Ultrasound-guided Fine-needle Aspiration Specimens Research Articles

Liquid-based cytology features of pancreatic acinar cell carcinoma: comparison with other non-ductal neoplasms of the pancreas.

Acinar cell carcinoma (ACC) is a rare malignant epithelial neoplasm, which shares many cytomorphological features with other non-ductal pancreatic neoplasms such as pancreatic neuroendocrine neoplasm (PanNEN) and solid-pseudopapillary neoplasm (SPN). Due to the relative rarity of these tumors, pathologists are less familiar with the cytological features, especially on liquid-based cytology (LBC) which has been relatively recently introduced for endoscopic ultrasound-guided fine needle aspiration specimens. We evaluated the detailed cytological features of 15 histologically confirmed ACC (7 conventional smears [CS], 8 LBC), and compared them with the LBC features of SPN (n = 9) and PanNEN (n = 9). Compared with CS, LBCs of ACC demonstrated significantly less bloody background. All ACCs demonstrated prominent nucleoli and macronucleoli on LBC. On comparison with the LBC features of SPN and PanNEN, most ACCs demonstrated a necrotic background with apoptotic debris while PanNEN and SPN did not show these features. Acinar structures were predominantly observed in ACC, while frequent pseudopapillary structures were seen only in SPN. Prominent nucleoli and macronucleoli were only seen in ACC. ACC had characteristic cytological features that could be observed on LBC preparations, such as high cellularity, necrotic/apoptotic background, nuclear tangles, acinar arrangement of cells, and macronucleoli. These findings also help distinguish ACC from PanNEN and SPN on LBC. It is important to be familiar with these features, as an accurate diagnosis on endoscopic ultrasound-guided fine needle aspiration cytology would have impact on the management of the patient.

Tissue processing of endoscopic ultrasound-guided fine-needle aspiration specimens from solid pancreatic lesions.

Now that tissue cores can be obtained using fine-needle biopsy (FNB) needles, the ways tissues are handled for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) are changing. Direct smear, touch smear of core tissues, and centrifugation have been used for cytological examinations, and liquid-based cytology (LBC), which allows immunostaining and genetic tests that use residual samples, is emerging as an alternative. We emphasize that liquid cytology (Cytospin™ cytology and LBC) is still important, because it enables the diagnosis of pancreatic ductal adenocarcinoma (PDAC) when cancerous cells are scarce in specimens. Cell blocks are being replaced by core tissues obtained via FNB needles. Recent reports indicate that rapid on-site evaluation (ROSE) is not necessary when FNB needles are used, and macroscopic on-site evaluation is used to evaluate specimen adequacy. Macroscopic findings of specimens are helpful in the diagnostic workup and for clarifying specimen-handling methods. In addition to the red strings and white cores observed in PDAC, mixed red and white strings, gray tissues, and gelatinous tissues are observed. Gray (necrotic) tissues and gelatinous (mucus) tissues are more suitable than histology for cell block or cytological processing. Tumor cells in neuroendocrine tumors (NETs) are numerous in red strings but cannot be observed macroscopically. ROSE might thus be necessary for lesions that may be NETs. Core tissues can be used for genetic tests, such as those used for KRAS mutations and comprehensive genomic profiling. Cytological materials, including slides and LBC specimens, can also be genetic test materials.

A case of pancreatic neuroendocrine tumor with amyloid deposition (insulinoma) predicted by rapid on-site evaluation of pancreatic endoscopic ultrasound-guided fine-needle aspiration specimen

A case of pancreatic neuroendocrine tumor with amyloid deposition (insulinoma) predicted by rapid on-site evaluation of pancreatic endoscopic ultrasound-guided fine-needle aspiration specimen

A New Preoperative Scoring System for Predicting Aggressiveness of Non-Functioning Pancreatic Neuroendocrine Neoplasms.

