Convex-probe endobronchial ultrasound-guided cryobiopsy for pleural tumors: three case reports

Introduction

Pleural tumors can arise from various malignancies, including primary pleural mesothelioma, direct invasion by lung cancer, metastases from epithelial tumors, and extranodal involvement of malignant lymphoma (1). Effective treatment strategies require precise histological differentiation, which in turn necessitates immunohistochemical staining and ancillary studies performed on adequate high-quality tissue specimens (2,3). Semi-rigid pleuroscopy and percutaneous image-guided needle biopsy are generally recommended as minimally invasive diagnostic modalities for pleural tumors (4). However, these approaches may be unsuitable in certain cases owing to lesion location, absence of pleural effusion, intrathoracic adhesions, or proximity to major vessels.

Endobronchial ultrasound-guided cryobiopsy (EBUS-cryo) is a novel sampling technique that combines a convex-probe ultrasound bronchoscope with a cryoprobe to obtain high-quality tissue specimens from mediastinal lymphadenopathy (5,6). Compared with conventional endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), EBUS-cryo yields larger and better-preserved tissue samples, potentially improving the diagnostic evaluation of both common malignancies and rare diseases.

This report presents three cases of pleural tumors in which the application of EBUS-cryo enabled definitive diagnosis and facilitated appropriate treatment strategies, despite diagnostic challenges associated with limited tissue acquisition using conventional biopsy techniques. We present this article in accordance with the CARE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-1-1426/rc).

Case presentation

Case 1

A 74-year-old man with a history of heavy smoking and occupational asbestos exposure during construction work for 5 years was referred to Osaka Metropolitan University Hospital for the evaluation of a 2-kg weight loss over 3 months and a small left-sided pleural effusion detected on chest radiography. Contrast-enhanced computed tomography (CT) revealed a 32.1-mm tumor on the mediastinal aspect of the left pleura, accompanied by bilateral pleural effusions, predominantly on the left side (Figure 1A and Figure S1A,S1B). Considering the proximity of the lesion to the descending aorta and left pulmonary vein, modest pleural effusion, and presumed advanced-stage malignancy, both semi-rigid and rigid pleuroscopy and percutaneous needle biopsy were deemed unsuitable for a definitive diagnosis. Therefore, EBUS-cryo was performed to obtain a histological diagnosis of the pleural tumor.

Figure 1 Imaging findings of a 74-year-old man with a history of heavy smoking and asbestos exposure. (A) Chest CT demonstrates a left pleural tumor measuring 32.1 mm in its longest diameter (arrowhead), situated between the descending aorta and left pulmonary vein. (B) The pleural tumor was visualized on EBUS imaging through the left lower bronchus. (C) EBUS-cryo was performed through the tract created by EBUS-TBNA. (D-F) Cryo-derived specimen shows loosely cohesive epithelioid cells with BRCA1-associated protein 1 deficiency, establishing a diagnosis of epithelioid mesothelioma. Magnification: 40× in (D); 200× in (E); 200× in (F); BRCA1-associated protein 1. Scale bar: 0.5 mm in (D); 0.2 mm in (E); 0.1 mm in (F). Staining method: HE staining (D,E); immunohistochemical staining for nuclear BRCA1-associated protein 1 (F). CT, computed tomography; EBUS, endobronchial ultrasound; EBUS-cryo, endobronchial ultrasound-guided cryobiopsy; EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration; HE, hematoxylin-eosin.

Following induction of conscious sedation with midazolam and fentanyl, a convex-probe ultrasound bronchoscope (BF-UC290F; Olympus, Tokyo, Japan) was orally inserted, and fiberoptic intubation was performed using an 8.5-mm uncuffed endotracheal tube (Portex^® Siliconized PVC Oral/Nasal Uncuffed Tracheal Tube, Smiths Medical, Minneapolis, MN, USA). The pleural tumor adjacent to the pulmonary artery was visualized from the left lower bronchus using EBUS, appearing as a heterogeneous lesion on morphological imaging (Figure 1B). Initially, three passes of EBUS-TBNA using a 25-gauge aspiration needle (NA-U401SX-4025N, Olympus) were performed on the lesion, yielding three tissue specimens. Rapid on-site evaluation (ROSE) using the obtained material demonstrated the presence of malignant cells. During EBUS-TBNA, to create a tract for subsequent EBUS-cryo and maximize tissue acquisition, the needle tip was repeatedly withdrawn to the level of the bronchial wall and then advanced.

