples obtained at the institution over a total of 36 months. Two periods were examined. Almost all of the aspirated material was smeared onto slides and fixed with 95% alcohol in the first period of the study. Subsequently, to improve diagnostic ability, a pair of slides was prepared from each needle pass: the first was air-dried, and the second was fixed in 95% alcohol. The remainder of the aspirate was kept for cell block analysis. In total, 462 samples were obtained from 260 patients. The overall sampling adequacy was 74% in the first and 81.1% in the second period (p < 0.05). Approximately 14% of the specimens included a sufficient number of cells for immunohistochemical cell block evaluation in the first period and 42% in the second period (p < 0.001). Histological subtyping of non-small cell lung carcinoma was determined in 18 (56.3%) and the primary origin of a tumor was determined based on morphological and immunohistochemical properties in 32 (84.2%) of the patients in the first and second periods, respectively. The access to a sufficient EBUS-TBNA sample and the ability to perform the appropriate preparation can impact the specific diagnosis and treatment of patient with a single procedure.

Flow cytometric analysis of lymphocyte profiles in mediastinal lymphadenopathy of sarcoidosis.

Lymphocyte profiles in mediastinal lymph nodes may reflect the immune status of patients with sarcoidosis. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is useful for the diagnosis of diseases with mediastinal lymphadenopathy including sarcoidosis. The purpose of this study was to determine lymphocyte profiles of lymph nodes in sarcoidosis by analyzing EBUS-TBNA samples. We prepared single cell suspensions from EBUS-TBNA samples of mediastinal lymph nodes from patients with sarcoidosis or lung cancer and analyzed surface markers (CD3, CD4, CD8, CD19, CD25) and FoxP3 expression in the resultant lymphocytes using flow cytometry. We studied 26 patients with sarcoidosis and 16 with lung cancer with mediastinal lymph node metastases. In sarcoidosis, the CD4/CD8 ratio was significantly more elevated in lymph nodes than in bronchoalveolar lavage fluid (P<0.001), although both were strongly correlated. The CD4/CD8 ratio was significantly higher in stage I than in stage II both in the BAL fluid and lymph nodes. When compared with lung cancer lymph node metastasis, the CD4/CD8 ratio was significantly higher in sarcoidosis, whereas the CD3/CD19 ratio was significantly higher in lung cancer. The proportion of regulatory T cells (CD4+, CD25+, FoxP3 high) did not differ between sarcoidosis and lung cancer samples. Lymphocyte profiles in mediastinal lymphadenopathy can be analyzed by flow cytometry of EBUS-TBNA samples. These findings might help elucidate the immunopathology of sarcoidosis.

Molecular Testing in EBUS-TBNA Specimens of Lung Adenocarcinoma: A Study of Concordance Between Cell Block Method and Liquid-Based Cytology in Appraising Sample Cellularity and EGFR Mutations.

Cytological endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimens of mediastinal lymph node metastasis are frequently used to perform concomitant diagnosis, staging and genetic testing in non-small-cell lung cancer (NSCLC). The purposes of this single-center retrospective study were to evaluate EBUS-TBNA samples' adequacy for molecular testingof epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), and to analyze the concordance between the cell block method and liquid-based cytology (LBC) in appraising the sample cellularity and in detecting EGFR mutation. We retrospectively examined 82 patients who underwent EBUS-TBNA from October 2012 to September 2015 and received a confirmed diagnosis of lymph node metastasis of lung adenocarcinoma. Each sample was processed using both cell block and LBC to carry out DNA analysis (adequacy criterion: tumor cell percentage >25%) and EGFR mutation testing. Fifty-four patients were male, 66 were current or former-smokers, and the median age was 67 years. The median size of sampled lymph nodes was 14.8 mm. Seventy-one and 66 samples were adequate to perform cell block and LBC, respectively. The κ-statistic (0.78) showed an excellent concordance. EGFR mutation was detected in eight patients using cell block and in seven using LBC, with a simple percentage agreement of 87.5%. ALK translocation was found in two patients. This study demonstrates the feasibility of EGFR mutation analysis with both cell block and LBC, with an excellent concordance between the two methods. Considering that the majority of advanced NSCLCs are diagnosed on cytology specimens, LBC is feasible and needs to be implemented for ancillary tests (immunocytochemistry, molecular analysis).

Rapid and Highly Sensitive Detection of Therapeutically Relevant Oncogenic Driver Mutations in EBUS-TBNA Specimens From Patients With Lung Adenocarcinoma

BackgroundFirst-line afatinib treatment prolongs overall survival in patients with metastatic non–small-cell lung cancer (NSCLC) harboring exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19) mutations. In contrast, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations are negative predictors for benefit from EGFR-targeting agents. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) allows for simultaneous interrogation for multiple mutations but has limitations (required tumor tissue amount, assay times). Reverse transcription polymerase chain reaction (RT-PCR) using light-Cycler technology (LCRT-PCR) can rapidly and sensitively detect somatic mutations from NSCLC patients. In the present study, we analyzed the feasibility of LCRT-PCR for rapid EGFRdelEx19 and KRAS exon 2 mutation detection in EBUS-TBNA samples and compared the LCRT-PCR and NGS results. Materials and MethodsA total of 48 EBUS-TBNA samples from 47 patients with a confirmed diagnosis of pulmonary adenocarcinoma were analyzed using LCRT-PCR (as previously described) and NGS (MiSeq; Illumina) using targeted resequencing and a customized multiplex PCR panel. The processing time was ∼1 week for the NGS and < 24 hours for the LCRT-PCR analyses. ResultsAll (100%) EGFRdelEx19 and KRAS exon 2 mutations detected by NGS were detected by LCRT-PCR. In addition, LCRT-PCR detected 2 KRAS exon 2 mutations and 3 EGFRdelEx19 mutations that were not detected by NGS. ConclusionLCRT-PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance (eg, EGFRdelEx19 and KRAS exon 2) in EBUS-TBNAs from NSCLC patients. It is of value as an initial assay for first-line treatment decisions.