The management of non-functioning pancreatic neuroendocrine neoplasms (NF-PanNENs) is still controversial. This study aimed to develop a new scoring system for treatment decisions at initial diagnosis based on the identification of the predictive factors for aggressive NF-PanNENs. Seventy-seven patients who had been pathologically diagnosed with NF-PanNENs were enrolled. We retrospectively reviewed 13 variables that could be assessed preoperatively. Univariate and multivariate stepwise logistic regression analyses were performed to identify factors for the aggressiveness of NF-PanNENs, and a scoring system was developed by assigning weighted points proportional to their β regression coefficient. Tumor size > 20 mm on contrast-enhanced computed tomography, tumor non-vascularity, and Ki-67 labeling index ≥5% on endoscopic ultrasound-guided fine-needle aspiration specimens were identified as independent factors for predicting the aggressiveness of NF-PanNENs. The new scoring system, developed using the identified factors, had an excellent discrimination ability, with area under the curve of 0.92 (95% CI, 0.85–0.99), and good calibration (p = 0.72, Hosmer-Lemeshow test). Ten-year overall survival rates in low-risk (0 point), intermediate-risk (1 to 2 points), and high-risk (3 to 4 points) groups were 100%, 90.9%, and 24.3%, respectively. This new scoring system would be useful for treatment decisions and prognostic prediction at initial diagnosis.

The Need for Histological Preparation of Endoscopic Ultrasound-guided Fine-needle Aspiration Specimens to Diagnose Rare Pancreatic Etiologies.

The Need for Histological Preparation of Endoscopic Ultrasound-guided Fine-needle Aspiration Specimens to Diagnose Rare Pancreatic Etiologies.

Establishment of pancreatic cancer organoids with endoscopic ultrasonography-guided fine-needle aspiration specimen: a prospective pilot study

Objective To study the feasibility of establishing human 3D pancreatic cancer organoids with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimen. Methods A total of 9 patients with suspected pancreatic masses were prospectively included in this study from June 2017 to January 2018. EUS-FNA was performed for initial diagnosis. The biopsy tissues were obtained from a COOK 22-gauge FNA needle for organoid establishment, and the growth status in vitro was observed. Results Nine specimens of pancreatic lesions obtained from EUS-FNA were enrolled. Successful establishment of organoids was achieved in 5 cases, which were all confirmed to be pancreatic cancer histopathologically. In the process of generating, the growth rate of organoids increased correspondingly. The pathological morphology of these organoids was similar to the corresponding pancreatic tissues in HE staining. Conclusion Pancreatic cancer organoids can be successfully created by means of EUS-FNA. Establishment of these organoids can potentially provide excellent models for patients with pancreatic cancer in guiding precision treatment. Key words: Carcinoma, pancreatic ductal; Endoscopic ultrasonography-guided fine-needle aspiration; Organoid; Precision medicine

Cell block processing is optimal for assessing endoscopic ultrasound fine needle aspiration specimens of pancreatic mucinous cysts

AimsThe cell block technique for assessing endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) specimens from pancreatic mucinous cystic lesions (MCLs) was systematically evaluated for the first time, including comparisons with three...

Instant messenger smartphone application for endosonographer/cytopathologist real-time interaction at a distance in EUS-FNA for solid pancreatic lesions.

Background and study aims Telecytopathology (TCP) may allow proper and timely evaluation of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens. The aim of this study was to evaluate the feasibility of TC through a multiplatform instant messenger smartphone application to evaluate specimens of EUS-FNA of pancreatic solid lesions. Patients and methods Twenty-three patients (14 male/9 female; median age: 56 yr.; age range: 33 – 86) with a solid pancreatic lesion were included. Exclusion criteria were as follows: age < 18 yr and predominantly cystic lesions. During each EUS-FNA, after each pass, the aspirated material was spread over a glass slide and was stained by the endoscopist. The glass slide was then reviewed on a microscope with a smartphone fitted in, and the most representative fields were captured and sent to the cytopathologist using WhatsApp Messenger. Results In initial evaluation using TCP rapid on-site evaluation (ROSE), adequate cellularity of the glass slide was detected in 16 of 23 patients (69.6 %). An initial diagnosis of malignancy (positive or suspicious) was possible in 14 of 23 patients (60.8 %). Conclusion The current study demonstrated the feasibility of a low-cost, Internet-based, telecytopathology system using WhatsApp Messenger to provide ROSE of EUS-FNA slides in patients with solid pancreatic lesions.