After completion of EBUS-TBNA, EBUS-cryo was performed thrice using a 1.1-mm cryoprobe (No. 20402-401, Erbe Elektromedizin GmbH, Tüebingen, Germany) through the needle-created tract, with approximately 7 s of freezing per pass, yielding three well-sized specimens (Figure 1C). Minor bleeding occurred, but was promptly controlled by bronchoscopic suction, and no additional complications were observed. Endoscopically, the entrance of the EBUS-cryo tract remained pinhole-sized.

TBNA-derived specimens suggested malignancy; however, a definitive diagnosis was difficult owing to blood contamination, artifacts, and limited specimen volume. The cryo-derived specimens contained sufficient tissue and demonstrated loosely cohesive atypical cells with eosinophilic cytoplasm on hematoxylin-eosin staining (Figure 1D,1E). Immunohistochemistry of the cryo-derived specimens showed negative results for thyroid transcription factor-1, carcinoembryonic antigen (CEA), and Napsin A. However, the results were positive for calretinin, Wilms tumor 1, and D2-40. Furthermore, immunohistochemistry confirmed the loss of nuclear BRCA1-associated protein 1 expression in the tumor cells (Figure 1F). Based on these findings, epithelioid pleural mesothelioma was diagnosed, and systemic chemotherapy was initiated.

Case 2

A 69-year-old man presented to his local physician with a 7-kg weight loss over 3 months. Chest CT revealed multiple small pleural tumors encasing the right lung, leading to his referral to our hospital. He had a history of light smoking but no significant medical conditions or asbestos exposure.

Serum tumor marker testing revealed elevated levels of cytokeratin 19 fragment (7.5 ng/mL) and soluble mesothelin-related protein (11.4 nmol/L), whereas CEA and pro-gastrin-releasing peptide levels remained within normal ranges. Contrast-enhanced CT and ¹⁸F-fluorodeoxyglucose positron emission tomography showed multiple right pleural tumors predominantly located along the mediastinum, with contrast enhancement and increased standardized uptake values. However, no pleural effusion or intrapulmonary lesions were identified (Figure 2A and Figure S1C).

Figure 2 Imaging findings of a 69-year-old man who presented with a chief complaint of weight loss. (A) Chest CT reveals multiple pleural tumors encircling the mediastinal aspect of the left lung, without associated pleural effusion. (B) EBUS-cryo was performed on the pleural tumor visualized by EBUS imaging from the right dorsal side of the trachea. (C) Following EBUS-cryo, only minimal bleeding was observed, and the puncture site appeared pinhole-sized upon endoscopy (arrowhead). (D-F) EBUS-cryo specimen showing both epithelioid and atypical spindle cells. Immunohistochemical findings support a histological diagnosis of biphasic mesothelioma. Magnification: ×40 in (D), epithelioid (right) and sarcomatoid (left) components; ×200 in (E), epithelial components; ×200 in (F), sarcomatoid components. Scale bar: 0.5 mm in (D); 0.1 mm in (E), 0.1 mm in (F). Staining method: HE staining (D-F). CT, computed tomography; EBUS, endobronchial ultrasound; EBUS-cryo, endobronchial ultrasound-guided cryobiopsy; HE, hematoxylin-eosin.

Semi-rigid pleuroscopy was considered inappropriate because of the absence of pleural effusion, and percutaneous image-guided needle biopsy was deemed unsuitable owing to the potential risk of lung injury. Consequently, tissue sampling via the trachea from sites in contact with the pleural tumors was considered appropriate for a definitive diagnosis; EBUS-cryo was performed under local anesthesia.

After fiberoptic intubation with an 8.5-mm uncuffed endotracheal tube using a convex-probe ultrasound bronchoscope (BF-UC290F), the pleural tumors were delineated as heterogeneous lesions on the right dorsal side of the trachea on EBUS morphological imaging. Color Doppler imaging revealed punctate vessels within the lesion, and EBUS elastography revealed blue areas. Following three EBUS-TBNA punctures using a 25-gauge needle on the pleural nodule, four EBUS-cryo-procedures (each with approximately 7 s of freezing) were performed through the tract created by TBNA (Figure 2B). Malignant cells were identified via ROSE from the TBNA-derived material, and adequate tissue specimens were obtained using both EBUS-TBNA and EBUS-cryo. No severe adverse events were observed, and the entrance of the EBUS-cryo tract remained pinhole-sized upon endoscopic examination (Figure 2C).

The TBNA-derived specimens contained atypical epithelioid cells. However, a definitive diagnosis was challenging because of an insufficient number of tumor cells, the presence of crush artifacts, and significant blood contamination. In contrast, the EBUS-cryo specimens predominantly showed tubular and papillary proliferation of atypical epithelioid cells, with sarcomatoid cells also observed in some areas (Figure 2D-2F). Immunohistochemical staining showed that the epithelioid component was negative for thyroid transcription factor-1 but positive for calretinin, Wilms tumor 1, and D2-40, whereas the sarcomatoid component was negative for all four markers. Accordingly, the EBUS-cryo specimens allowed a histological diagnosis of biphasic pleural mesothelioma to be established, and systemic chemotherapy was subsequently initiated.

Case 3

An 82-year-old man was referred to our hospital for further evaluation after a routine health check-up revealed an infiltrative shadow in the left upper lung field and pleural effusion on chest radiography. He had undergone radiofrequency ablation for hepatocellular carcinoma at 80 years of age and remained in remission, with no other significant medical history. However, he reported a history of heavy smoking and asbestos exposure.

Contrast-enhanced chest CT revealed multiple pleural lesions with moderate left pleural effusion and an infiltrative shadow in the left upper lobe. Pleural tumors were most prominent along the mediastinum in the upper third of the thorax, with additional tumors in several areas of the middle and lower thirds (Figure 3A and Figure S1D). Serum levels of CEA, cytokeratin 19 fragment, pro-gastrin-releasing peptide, and soluble mesothelin-related protein remained within normal ranges. Thoracentesis revealed exudative pleural effusion; however, cytological examination results were negative.

Figure 3 Imaging findings of an 82-year-old man with a history of heavy smoking and asbestos exposure. (A) Chest CT shows a dominant mediastinal pleural tumor (arrowhead) with infiltrative opacities in the left upper lobe. (B) Semirigid pleuroscopy fails to establish a definitive diagnosis. (C) EBUS-cryo was performed from the left posterior side of the trachea. (D,E) EBUS-cryo specimen shows diffuse proliferation of small- to medium-sized lymphoid cells, accompanied by mild plasma cell infiltration. Magnification: 40× in (D); 200× in (E). (F) Immunohistochemical staining of the EBUS-cryo specimen supports a diagnosis of B-cell lymphoma. Magnification: 200× in (F), CD20. Scale bar: 0.5 mm in (D); 0.1 mm in (E); 0.1 mm in (F). Staining method: HE staining (D,E); CD20 staining (F). CT, computed tomography; EBUS-cryo, endobronchial ultrasound-guided cryobiopsy; HE, hematoxylin-eosin.

Based on the patient’s history of asbestos exposure and CT findings suggestive of pleural mesothelioma, semi-rigid pleuroscopy was performed to obtain a definitive diagnosis. A full-thickness pleural specimen was retrieved using an insulated-tip diathermic knife and cryoprobe. The size of the tissue specimen was sufficient for histopathological evaluation. However, histology showed only inflammatory changes and did not yield a definitive diagnosis (Figure 3B and Figure S1E,S1F). Given the patient’s advanced age and preference to avoid invasive procedures, a surgical biopsy was not pursued. As an alternative approach, EBUS-cryo was performed to sample the area of pleural thickening located posterior to the left trachea.