Diagnostic ability of artificial intelligence using deep learning analysis of cyst fluid in differentiating malignant from benign pancreatic cystic lesions

The diagnosis of pancreatic cystic lesions remains challenging. This study aimed to investigate the diagnostic ability of carcinoembryonic antigen (CEA), cytology, and artificial intelligence (AI) by deep learning using cyst fluid in differentiating malignant from benign cystic lesions. We retrospectively reviewed 85 patients who underwent pancreatic cyst fluid analysis of surgical specimens or endoscopic ultrasound-guided fine-needle aspiration specimens. AI using deep learning was used to construct a diagnostic algorithm. CEA, carbohydrate antigen 19-9, carbohydrate antigen 125, amylase in the cyst fluid, sex, cyst location, connection of the pancreatic duct and cyst, type of cyst, and cytology were keyed into the AI algorithm, and the malignant predictive value of the output was calculated. Area under receiver-operating characteristics curves for the diagnostic ability of malignant cystic lesions were 0.719 (CEA), 0.739 (cytology), and 0.966 (AI). In the diagnostic ability of malignant cystic lesions, sensitivity, specificity, and accuracy of AI were 95.7%, 91.9%, and 92.9%, respectively. AI sensitivity was higher than that of CEA (60.9%, p = 0.021) and cytology (47.8%, p = 0.001). AI accuracy was also higher than CEA (71.8%, p < 0.001) and cytology (85.9%, p = 0.210). AI may improve the diagnostic ability in differentiating malignant from benign pancreatic cystic lesions.

Abstract 3331: Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis

Abstract Pancreatic cancer is a highly lethal malignancy despite recent clinical advances. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop noninvasive biomarkers for detection. Epigenetic alterations have been recognized as a common hallmark of many types of human cancers as well as pancreatic cancer. The cell-free DNA (cfDNA) in blood possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first we performed genome-wide DNA methylation analysis of 38 endoscopic ultrasound-guided fine-needle aspiration specimens of pancreatic cancer using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. Analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated in at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status of these markers in the validation cohort (n=46) by bisulfite pyrosequencing or quantitative methylation-specific PCR and found that 42/46 (91.3%) cases were methylated in at least one marker. Finally, we analyzed methylation status in cfDNA from pancreatic cancer patients. We performed bisulfite sequencing using next-generation sequencer and detected methylation in only 5/13 (38.5%) samples in one of the markers. Since the amount of DNA from cancer tissue is very small, we need to develop more a sensitive technique to detect aberrant DNA methylation to diagnose cancer patients. Now we are developing a new sensitive method to detect DNA methylation in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Miho Suzuki, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3331.

Endobronchial ultrasound-guided transbronchial fine needle aspiration: Determinants of adequacy.

This study aims to evaluate the factors influencing the adequacy of endobronchial ultrasound-guided transbronchial fine needle aspiration specimens. A retrospective analysis of 1,700 endobronchial ultrasound-guided transbronchial fine needle aspiration samples obtained from 822 patients (500 males, 322 females; mean age 56±13 years; range 16 to 83 years) was performed between March 2011 and March 2014 at our center. Variables potentially associated with sampling adequacy, such as all cytological materials and procedure notes (lymph node and/or lesion size, localization, needle pass number, and slide number) were examined. The overall specimen adequacy was 79.8%. The specimen adequacy was associated with needle pass number (p≤0.001). Adequacy rate was 66.9% for one needle pass and 85.8% for three needle passes. According to the sampling regions, adequacy rates showed a difference [69.2%-85.8%; (p≤0.005)]. In the multivariate logistic regression analysis of subcarinal (7) lymph node station, patient age (odds ratio, 0.983; 95% confidence interval, 0.966-1.000; p=0.049) and number of slides (odds ratio, 1.240; 95% confidence interval, 1.062-1.448; p=0.006) were independent determining factors of specimen adequacy. While independent determinants of specimen adequacy for the right paratracheal (4R) region were lymph node size (odds ratio, 1.486; 95% confidence interval, 0.973-2.268; p=0.067) and number of slides (odds ratio, 1.418; 95% confidence interval, 1.146-1.756; p=0.001), they were lymph node size (odds ratio, 1.594; 95% confidence interval, 0.960-2.645; p=0.071) and number of needle passes (odds ratio, 2.277; 95% confidence interval, 1.360-3.811; p=0.002) for the right interlobar (11R) region. Independent determinant of specimen adequacy for the left paratracheal (4L) lymph node station was the number of needle passes (odds ratio, 1.656; 95% confidence interval, 0.955-2.869; p=0.072). During endobronchial ultrasound-guided transbronchial fine needle aspirations, particularly when rapid on site evaluation cannot be applied, consideration of factors affecting adequacy according to lymph node localizations may increase the chance for obtaining materials with suitable quality for cytologic evaluation.