Bronchoscopy was conducted under conscious sedation using midazolam and fentanyl. Following fiberoptic intubation with an 8.5-mm cuffless endotracheal tube under EBUS guidance (BF-UC290F), the pleural tumor on the left posterior tracheal wall was visualized as a slightly indistinct internally heterogeneous lesion with several calcifications. After three EBUS-TBNA passes using a 25-gauge needle, a 1.1-mm cryoprobe was inserted through the same puncture site under endoscopic and/or EBUS visualization, and four EBUS-cryo procedures with 7-s freezing cycles were performed (Figure 3C). ROSE of the TBNA-derived material showed clusters of atypical lymphocytes, and sufficient tissue samples were obtained using both EBUS-TBNA and EBUS-cryo. Minor bleeding occurred, but was promptly controlled with suction.

TBNA-derived specimens did not allow a definitive diagnosis owing to specimen damage and significant blood contamination. In contrast, cryo-derived specimens showed diffuse proliferation of small- to medium-sized lymphoid cells with slightly irregular nuclei and pale cytoplasm, accompanied by scattered plasma cells (Figure 3D,3E). Immunohistochemical staining demonstrated that the lymphoid cells were negative for CD3 but positive for CD20 and CD79a (Figure 3F). Light chain restriction with λ chain dominance was also confirmed.

These findings supported a diagnosis of B-cell malignant lymphoma, most consistent with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) lymphoma. Considering the CT findings and extreme rarity of primary pleural MALT lymphoma, we inferred that the primary intrapulmonary lymphoma lesion—seen as an infiltrative opacity in the left upper lobe—had extended into the pleura through direct invasion or lymphatic spread, resulting in the pleural lesions. Chemotherapy was promptly initiated after disease staging.

Ethical statement

All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Ethics Committee of Osaka Metropolitan University Graduate School of Medicine (approval No. 4364). Written informed consent was obtained from the patients for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

This study demonstrates that applying EBUS-cryo to pleural tumors enables a minimally invasive definitive diagnosis and helps guide appropriate treatment strategies. EBUS-cryo provides anatomical access to pleural lesions located along the mediastinal aspect or in the upper lung fields—areas that are often difficult to access using conventional biopsy techniques. The method also allows precise sampling of pleural lesions under real-time ultrasound guidance while avoiding adjacent major vessels, as seen in case 1. Furthermore, the large non-crushed tissue specimens obtained via cryobiopsy can facilitate the diagnosis of rare pleural diseases, including pleural mesothelioma and MALT lymphoma.

Pleural tumors are predominantly metastatic lesions originating from epithelial malignancies such as lung or breast cancer. However, they may also arise from rare malignant conditions such as pleural mesothelioma, pleural involvement of lymphoma, or synovial sarcoma. In addition, benign tumors, including solitary fibrous tumor of the pleura, lymphangioma, and schwannoma, can present as pleural masses (1,7).

Typically, clinicians can establish a treatment strategy for metastatic pleural tumors if: (I) a potential primary lesion of a common epithelial tumor is identified on imaging studies; (II) pleural effusion is present; and (III) cytology findings are consistent. However, when these criteria are not met, the limited diagnostic sensitivity of pleural cytology and cell-block specimens, particularly for uncommon tumors, poses a major challenge. Additionally, in some tumors, such as pleural mesothelioma, the prognosis and treatment strategies vary considerably depending on the histological subtype (8). Therefore, adequate tissue sampling via pleural biopsy is essential for securing a definitive diagnosis and determining an appropriate treatment plan.

Since EBUS-cryo has been reported as a useful technique for diagnosing mediastinal lymphadenopathy, its clinical applications have been progressively expanded to various intrathoracic tumor lesions by leveraging the ability of convex-probe EBUS bronchoscopes to efficiently access lesions adjacent to the central airways. These include lesions involving the lungs, heart, and pulmonary artery (9-12). EBUS-cryo can obtain higher-quality specimens compared with TBNA and has a lower risk of bleeding than conventional cryobiopsy techniques. Notably, EBUS-cryo-derived specimens are larger [present cases: median area, 5.48 mm² (range, 4.28–9.22 mm²); mean ± standard deviation (SD) area, 6.13±1.70 mm²] and less affected by crush artifacts, allowing for more accurate histopathological assessment.

Several malignant diseases that cause pleural lesions require immunohistochemical staining and ancillary testing for definitive diagnosis and histological subtype classification. Although EBUS-TBNA carries a risk of yielding insufficient material for these detailed analyses, EBUS-cryo can offer a reliable alternative. It provides ample well-preserved tissue suitable for comprehensive pathological and molecular evaluation—an essential factor in diagnosing rare diseases. Accordingly, as demonstrated in the present cases, EBUS-cryo should be considered when preoperative imaging reveals atypical or inconclusive findings. Conversely, when common lung cancer is preoperatively suspected, a shorter freezing time may be considered to minimize the risk of adverse events, including fistula formation and mediastinitis (13).

When performing EBUS-cryo, the method used to create the cryoprobe insertion site is critical. Previously reported techniques include making an airway incision using a high-frequency device or creating a tract with a TBNA needle. In this study, pleural mesothelioma and lymphoma were included in the preoperative differential diagnosis; therefore, we used a thin 25-gauge needle to create the EBUS-cryo tract. This approach minimized the insertion hole size and reduced the risk of tumor seeding at the biopsy site (14). Although a thin needle yields a smaller amount of TBNA-derived specimens, the post-biopsy puncture site remains pinhole-sized even after EBUS-cryo (15). Moreover, its higher flexibility than the thicker needles used in conventional EBUS-TBNA allows access to a wider range of thoracic sites, thereby broadening the potential indications for EBUS-cryo. In cases where post-biopsy seeding is a concern, or when lesions are located in areas difficult to access with a conventional 22-gauge needle, EBUS-TBNA using a 25-gauge needle may be an appropriate method for creating a tract before performing EBUS-cryo.

The gold standard diagnostic approach for pleural malignancies is rigid pleuroscopy performed under general anesthesia. In contrast, semi-rigid pleuroscopy performed under local anesthesia and image-guided needle biopsy are commonly used in routine clinical practice as less invasive alternatives (16). Although rigid pleuroscopy offers reliable diagnostic accuracy, its invasiveness often limits its use in patients with poor general condition resulting from the malignancy itself or coexisting comorbidities. Conversely, semi-rigid pleuroscopy and image-guided needle biopsy are safe and minimally invasive while maintaining a high diagnostic yield. However, lesions located in the upper third of the lung field or along the mediastinum, as well as those adjacent to major vessels, may fall beyond the scope of these techniques.

EBUS-cryo provides access to pleural lesions located in the upper third of the lung fields and along the mediastinal aspect. Therefore, it can serve as a complementary diagnostic modality that addresses the limitations of conventional biopsy techniques. Recently, a thinner EBUS scope has been introduced into clinical practice. This scope features an outer diameter of 5.9 mm (approximately 0.7 mm thinner than the conventional model used in this study), a tapered tip that facilitates insertion into peripheral bronchi, and improved bending angles for enhanced maneuverability (17). The introduction of thinner EBUS scopes is expected to widen the indications of EBUS-cryo, allowing clinicians to sample lesions across a broader spectrum of intrathoracic regions using a reliable and minimally invasive biopsy approach.

However, this study has certain limitations. These include the small sample size comprising only three cases and the absence of a direct comparison between EBUS-cryo and conventional biopsy techniques. Additionally, only a thin 25-gauge needle was used for EBUS-TBNA, and the feasibility of EBUS-cryo for pleural tumors using needles of other gauges is yet to be verified. Finally, MTAP immunohistochemistry and CDKN2A fluorescence in situ hybridization, which support the diagnosis of pleural mesothelioma, are not routinely performed at our institution and were therefore not included in this study.

Conclusions

The expanded application of EBUS-cryo for the diagnosis of pleural tumors has the potential to establish this method as a precise and minimally invasive biopsy technique.

Acknowledgments

We thank the staff in the endoscopy room for their assistance during examinations.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-1-1426/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-1-1426/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-1-1426/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Ethics Committee of Osaka Metropolitan University Graduate School of Medicine (approval No. 4364). Written informed consent was obtained from the patients for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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