Cytological characteristics of atypical cells in endoscopic ultrasound-guided fine-needle aspiration specimens obtained from the pancreas

Cytological characteristics of atypical cells in endoscopic ultrasound-guided fine-needle aspiration specimens obtained from the pancreas

Immunohistochemical analysis of IMP3 and p53 expression in endoscopic ultrasound-guided fine needle aspiration and resected specimens of pancreatic diseases

BackgroundInsulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3) is a valuable marker that distinguishes malignant from benign lesions and predicts prognosis. MethodsFirst, we evaluated IMP3 expression in 77 resected specimens of pancreatic ductal adenocarcinoma (PDAC), intraductal papillary mucinous neoplasm (IPMN), and chronic pancreatitis (CP). Eleven PDAC patients preoperatively underwent endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Survival analysis of IMP3 and clinicopathological factors was performed. IMP3 and p53 expression was evaluated in another 127 EUS-FNA samples of solid pancreatic masses to compare the diagnostic value of routine and immunohistochemical staining. ResultsIMP3 expression was detected in 72.3%, 50%, 20%, and 0% of PDAC, malignant IPMN, benign IPMN, and CP, respectively. Evaluation of IMP3 expression in EUS-FNA specimens coincided with that in resected specimens in 10 of 11. IMP3 expression correlated with tumor differentiation in PDAC samples (p = .006) and with poor prognosis through univariate analysis (p = .045). Tumor differentiation and lymph node metastasis were significantly associated with poor prognosis through multivariate analysis. In EUS-FNA specimens, the sensitivity, specificity, and accuracy of cytohistological analysis were 80.8%, 100%, and 85.0%, respectively. IMP3 and p53 expression were detected in 80.8% and 44.9% of malignant and 0% and 5% of benign lesions. Combined with IMP3 immunostaining, the sensitivity, specificity and accuracy of cytohistological analysis significantly increased to 87.9%, 100%, and 90.8% (p = .016), respectively. Meanwhile, p53 staining had no impact on the results. ConclusionsIMP3 immunohistochemical staining can improve the diagnostic accuracy of EUS-FNA for malignant pancreatic tumors.

Young Investigator Challenge: A novel, simple method for cell block preparation, implementation, and use over 2 years.

The cell block is an essential adjunct to conventional cytopreparatory techniques. The need for molecular analysis and immunostains will increase the need for successful cell block preparation. Even with this need, to the authors' knowledge very little has changed regarding the way in which cell blocks are produced. The authors developed A Formalin-Fixed, paraffin Embedded Cytology cell block Technique (AFFECT) that uses a cytospin centrifuge and funnel to deposit a cell pellet into a well on a piece of open-cell, absorbent foam. The foam and the pellet are then sent through normal processing. Herein, the authors present the implementation of this method and some of their experience with its performance over the course of 2 years. Although a comparison of the methods indicated good correlation for the production of a cell block between AFFECT and the agarose method, the AFFECT blocks demonstrated markedly improved cellular morphology. Over the first 6 months of use, AFFECT produced a successful cell block in 74% of cases overall, and in 65% of cases with a cell pellet measuring ≤0.1 mL. The year preceding the implementation of AFFECT and its first year of use were compared for endoscopic and bronchoscopic ultrasound-guided fine-needle aspiration specimens, and demonstrated an improved success rate. The authors developed a novel method of cell block preparation that demonstrates improved histology and has increased the success rate of cell block production compared with the agarose method. Cancer Cytopathol 2016;124:885-892. © 2016 American Cancer Society.

Fine-Needle Aspiration with Immunohistochemistry Using a Modified Scrape Cell Block Technique for the Diagnosis of Thyroid and Parathyroid Nodules

Objective: To evaluate the diagnostic accuracy of a modified scrape cell block (SCB) technique in a large series of patients. The technique was especially developed and tested for fine-needle aspiration of thyroid and parathyroid nodules. Study Design: Eighty-two ultrasound-guided fine-needle aspiration specimens with the sonographic aspect of a thyroid (n = 33) or a possible parathyroid nodule (n = 49) were studied. Immunohistochemistry (IHC) was used on cell blocks containing plasma, thromboplastin, and selected 3-dimensional cell aggregates scraped off Papanicolaou-stained smears. Antibodies for chromogranin A, thyroglobulin, parathyroid hormone, calcitonin, and carcinoembryonic antibody (CEA) were used. In cases of reduced immunosensitivity or suspected metastases or rare primary tumors, additional IHC markers were employed. Results: Chromogranin A was expressed in all 28 parathyroid adenomas (PA), in 7 of 8 hyperplastic parathyroid glands, and in 13 of 14 medullary thyroid carcinomas (MTC). When combining positivity for chromogranin A and calcitonin/CEA, the specificity for the detection of MTC was 100%. Parathyroid hormone was expressed in 26 of 36 parathyroid nodules (72.2%). When combining follicular microarchitecture and expression of chromogranin A, the specificity for the detection of parathyroid tissue was 97%. Conclusion: With the modified SCB technique, accurate cytological diagnoses were obtained in 97.6% of 82 patients.

Use of the BRAF V600E mutation in fine-needle aspirations of thyroid nodules

Objective To evaluate the diagnostic and prognostic value of BRAF V600E mutation screening of ultrasound-guided fine-needle aspiration (FNA) specimens in patients with thyroid nodule. Methods The BRAF V600E mutation status were assessed in FNA specimens of 104 patients with thyroid nodules before operations. The BRAF mutation status, clinical, and pathology records of the patients were reviewed and the associations between these characteristics and papillary thyroid cancer(PTC) were analyzed. Results Seventy-one PTC and 14 benign thyroid nodules were included in this study. BRAF V600E mutations were found in 57/71(80%) PTC. All benign thyroid nodules had no BRAF V600E mutation. The sensitivity, specificity, positive predictive value and negative predictive value of BRAF V600E mutations in differentiation between PTC and benign thyroid nodules were 80%, 100%, 100% and 50% (P<0.001). In 44 patients with PTC who underwent surgery, the central compartment lymph node metastases and extrathyroidal invasion were not significantly different between BRAF-positive and BRAF-negative PTC(P=0.283 and 0.307). Conclusions BRAF V600E mutation may be a potential tool to facilitate ultrasound in diagnosis of PTC. In patients with PTC, the presence of the BRAFV600E mutation was not significantly associated with prognostic factors. Key words: Ultrasonography; Thyroid neoplasms; Biopsy, fine-needle; BRAF mutation

Mucin Expression in Endoscopic Ultrasound-Guided Fine-Needle Aspiration Specimens Is a Useful Prognostic Factor in Pancreatic Ductal Adenocarcinoma

The aim of this study was to further examine the utility of mucin (MUC) expression profiles as prognostic factors in pancreatic ductal adenocarcinoma (PDAC). Mucin expression was examined by immunohistochemistry analysis in endoscopic ultrasound-guided fine-needle aspiration specimens obtained from 114 patients with PDAC. The rate of expression of each MUC was compared with clinicopathologic features. The expression rates of MUCs in cancer lesions were MUC1, 87.7%; MUC2, 0.8%; MUC4, 93.0%; MUC5AC, 78.9%; MUC6, 24.6%; and MUC16, 67.5%. MUC1 and MUC4 were positive, and MUC2 was negative in most PDACs. Patients with advanced stage of PDAC with MUC5AC expression had a significantly better outcome than those who were MUC5AC-negative (P = 0.002). With increasing clinical stage, total MUC6 expression decreased (P for trend = 0.001) and MUC16 cytoplasmic expression increased (P for trend = 0.02). The prognosis of patients with MUC16 cytoplasmic expression was significantly poorer than those without this expression. Multivariate survival analysis revealed that MUC16 cytoplasmic expression was a significant independent predictor of a poor prognosis after adjusting for the effects of other prognostic factors (P = 0.002). Mucin expression profiles in ultrasound-guided fine-needle aspiration specimens have excellent diagnostic utility and are useful predictors of outcome in patients with PDAC.

Low interobserver agreement in cytology grading of mucinous pancreatic neoplasms.

Identifying high-grade features in patients with pancreatic mucinous neoplasms (MNs) is important for patient management. The reproducibility of MN cytology grading has been evaluated to a limited extent. In the current study, the authors evaluated interobserver variability in grading MNs and the identification of neoplastic mucin in endoscopic ultrasound-guided fine-needle aspiration specimens. A 54-case grading set was created from histologically confirmed MNs (44 MNs) and nonmucinous lesions with abundant gastrointestinal contamination (10 nonmucinous lesions). Six observers received a tutorial, reviewed prescreened slides, and recorded: 1) a diagnosis according to a 6-tiered system (TS) (nondiagnostic, atypical [ATP], mucinous cyst low grade [MCLG], mucinous cyst high grade, suspicious for adenocarcinoma, and positive for adenocarcinoma); 2) the cyst fluid carcinoembryonic antigen diagnosis (CEADX); and 3) the presence of neoplastic musin. Interobserver agreement (IOA) was evaluated by calculation of kappa coefficients (Kappa). Diagnostic accuracy was not evaluated. The IOA was lowest for the 6-TS (Kappa, 0.13; P<.001). The CEADX was available for 18 cases (33%), including 6 of 24 MCLG cases (25%). CEADX modestly improved IOA for combined tiers of the 6-TS with ATP and MCLG as separate categories. The highest IOA was noted with a 3-TS (nondiagnostic, ATP/MCLG, and mucinous cyst high grade/suspicious for adenocarcinoma/positive for adenocarcinoma [Kappa, 0.28; P<.001]) and various 4-TS (Kappa, 0.22-0.23). IOA was found to be low for neoplastic mucin (Kappa = 0.15; P<.001). In a study using simulated cytology practice, observers demonstrated fair IOA for grading MNs and low IOA for identifying neoplastic mucin. Knowledge of the cyst fluid CEA level was found to modestly improve the IOA for low-grade lesions.

The Frequency and Cancer Risk Associated with the Suspicious Cytologic Diagnostic Category in Endoscopic Ultrasound-Guided Fine-Needle Aspiration Specimens of Solid Pancreatic Lesions: A Meta-Analysis

The Frequency and Cancer Risk Associated with the Suspicious Cytologic Diagnostic Category in Endoscopic Ultrasound-Guided Fine-Needle Aspiration Specimens of Solid Pancreatic Lesions: A Meta-Analysis

Reply to verification bias in diagnostic accuracy studies for fine‐needle aspiration cytology

We would like to thank Dr. Schmidt for highlighting the importance of verification bias. We agree that this is a frequent drawback of studies examining sensitivity and specificity based on histological follow-up. The use of histological follow-up usually lowers the number of true-positive cases in endobronchial ultrasound-guided fine-needle aspiration (EBUS-FNA) specimens given that EBUS-FNAs with a positive diagnosis usually do not go on for confirmatory histological sampling. This is in contrast to other types of diagnostic accuracy studies in which a positive screening cytology finding will usually result in a gold-standard procedure, such as surgical resection, and thus more positive cytology cases will usually have histological follow-up than negative cases. For ethical reasons and good clinical practice, verification bias therefore cannot be truly eliminated in this clinical setting because it is often not feasible to perform an invasive mediastinoscopy after a positive EBUS diagnosis in which sufficient cellular material has been collected. In our study,1 there were 420 satisfactory EBUS-FNAs, 138 of which (33%) had a negative cytological diagnosis and 216 of which (51%) had a positive cytological diagnosis, but among the 97 histologically verified samples with a positive or negative EBUS-FNA diagnosis, 81 (84%) were initially negative and 16 (16%) were initially positive. Therefore, overall, more negative samples were followed with biopsy, and this verification bias may actually underestimate the study's sensitivity using histological follow-up.1 In prior, smaller studies that examined the sensitivity and specificity of EBUS-FNA based on cases with histological follow-up, the results were found to be similar, with sensitivity ranging from approximately 69% to 89%.2-5 In addition, the few EBUS studies incorporating clinical follow-up have demonstrated that the sensitivity increases to approximately ≥ 95%, given that patients with EBUS-FNAs that were positive for malignancy and never underwent surgery were included as having true-positive results based on clinical and radiological parameters.3, 4 We would like to thank Dr. Schmidt for his valuable comments and for highlighting the importance of verification bias. No specific funding was disclosed. The authors made no disclosures. Arivarasan Karunamurthy, MD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Guoping Cai, MD Department of Pathology Yale University School of Medicine New Haven, Connecticut Sanja Dacic, MD, PhD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Walid E. Khalbuss, MD, PhD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Liron Pantanowitz, MD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Sara E. Monaco, MD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania