Optimizing Tissue Sampling During Medical Pleuroscopy for Diagnosis of Malignant Pleural Effusion Due to Lung Cancer: Study Criopleura

Optimizing Tissue Sampling During Medical Pleuroscopy for Diagnosis of Malignant Pleural Effusion Due to Lung Cancer: Study Criopleura | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optimizing Tissue Sampling During Medical Pleuroscopy for Diagnosis of Malignant Pleural Effusion Due to Lung Cancer: Study Criopleura Irene Lojo-Rodríguez, Maribel Botana-Rial, Ana González-Piñeiro, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7103432/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Cryobiopsy has emerged in recent years as a tool of growing interest in the diagnosis of non-small cell lung cancer (NSCLC). We conducted the first study with the primary objective of analyzing the techniques of biopsy during semirigid pleuroscopy to compare the quality of cryobiopsies versus conventional forceps biopsies, including expected tissue outcomes and the feasibility of histological characterization and molecular testing, of malignant pleural effusion (MPE). Prospective study including 14 caucasian patients with MPE due to NSCLC who underwent semirigid pleuroscopy with cryobiopsies. The median biopsy size for conventional flexible forceps and cryoprobe was 2.5 mm (1.5–3.2 mm) and 5.5 mm (3.8–7.6 mm), respectively (p = 0.07). The number of biopsies also differed: flexible forceps: 5 (4-6.25) biopsies vs cryoprobe: 3 ( 3 – 4 ) biopsies(p = 0.01). The tumor/non-tumor ratio in the conventional forceps sample was 2.4 (1.2–5.9), while in the cryoprobe sample, it was 3.6 (1.2–10) (p = 0.09). Only in one case, the samples obtained during semirigid pleuroscopy were insufficient for molecular diagnosis. The incorporation of cryobiopsy into semirigid pleuroscopy could reduce the number of biopsies required, the sample size was significantly larger, as was the tumor/non-tumor ratio. This technique could shortening procedure time and facilitating tissue collection without increasing procedural risks. Biological sciences/Cancer Health sciences/Medical research Health sciences/Oncology biomarker diagnostic cryobiopsy semirigid pleuroscopy histology pathological finding INTRODUCTION Lung adenocarcinoma is the most common cause of malignant pleural efusiones (MPE), representing an advanced disease at the time of diagnosis 1,2. In order to improve the prognosis of patients; a complete characterization of the tumor needs to performed, with the determination of all the molecular alterations that can be targeted by novel therapies. Therefore, using a biopsy method that provides safe and adequate lung tissue sampling is very imporantant. Medical pleuroscopy is the main procedure for the diagnosis of MPE 1,2. Cryobiopsy has emerged in recent years as a tool of growing interest in the diagnosis of non-small cell lung cancer (NSCLC) 3,4. Two recently published systematic reviews, about the diagnostic yield and safety of pleural cryobiopsy during medical thoracoscopy to diagnose pleural effusion one, one of them by our group, demonstrated that it is a safe and effective technique5,6. Early studies, which included a small number of patients, showed that pleural cryobiopsy had a high diagnostic yield, in some cases up to 100%7–10. Another aspect of increasing interest is going be that the obtained samples may also allow for the molecular characterization of the tumor. So far, only two publications have analyzed this aspect8,10. A limitation of both studies were that only a single specific molecular determination was performed on the samples obtained with the cryoprobe, specifically the EGFR gene mutation. Optimizing the diagnosis of lung cancer represents a challenge, as well as a necesary, for improving the low survival of patients with MPE. When performing semirigid pleuroscopy and comparing it with rigid medical thoracoscopy, a greater number of biopsies is required to achieve a diagnosis due to the small size of the fórceps 2. In the era of personalized medicine, molecular diagnosis is especially relevant, making it essential to improve biopsy techniques. Therefore, we conducted the first study with the primary objective of analyzing the techniques of biopsy during semirigid pleuroscopy to compare the quality of cryobiopsies versus conventional forceps biopsies, including expected tissue outcomes and the feasibility of histological characterization and molecular testing, of malignant pleural effusion (MPE) due to NSCLC. RESULTS Data from 19 patients who underwent semirigid pleuroscopy with conventional forceps and cryoprobe biopsies were analyzed. The baseline characteristics of the patients are shown in Table 1. Regarding the procedural characteristics, the drainage amount (ml) was 1,900 ml (1,050 − 2,400). The pleural abnormalities found during pleuroscopy were: plaques in 12 patients (63.2%), nodules in 2 patients (10.5%) and polyps in 5 patients (26.3%). The duration of subsequent chest drainage (days) was 3 (2–3) days and the length of stay after the procedure (days) was 6 (4–10) days. The dose of sedative drugs used during the procedure was 6 (5–7) micrograms of midazolam and 100 (50–100) micrograms of fentanyl. The 30-day mortality was 2 cases (10.5%), and the overall survival was 4.5 (2-31.5) months. There were no severe complications observed during the procedure, nor minor bleeding that required hemostatic control. Pain was reported in 2 patients (10.5%). The patients with MPE due to NSCLC included in the study were 14 cases. The Table 2 presents some of their clinical, histological, and TNM characteristics. Table 2 Clinical, histological, and prognostic characteristics of patients with malignant pleural effusion due to NSCLC Patient Age (years) Sex Histology TNM 1 69 Male ADC T3N0 2 65 Male ADC T3N1 3 65 Male ADC TxN0 4 78 Female ADC T3N2 5 79 Male ADC TxN0 6 60 Male ADC T4N3 7 63 Male ADC TxN3 8 80 Male ADC T4N3 9 73 Male ADC T1N3 10 70 Female ADC T2aN2 11 63 Male ADC T4N0 12 68 Male ADC TxN1 13 61 Male Large cell carcinoma T1N2 14 60 Male ADC TxN0 Definition of abbreviation = Adenocarcinoma (ADC) In this group, the median biopsy size for conventional flexible forceps and cryoprobe was 2.5 mm (1.5–3.2 mm) and 5.5 mm (3.8–7.6 mm), respectively (p = 0.07). The number of biopsies also differed: flexible forceps: 5 (4-6.25) biopsies vs cryoprobe: 3 (3–4) biopsies(p = 0.01). The percentage of viable tumor in samples obtained by flexible forceps and cryoprobe was 55% (15–80%) and 55% (10–80%), respectively (p = 0.9). The tumor/non-tumor ratio in the conventional forceps sample was 2.4 (1.2–5.9), while in the cryoprobe sample, it was 3.6 (1.2–10) (p = 0.09). In all patients, a morphological and immunohistochemical diagnosis was possible using both conventional forceps and cryoprobe biopsies. Only in one case, the samples obtained during semirigid pleuroscopy with both cryoprobe and forceps were insufficient for molecular diagnosis, requiring a surgical video-assisted thoracoscopic surgery. In this case, the pleuroscopy revealed plaque-like lesions. DISCUSSION Our study is the first published Spanish study to analyze the quality of cryoprobe obtained samples to optimize the molecular diagnosis of MPE due to NSCLC. When pleural biopsies were obtained using a cryoprobe, although there were no differences in the percentage of viable tumor cells, the sample size was significantly larger, as was the tumor/non-tumor ratio. Additionally, fewer biopsies were required when using the cryoprobe. A limitation of semirigid pleuroscopy is its size and flexibility 1,2. Although the semirigid pleuroscope is more manageable than the rigid one, its smaller diameter limits the ability to obtain larger biopsies, potentially affecting the amount of tissue available for molecular studies. Furthermore, its lower rigidity can make biopsy collection more challenging. In a small study conducted by our group on the diagnostic performance of semirigid thoracoscopy for the molecular characterization of MPD due to NSCLC, molecular testing was performed in 14 adenocarcinomas. Sufficient and adequate material was obtained to determine EGFR mutation status in 100% of cases, but only in 90% of cases for ALK translocation15. EGFR mutations were identified in two patients, while no cases of ALK translocation were detected. EGFR mutation detection was possible in all cases, but the yield for ALK translocation analysis was lower 15. Many revolutionary advances have recently been made in the management of NSCLC. There have been a number of approvals of molecular-targeted therapy for subgroups of NSCLC patients with sensitizing EGFR, ALK, ROS1, RET, BRAF V600E, MET, or NTRK alterations 21. In addition to genetic examination, NSCLC are usually subjected to the analysis of PD-L1 protein expression in order to direct the use of immune checkpoint inhibitors 20,21. Cryobiopsy, a relatively novel bronchoscopic technique, has gained prominence in recent years due to its ability to procure larger, higher-quality tissue samples with fewer crush artifacts compared to traditional forceps biopsies 3,4. This method has demonstrated promising diagnostic yields in lung cancer 3. However, none of the published studies on MPE and lung cancer have primarily focused on analyzing the effectiveness of pleural cryobiopsies for mutation studies and other molecular alterations. In studies where this aspect was analyzed secondarily, only EGFR gene mutations had been investigated 8,10. In one study including 14 cases of MPE, four of which were lung adenocarcinomas, obtaining pleural biopsies with a cryoprobe allowed DNA isolation for EGFR mutation analysis in all samples using a commercially available kit8. In another study, samples were valid for EGFR mutation testing, but these results were not compared with the molecular diagnostic yield of biopsies obtained using conventional fórceps 10. Another study conducted in an Asian population included six patients with NSCLC, all of whom underwent genetic panel testing to detect driver gene mutations. Sufficient specimens were obtained for appropriate genetic analysis in all cases 14. However, a limitation of this study was the lack of specification regarding which molecular alterations were analyzed. In our study, all the necessary molecular analyses, including PDL1 expression, were successfully performed. There is ongoing debate regarding the ideal instrument for medical thoracoscopy. Bansal S et al. compared rigid mini-thoracoscopy with semirigid thoracoscopy and found that biopsy size was larger in the mini-thoracoscopy group (16.1 ± 4.5 mm vs. 8.3 ± 2.9 mm; P < 0.001)16. However, the diagnostic yield of pleural biopsy was similar between the two groups. When comparing rigid versus semirigid thoracoscopy, differences have been observed. In 2016, Wurps H et al. conducted a prospective study with 80 patients to compare parietal pleural biopsies obtained using rigid forceps, flexible forceps, and a cryoprobe during medical thoracoscopy 17. Rigid biopsies were significantly larger than both cryobiopsies and flexible biopsies (P < 0.001), while cryobiopsies were significantly larger than flexible biopsies (P < 0.01). The diagnostic yield of cryobiopsies was lower than that of rigid biopsies but not inferior to flexible biopsies17. In our study, although the tumor percentage was similar in both biopsy types, cryobiopsies were larger, and fewer samples were required with cryobiopsy. It is important to consider that tissue remains the standard material for NSCLC diagnosis, and there is a continuous search for new molecular markers in MPE, with the primary limiting factor being the availability of viable samples. In this context, cryobiopsy of mediastinal lymph nodes (cryoEBUS) has been described as improving diagnostic accuracy. Although cryoEBUS did not significantly enhance lung cancer diagnosis compared to EBUS-TBNA, it appeared to provide superior samples for complete molecular characterization, including PDL1 studies, while requiring fewer passes per lymph node4. Studies comparing the diagnostic yield of cryobiopsy by radial endobronchial ultrasound with that of conventional biopsy have shown that cryobiopsy improves diagnostic yield and obtains larger samples in patients with peripheral pulmonary lesions due to lung cancer 18,19. As discussed earlier, the main limitation of semirigid pleuroscopy is the small sample size, which necessitates obtaining more biopsies. Additionally, when using flexible forceps, it can be difficult to biopsy lesions located tangentially to the instrument 1,2,5. However, a notable advantage of the semirigid pleuroscope is its ease of use compared to the rigid thoracoscope, making it a favorable option for Interventional Pulmonology Units. The incorporation of cryobiopsy into semirigid pleuroscopy could reduce the number of biopsies required, thereby shortening procedure time and facilitating tissue collection without increasing procedural risks. Pleural cryobiopsy has been shown to be a technique with a low complication rate, as reported in recent systematic reviews on its efficacy and safety 4,5. In our study, despite performing biopsies with both a cryoprobe and conventional flexible forceps, no major complications were observed, nor were there increases in hospital stays or chest drainage duration. These findings are consistent with those reported in the literatura 22. The primary limitation of this study is the small number of patients with MPE due to NSCLC, although it is the first study conducted in a Caucasian population for this purpose. Additionally, as previously mentioned, this is an innovative technique that will require further standardization and refinement for integration into clinical practice. Incorporating this technique into semirigid pleuroscopy could enhance procedural methodology, reduce procedure time by requiring fewer biopsies, and do so without increasing complication rates. Pleural cryobiopsy has been demonstrated to be a safe and effective diagnostic tool during pleuroscopy semirigid, providing adequate samples for all molecular testing and for the histological characterization of patients with MPE due to lung cancer. METHODS Study population Prospective study including all patients with a diagnosis or high suspicion of MPE due to NSCLC who underwent semirigid pleuroscopy with cryobiopsies between November 1, 2019, and November 30, 2024, at the Bronchopleural Techniques Unit of Hospital in Spain. The inclusion criteria were: age > 18 years; caucasian population and patients with MPE due to NSCLC confirmed by cytology or histology Exclusion criteria included were: patients with benign pleural effusion; patients with paramalignant pleural effusion without evidence of malignant cells in the pleural cavity; other etiologies different from MPE and contraindication for pleural biopsy Semirigid Pleuroscopy Procedure and Pleural Biopsy All procedures were performed according to clinical practice guideline recommendations 2,11. Pleuroscopy was conducted under aseptic conditions in the endoscopy suite, with the patient under spontaneous respiration, conscious sedation (midazolam ± fentanyl), and local anesthesia (2% mepivacaine). The semirigid pleuroscope (LTF-160 autoclave, Olympus, Tokyo, Japan) was used for the procedures. Ultrasound was performed on all patients before trocar insertion to determine the entry point. The pleural fluid was drained at the beginning, and the pleural cavity was inspected under direct visualization. All pleural biopsies were performed on parietal pleural lesions. Before biopsy collection, 5 ml of 2% lidocaine was applied under direct visualization through the working channel of the pleuroscope to the selected area. Initially, 4 to 8 pleural biopsies were taken using flexible conventional forceps. Subsequently, biopsies were collected using a flexible cryoprobe (ERBOCRYOCA, ERBE, Tübingen, Germany) with an external diameter of 1.7 mm (2 to 5 biopsies). The probe tip, which froze tissue using CO₂ gas, was placed on the lesion and frozen for 5–7 seconds. The adhered sample was then extracted. Samples obtained by both conventional forceps and cryoprobe were placed in separate formalin containers and sent to the Pathology Department for histological analysis. Following the procedure, a 24Fr chest drain was placed Study Variables : The following variables were recorded: epidemiological characteristics of patient (age, sex, smoking history), functional characteristics (general health status using the ECOG performance scale), radiological characteristics (size of pleural effusion on chest radigraphic, and characteristics of pleural lesions and/or thickening on CT scan. Others variables were also analyzed as tumor classification: Histological tumor type and TNM classification12 at the time of DPM diagnosis or at the time of primary tumor diagnosis in cases of disease progression. The prognostic and survival variables were the predictive variables from the LENT prognostic scale13. We recorded the 30-day mortality rate and overall survival. The procedure-related variables were: volume of pleural fluid drained, pleural findings during pleuroscopy, duration of chest drainage (days) and the length of hospital stay (days). About the procedure safety, we classified bleeding complications as follows: mild (self-limiting), moderate (requiring topical vasoconstrictors such as adrenaline) and severe (requiring intervention with electrocautery or argon plasma). Other complications, including pain. We analized the diagnostic yield and molecular analysis as follows: comparison of histological diagnostic yield between cryobiopsy and conventional forceps biopsies. The pathological findings in both biopsy samples, including: numer, sample size (mm). sample volume (mm²) and percentage of viable vs. non-viable tumor cells. The results of molecular studies were also analyzed (detection of genomic alterations EGFR, ROS1,BRAF, ALK, RET, MET, NTRK and PD-L1 expression) Ethical aspects : The study was conducted following the Helsinki Declaration guidelines. The data were pseudonymized for biomedical research purposes. The study has been approved by the provincial research ethics committee of Galicia (Pontevedra-Vigo-Ourense code: 2019/293). All participants provided written informed consent to participate in this study and to allow the publication of their data and sample results. Statistical analyses Categorical variables were presented as counts and percentages, and continuous variables were presented as mean and standard derivation (SD). Statistical analyses were performed using SPSS Statistic 21 (IBM Corp., Armonk, NY). Pearson’s chi-square or Fisher exact test was used to compare proportions, as appropriate. For continuous data, differences between groups were determined using Students t-test or Mann-Whitney U test for parametric or non-parametric data, respectively. P < 0.05 was considered statistically significant. Declarations Authors contributions All authors should have made sustancial contributions to all of the folllowing (the concepcion and design of the study, or acquisition of data, or analysis and interpretation of data, drafting the article and revising it critically for impostant intellectual concept. Author contributions: - conception and design: MBR, AFV - analysis, acquisition, analysis, interpretation of the data: MBR, ILR, AGP, AGM, CRH, ECV - the drafting of the paper or revising it critically for intellectual content: AFV, MND, ECV - The final approval of the versión: all authors All authors agree to be accountable for all aspects of the work. Data availability statement: The datasets used and/or analysed during the current study available from the corresponding author ( [email protected] ) on reasonable request. Declaration of conflicting interest: The authors declare that there are no conflicts of interest Funding: This research received a specific grant from public agency: Spanish Society of Pulmonology (SEPAR) and Integrated Research Projects of Interventional Pulmonology (PII NI) in 2019. References Kapp CM, Lee HJ. Malignant Pleural Effusions. Clin Chest Med. 2021;42:687-696 Avasarala SK, Lentz RJ, Maldonado F. Medical Thoracoscopy. Clin Chest Med. 2021;42:751-766 Simon M, Simon I,Andrei Tent P, Adina Todea D, Haranguș A. Cryobiopsy in Lung Cancer Diagnosis-A Literature Review. Medicina (Kaunas) 2021 19;57:393. Botana-Rial M, Lojo-Rodríguez I, Leiro-Fernández V, Ramos-Hernández C, González-Montaos A, Pazos-Area L, Núñez-Delgado M, Fernández-Villar A. Is the diagnostic yield of mediastinal lymph node cryobiopsy (cryoEBUS) better for diagnosing mediastinal node involvement compared to endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)? A systematic review. Respir Med. 2023;218:10738 Rial MB, Rodríguez IL, Roibás CM, Fernández VL, Delgado MN, Barreira ÁS, Torrado AP, Villar AF. Diagnostic Yield and Safety of Pleural Cryobiopsy during Medical Thoracoscopy to Diagnose Pleural Effusion. A Systematic Review and Meta-Analysis. Arch Bronconeumol 2020 ;56:784-791. Shafiq M, Sethi J, Ali MS, Ghori UK, Saghaie T, Folch E. Pleural Cryobiopsy: A Systematic Review and Meta-Analysis. Chest. 2020;157:223-230 Thomas R, et al. Pleuroscopic cryoprobe biopsies of the pleura: a feasibility and safety study. Rozman A, Camlek L, Marc Malovrh M, Kern I, Schönfeld N. Feasibility and safety of parietal pleural cryobiopsy during semi-rigid thoracoscopy. Clin Respir J 2016;10:574-8. Pathak V, E, et al. Safety and feasibility of pleural cryobiopsy compared to forceps biopsy during semirigid pleuroscopy. Lung 2017;195:371-375 Tousheed SZ, Manjunath PH, Chandrasekar S, Murali Mohan BV, Kumar H, Hibare KR, Ramanjaneya R. Cryobiopsy of the Pleura: An Improved Diagnostic Tool. J Bronchology Interv Pulmonol 2018;25:37-41 Shaikh F, Lentz RJ, Feller-Kopman D, Maldonado F. Medical thoracoscopy in the diagnosis of pleural disease: a guide for the clinician. Expert Rev Respir Med 2020;14:987-1000. º clasificación TNM; Goldstraw P et al.J Thorac Oncol 2016;11:39 Clive AO, Kahan BC, Hooper CE, Bhatnagar R, Morley AJ, Zahan-Evans N, Bintcliffe OJ, Boshuizen RC, Fysh ET, Tobin CL, Medford AR, Harvey JE, van den Heuvel MM, Lee YC, Maskell NA. Predicting survival in malignant pleural effusion: development and validation of the LENT prognostic score. Thorax. 2014 Dec;69(12):1098-104. Kamatani M, Awano N, Inomata M, Kuse N, et al. Diagnostic yields and safety of thoracoscopic cryobiopsies in Japan: A single-center retrospective observational study. Respir Investig 2024;62:617-622 Botana M, A et al. Rendimiento diagnóstico de la toracoscopia semirrígida para la caracterización molecular de derrames pleurales malignos de origen pulmonar. Arch Bronconeumol 2018;54:489-91 Bansal S, Mittal S, Tiwari P, Jain D, Arava S, Hadda V, et al. Rigid Mini-Thoracoscopy Versus Semirigid Thoracoscopy in Undiagnosed Exudative Pleural Effusion. The MINT Randomized Controlled Trial. J Bronchology Interv Pulmonol 2020;27:163-171. Wurps H, Schönfeld N, Bauer TT, Bock M, Duve C, Sauer R, Mairinger T, Griff S. Intra-patient comparison of parietal pleural biopsies by rigid forceps, flexible forceps and cryoprobe obtained during medical thoracoscopy: a prospective series of 80 cases with pleural effusion. BMC Pulm Med 2016 Jul;16:98. Udagawa H, Kirita K, Naito T, Nomura S, Ishibashi M, et al. (2020) Feasibility and utility of transbronchial cryobiopsy in precisión medicine for lung cancer: prospective single-arm study. Cancer Sci 111:2488- 2498. Arimura K, Tagaya E, Akagawa H, Nagashima Y, Shimizu S, et al. (2019) Cryobiopsy with endobronchial ultrasonography using a guide sheath for peripheral pulmonary lesions and DNA analysis by next generation sequencing and rapi don-site evaluation. Respir Investig 57:150-156. Alexander M, Kim SY, Cheng H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020;198:897-907. Alduais Y, Zhang H, Fan F, Chen J, Chen B. Non-small cell lung cancer (NSCLC): A review of risk factors, diagnosis, and treatment. Medicine (Baltimore) 2023;102:e32899. Safety and accuracy of semirigid pleuroscopy performed by pulmonary fellows at a major university hospital: our initial experience. Adil Shujaat , Abubakr A Bajwa, Faisal Usman, Lisa Jones, James D Cury. J Bronchology Interv Pulmonol 2013;20:213-23. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Aug, 2025 Reviews received at journal 06 Aug, 2025 Reviews received at journal 02 Aug, 2025 Reviewers agreed at journal 02 Aug, 2025 Reviews received at journal 02 Aug, 2025 Reviewers agreed at journal 02 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 30 Jul, 2025 Editor assigned by journal 30 Jul, 2025 Editor invited by journal 22 Jul, 2025 Submission checks completed at journal 16 Jul, 2025 First submitted to journal 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Predoctoral student of Santiago de Compostela University","correspondingAuthor":false,"prefix":"","firstName":"Almudena","middleName":"","lastName":"González-Montaos","suffix":""},{"id":494680824,"identity":"cd24d7f3-d140-453a-967c-8869eeb0fbdc","order_by":5,"name":"Cristina Ramos-Hernández","email":"","orcid":"","institution":"Alvaro Cunqueiro Hospital, Sanitary Research Institute Galicia Sur (IISGS). a Predoctoral student of Santiago de Compostela University","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Ramos-Hernández","suffix":""},{"id":494680825,"identity":"98424e1e-e3c7-43c0-830e-ca68510e7d96","order_by":6,"name":"Manuel Nuñez-Delgado","email":"","orcid":"","institution":"Alvaro Cunqueiro Hospital, Sanitary Research Institute Galicia Sur (IISGS). a Predoctoral student of Santiago de Compostela University","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Nuñez-Delgado","suffix":""},{"id":494680826,"identity":"3a32ff8c-f8d6-413a-8471-24866ba4fdbf","order_by":7,"name":"Alberto Fernández-Villar","email":"","orcid":"","institution":"Alvaro Cunqueiro Hospital, Sanitary Research Institute Galicia Sur (IISGS)","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Fernández-Villar","suffix":""}],"badges":[],"createdAt":"2025-07-11 16:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7103432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7103432/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-21170-8","type":"published","date":"2025-10-27T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95039917,"identity":"771bfad2-13e5-49e8-8fda-37718e2f205c","added_by":"auto","created_at":"2025-11-03 16:05:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":505706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103432/v1/5ffe4787-1ce3-4b3b-afa2-fb2ceee4e146.pdf"},{"id":88276274,"identity":"a443b020-73d9-4e85-99be-7f44ca37dc31","added_by":"auto","created_at":"2025-08-04 18:21:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15462,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7103432/v1/ea12aaa2915efcda6bd9ac7b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOptimizing Tissue Sampling During Medical Pleuroscopy for Diagnosis of Malignant Pleural Effusion Due to Lung Cancer: Study Criopleura\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLung adenocarcinoma is the most common cause of malignant pleural efusiones (MPE), representing an advanced disease at the time of diagnosis 1,2. In order to improve the prognosis of patients; a complete characterization of the tumor needs to performed, with the determination of all the molecular alterations that can be targeted by novel therapies. Therefore, using a biopsy method that provides safe and adequate lung tissue sampling is very imporantant. Medical pleuroscopy is the main procedure for the diagnosis of MPE 1,2.\u003c/p\u003e\u003cp\u003eCryobiopsy has emerged in recent years as a tool of growing interest in the diagnosis of non-small cell lung cancer (NSCLC) 3,4. Two recently published systematic reviews, about the diagnostic yield and safety of pleural cryobiopsy during medical thoracoscopy to diagnose pleural effusion one, one of them by our group, demonstrated that it is a safe and effective technique5,6. Early studies, which included a small number of patients, showed that pleural cryobiopsy had a high diagnostic yield, in some cases up to 100%7\u0026ndash;10. Another aspect of increasing interest is going be that the obtained samples may also allow for the molecular characterization of the tumor. So far, only two publications have analyzed this aspect8,10. A limitation of both studies were that only a single specific molecular determination was performed on the samples obtained with the cryoprobe, specifically the EGFR gene mutation.\u003c/p\u003e\u003cp\u003eOptimizing the diagnosis of lung cancer represents a challenge, as well as a necesary, for improving the low survival of patients with MPE. When performing semirigid pleuroscopy and comparing it with rigid medical thoracoscopy, a greater number of biopsies is required to achieve a diagnosis due to the small size of the f\u0026oacute;rceps 2.\u003c/p\u003e\u003cp\u003eIn the era of personalized medicine, molecular diagnosis is especially relevant, making it essential to improve biopsy techniques. Therefore, we conducted the first study with the primary objective of analyzing the techniques of biopsy during semirigid pleuroscopy to compare the quality of cryobiopsies versus conventional forceps biopsies, including expected tissue outcomes and the feasibility of histological characterization and molecular testing, of malignant pleural effusion (MPE) due to NSCLC.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eData from 19 patients who underwent semirigid pleuroscopy with conventional forceps and cryoprobe biopsies were analyzed. The baseline characteristics of the patients are shown in Table\u0026nbsp;1.\u003c/p\u003e\n\u003cdiv\u003e\n\u003c/div\u003e\n\u003cp\u003eRegarding the procedural characteristics, the drainage amount (ml) was 1,900 ml (1,050 − 2,400).\u003c/p\u003e\n\u003cp\u003eThe pleural abnormalities found during pleuroscopy were: plaques in 12 patients (63.2%), nodules in 2 patients (10.5%) and polyps in 5 patients (26.3%). The duration of subsequent chest drainage (days) was 3 (2–3) days and the length of stay after the procedure (days) was 6 (4–10) days.\u003c/p\u003e\n\u003cp\u003eThe dose of sedative drugs used during the procedure was 6 (5–7) micrograms of midazolam and 100 (50–100) micrograms of fentanyl. The 30-day mortality was 2 cases (10.5%), and the overall survival was 4.5 (2-31.5) months.\u003c/p\u003e\n\u003cp\u003eThere were no severe complications observed during the procedure, nor minor bleeding that required hemostatic control. Pain was reported in 2 patients (10.5%).\u003c/p\u003e\n\u003cp\u003eThe patients with MPE due to NSCLC included in the study were 14 cases. The Table\u0026nbsp;2 presents some of their clinical, histological, and TNM characteristics.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eClinical, histological, and prognostic characteristics of patients with malignant pleural effusion due to NSCLC\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHistology\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTNM\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT3N0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT3N1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTxN0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT3N2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTxN0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4N3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTxN3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4N3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1N3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT2aN2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4N0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTxN1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarge cell carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1N2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTxN0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cem\u003eDefinition of abbreviation = Adenocarcinoma (ADC)\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn this group, the median biopsy size for conventional flexible forceps and cryoprobe was 2.5 mm (1.5–3.2 mm) and 5.5 mm (3.8–7.6 mm), respectively (p = 0.07). The number of biopsies also differed: flexible forceps: 5 (4-6.25) biopsies vs cryoprobe: 3 (3–4) biopsies(p = 0.01). The percentage of viable tumor in samples obtained by flexible forceps and cryoprobe was 55% (15–80%) and 55% (10–80%), respectively (p = 0.9). The tumor/non-tumor ratio in the conventional forceps sample was 2.4 (1.2–5.9), while in the cryoprobe sample, it was 3.6 (1.2–10) (p = 0.09).\u003c/p\u003e\n\u003cp\u003eIn all patients, a morphological and immunohistochemical diagnosis was possible using both conventional forceps and cryoprobe biopsies. Only in one case, the samples obtained during semirigid pleuroscopy with both cryoprobe and forceps were insufficient for molecular diagnosis, requiring a surgical video-assisted thoracoscopic surgery. In this case, the pleuroscopy revealed plaque-like lesions.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study is the first published Spanish study to analyze the quality of cryoprobe obtained samples to optimize the molecular diagnosis of MPE due to NSCLC. When pleural biopsies were obtained using a cryoprobe, although there were no differences in the percentage of viable tumor cells, the sample size was significantly larger, as was the tumor/non-tumor ratio. Additionally, fewer biopsies were required when using the cryoprobe.\u003c/p\u003e\u003cp\u003eA limitation of semirigid pleuroscopy is its size and flexibility 1,2. Although the semirigid pleuroscope is more manageable than the rigid one, its smaller diameter limits the ability to obtain larger biopsies, potentially affecting the amount of tissue available for molecular studies. Furthermore, its lower rigidity can make biopsy collection more challenging.\u003c/p\u003e\u003cp\u003eIn a small study conducted by our group on the diagnostic performance of semirigid thoracoscopy for the molecular characterization of MPD due to NSCLC, molecular testing was performed in 14 adenocarcinomas. Sufficient and adequate material was obtained to determine EGFR mutation status in 100% of cases, but only in 90% of cases for ALK translocation15. EGFR mutations were identified in two patients, while no cases of ALK translocation were detected. EGFR mutation detection was possible in all cases, but the yield for ALK translocation analysis was lower 15. Many revolutionary advances have recently been made in the management of NSCLC. There have been a number of approvals of molecular-targeted therapy for subgroups of NSCLC patients with sensitizing EGFR, ALK, ROS1, RET, BRAF V600E, MET, or NTRK alterations 21. In addition to genetic examination, NSCLC are usually subjected to the analysis of PD-L1 protein expression in order to direct the use of immune checkpoint inhibitors 20,21.\u003c/p\u003e\u003cp\u003eCryobiopsy, a relatively novel bronchoscopic technique, has gained prominence in recent years due to its ability to procure larger, higher-quality tissue samples with fewer crush artifacts compared to traditional forceps biopsies 3,4. This method has demonstrated promising diagnostic yields in lung cancer 3.\u003c/p\u003e\u003cp\u003eHowever, none of the published studies on MPE and lung cancer have primarily focused on analyzing the effectiveness of pleural cryobiopsies for mutation studies and other molecular alterations. In studies where this aspect was analyzed secondarily, only EGFR gene mutations had been investigated 8,10. In one study including 14 cases of MPE, four of which were lung adenocarcinomas, obtaining pleural biopsies with a cryoprobe allowed DNA isolation for EGFR mutation analysis in all samples using a commercially available kit8. In another study, samples were valid for EGFR mutation testing, but these results were not compared with the molecular diagnostic yield of biopsies obtained using conventional fórceps 10.\u003c/p\u003e\u003cp\u003eAnother study conducted in an Asian population included six patients with NSCLC, all of whom underwent genetic panel testing to detect driver gene mutations. Sufficient specimens were obtained for appropriate genetic analysis in all cases 14. However, a limitation of this study was the lack of specification regarding which molecular alterations were analyzed. In our study, all the necessary molecular analyses, including PDL1 expression, were successfully performed.\u003c/p\u003e\u003cp\u003eThere is ongoing debate regarding the ideal instrument for medical thoracoscopy. Bansal S et al. compared rigid mini-thoracoscopy with semirigid thoracoscopy and found that biopsy size was larger in the mini-thoracoscopy group (16.1 ± 4.5 mm vs. 8.3 ± 2.9 mm; P \u0026lt; 0.001)16. However, the diagnostic yield of pleural biopsy was similar between the two groups.\u003c/p\u003e\u003cp\u003eWhen comparing rigid versus semirigid thoracoscopy, differences have been observed. In 2016, Wurps H et al. conducted a prospective study with 80 patients to compare parietal pleural biopsies obtained using rigid forceps, flexible forceps, and a cryoprobe during medical thoracoscopy 17. Rigid biopsies were significantly larger than both cryobiopsies and flexible biopsies (P \u0026lt; 0.001), while cryobiopsies were significantly larger than flexible biopsies (P \u0026lt; 0.01). The diagnostic yield of cryobiopsies was lower than that of rigid biopsies but not inferior to flexible biopsies17. In our study, although the tumor percentage was similar in both biopsy types, cryobiopsies were larger, and fewer samples were required with cryobiopsy.\u003c/p\u003e\u003cp\u003eIt is important to consider that tissue remains the standard material for NSCLC diagnosis, and there is a continuous search for new molecular markers in MPE, with the primary limiting factor being the availability of viable samples.\u003c/p\u003e\u003cp\u003eIn this context, cryobiopsy of mediastinal lymph nodes (cryoEBUS) has been described as improving diagnostic accuracy. Although cryoEBUS did not significantly enhance lung cancer diagnosis compared to EBUS-TBNA, it appeared to provide superior samples for complete molecular characterization, including PDL1 studies, while requiring fewer passes per lymph node4.\u003c/p\u003e\u003cp\u003eStudies comparing the diagnostic yield of cryobiopsy by radial endobronchial ultrasound with that of conventional biopsy have shown that cryobiopsy improves diagnostic yield and obtains larger samples in patients with peripheral pulmonary lesions due to lung cancer 18,19.\u003c/p\u003e\u003cp\u003eAs discussed earlier, the main limitation of semirigid pleuroscopy is the small sample size, which necessitates obtaining more biopsies. Additionally, when using flexible forceps, it can be difficult to biopsy lesions located tangentially to the instrument 1,2,5. However, a notable advantage of the semirigid pleuroscope is its ease of use compared to the rigid thoracoscope, making it a favorable option for Interventional Pulmonology Units. The incorporation of cryobiopsy into semirigid pleuroscopy could reduce the number of biopsies required, thereby shortening procedure time and facilitating tissue collection without increasing procedural risks.\u003c/p\u003e\u003cp\u003ePleural cryobiopsy has been shown to be a technique with a low complication rate, as reported in recent systematic reviews on its efficacy and safety 4,5. In our study, despite performing biopsies with both a cryoprobe and conventional flexible forceps, no major complications were observed, nor were there increases in hospital stays or chest drainage duration. These findings are consistent with those reported in the literatura 22.\u003c/p\u003e\u003cp\u003eThe primary limitation of this study is the small number of patients with MPE due to NSCLC, although it is the first study conducted in a Caucasian population for this purpose. Additionally, as previously mentioned, this is an innovative technique that will require further standardization and refinement for integration into clinical practice. Incorporating this technique into semirigid pleuroscopy could enhance procedural methodology, reduce procedure time by requiring fewer biopsies, and do so without increasing complication rates.\u003c/p\u003e\u003cp\u003ePleural cryobiopsy has been demonstrated to be a safe and effective diagnostic tool during pleuroscopy semirigid, providing adequate samples for all molecular testing and for the histological characterization of patients with MPE due to lung cancer.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eStudy population\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProspective study including all patients with a diagnosis or high suspicion of MPE due to NSCLC who underwent semirigid pleuroscopy with cryobiopsies between November 1, 2019, and November 30, 2024, at the Bronchopleural Techniques Unit of Hospital in Spain.\u003c/p\u003e\u003cp\u003eThe inclusion criteria were: age \u0026gt; 18 years; caucasian population and patients with MPE due to NSCLC confirmed by cytology or histology\u003c/p\u003e\u003cp\u003eExclusion criteria included were: patients with benign pleural effusion; patients with paramalignant pleural effusion without evidence of malignant cells in the pleural cavity; other etiologies different from MPE and contraindication for pleural biopsy\u003c/p\u003e\u003cp\u003e\u003cb\u003eSemirigid Pleuroscopy Procedure and Pleural Biopsy\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All procedures were performed according to clinical practice guideline recommendations 2,11. Pleuroscopy was conducted under aseptic conditions in the endoscopy suite, with the patient under spontaneous respiration, conscious sedation (midazolam ± fentanyl), and local anesthesia (2% mepivacaine). The semirigid pleuroscope (LTF-160 autoclave, Olympus, Tokyo, Japan) was used for the procedures. Ultrasound was performed on all patients before trocar insertion to determine the entry point. The pleural fluid was drained at the beginning, and the pleural cavity was inspected under direct visualization. All pleural biopsies were performed on parietal pleural lesions. Before biopsy collection, 5 ml of 2% lidocaine was applied under direct visualization through the working channel of the pleuroscope to the selected area. Initially, 4 to 8 pleural biopsies were taken using flexible conventional forceps. Subsequently, biopsies were collected using a flexible cryoprobe (ERBOCRYOCA, ERBE, Tübingen, Germany) with an external diameter of 1.7 mm (2 to 5 biopsies). The probe tip, which froze tissue using CO₂ gas, was placed on the lesion and frozen for 5–7 seconds. The adhered sample was then extracted.\u003c/p\u003e\u003cp\u003eSamples obtained by both conventional forceps and cryoprobe were placed in separate formalin containers and sent to the Pathology Department for histological analysis. Following the procedure, a 24Fr chest drain was placed\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy Variables\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe following variables were recorded: epidemiological characteristics of patient (age, sex, smoking history), functional characteristics (general health status using the ECOG performance scale), radiological characteristics (size of pleural effusion on chest radigraphic, and characteristics of pleural lesions and/or thickening on CT scan.\u003c/p\u003e\u003cp\u003eOthers variables were also analyzed as tumor classification: Histological tumor type and TNM classification12 at the time of DPM diagnosis or at the time of primary tumor diagnosis in cases of disease progression.\u003c/p\u003e\u003cp\u003eThe prognostic and survival variables were the predictive variables from the LENT prognostic scale13. We recorded the 30-day mortality rate and overall survival.\u003c/p\u003e\u003cp\u003eThe procedure-related variables were: volume of pleural fluid drained, pleural findings during pleuroscopy, duration of chest drainage (days) and the length of hospital stay (days).\u003c/p\u003e\u003cp\u003eAbout the procedure safety, we classified bleeding complications as follows: mild (self-limiting), moderate (requiring topical vasoconstrictors such as adrenaline) and severe (requiring intervention with electrocautery or argon plasma). Other complications, including pain.\u003c/p\u003e\u003cp\u003eWe analized the diagnostic yield and molecular analysis as follows: comparison of histological diagnostic yield between cryobiopsy and conventional forceps biopsies. The pathological findings in both biopsy samples, including: numer, sample size (mm). sample volume (mm²) and percentage of viable vs. non-viable tumor cells. The results of molecular studies were also analyzed (detection of genomic alterations EGFR, ROS1,BRAF, ALK, RET, MET, NTRK and PD-L1 expression)\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthical aspects\u003c/b\u003e:\u003c/p\u003e\u003cp\u003e The study was conducted following the Helsinki Declaration guidelines. The data were pseudonymized for biomedical research purposes. The study has been approved by the provincial research ethics committee of Galicia (Pontevedra-Vigo-Ourense code: 2019/293). All participants provided written informed consent to participate in this study and to allow the publication of their data and sample results.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCategorical variables were presented as counts and percentages, and continuous variables were presented as mean and standard derivation (SD). Statistical analyses were performed using SPSS Statistic 21 (IBM Corp., Armonk, NY). Pearson’s chi-square or Fisher exact test was used to compare proportions, as appropriate. For continuous data, differences between groups were determined using Students t-test or Mann-Whitney U test for parametric or non-parametric data, respectively. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors should have made sustancial contributions to all of the folllowing (the concepcion \u003c/p\u003e\n\u003cp\u003eand design of the study, or acquisition of data, or analysis and interpretation of data, drafting \u003c/p\u003e\n\u003cp\u003ethe article and revising it critically for impostant intellectual concept. \u003c/p\u003e\n\u003cp\u003eAuthor contributions: \u003c/p\u003e\n\u003cp\u003e- conception and design: MBR, AFV\u003c/p\u003e\n\u003cp\u003e- analysis, acquisition, analysis, interpretation of the data: MBR, ILR, AGP, AGM, CRH, ECV\u003c/p\u003e\n\u003cp\u003e- the drafting of the paper or revising it critically for intellectual content: AFV, MND, ECV\u003c/p\u003e\n\u003cp\u003e- The final approval of the versión: all authors\u003c/p\u003e\n\u003cp\u003eAll authors agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e The datasets used and/or analysed during the current study available from the corresponding author ([email protected]) on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interest:\u003c/strong\u003e The authors declare that there are no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research received a specific grant from public agency: Spanish Society of Pulmonology (SEPAR) and Integrated Research Projects of Interventional Pulmonology (PII NI) in 2019.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKapp CM, Lee HJ. Malignant Pleural Effusions. Clin Chest Med. 2021;42:687-696\u003c/li\u003e\n\u003cli\u003eAvasarala SK, Lentz RJ, Maldonado F. Medical Thoracoscopy. Clin Chest Med. 2021;42:751-766\u003c/li\u003e\n\u003cli\u003eSimon M, Simon I,Andrei Tent P, Adina Todea D, Haranguș A. Cryobiopsy in Lung Cancer Diagnosis-A Literature Review. Medicina (Kaunas) 2021 19;57:393. \u003c/li\u003e\n\u003cli\u003eBotana-Rial M, Lojo-Rodr\u0026iacute;guez I, Leiro-Fern\u0026aacute;ndez V, Ramos-Hern\u0026aacute;ndez C, Gonz\u0026aacute;lez-Montaos A, Pazos-Area L, N\u0026uacute;\u0026ntilde;ez-Delgado M, Fern\u0026aacute;ndez-Villar A. Is the diagnostic yield of mediastinal lymph node cryobiopsy (cryoEBUS) better for diagnosing mediastinal node involvement compared to endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)? A systematic review. Respir Med. 2023;218:10738\u003c/li\u003e\n\u003cli\u003eRial MB, Rodr\u0026iacute;guez IL, Roib\u0026aacute;s CM, Fern\u0026aacute;ndez VL, Delgado MN, Barreira \u0026Aacute;S, Torrado AP, Villar AF. Diagnostic Yield and Safety of Pleural Cryobiopsy during Medical Thoracoscopy to Diagnose Pleural Effusion. A Systematic Review and Meta-Analysis. Arch Bronconeumol 2020 ;56:784-791.\u003c/li\u003e\n\u003cli\u003eShafiq M, Sethi J, Ali MS, Ghori UK, Saghaie T, Folch E. Pleural Cryobiopsy: A Systematic Review and Meta-Analysis. Chest. 2020;157:223-230\u003c/li\u003e\n\u003cli\u003eThomas R, et al. Pleuroscopic cryoprobe biopsies of the pleura: a feasibility and safety study.\u003c/li\u003e\n\u003cli\u003eRozman A, Camlek L, Marc Malovrh M, Kern I, Sch\u0026ouml;nfeld N. Feasibility and safety of parietal pleural cryobiopsy during semi-rigid thoracoscopy. Clin Respir J 2016;10:574-8.\u003c/li\u003e\n\u003cli\u003ePathak V, E, et al. Safety and feasibility of pleural cryobiopsy compared to forceps biopsy during semirigid pleuroscopy. Lung 2017;195:371-375\u003c/li\u003e\n\u003cli\u003eTousheed SZ, Manjunath PH, Chandrasekar S, Murali Mohan BV, Kumar H, Hibare KR, Ramanjaneya R. Cryobiopsy of the Pleura: An Improved Diagnostic Tool. J Bronchology Interv Pulmonol 2018;25:37-41\u003c/li\u003e\n\u003cli\u003eShaikh F, Lentz RJ, Feller-Kopman D, Maldonado F. Medical thoracoscopy in the diagnosis of pleural disease: a guide for the clinician. Expert Rev Respir Med 2020;14:987-1000.\u003c/li\u003e\n\u003cli\u003e\u0026ordm; clasificaci\u0026oacute;n TNM; Goldstraw P et al.J Thorac Oncol 2016;11:39\u003c/li\u003e\n\u003cli\u003eClive AO, Kahan BC, Hooper CE, Bhatnagar R, Morley AJ, Zahan-Evans N, Bintcliffe OJ, Boshuizen RC, Fysh ET, Tobin CL, Medford AR, Harvey JE, van den Heuvel MM, Lee YC, Maskell NA. Predicting survival in malignant pleural effusion: development and validation of the LENT prognostic score. Thorax. 2014 Dec;69(12):1098-104.\u003c/li\u003e\n\u003cli\u003eKamatani M, Awano N, Inomata M, Kuse N, et al. Diagnostic yields and safety of thoracoscopic cryobiopsies in Japan: A single-center retrospective observational study. Respir Investig 2024;62:617-622\u003c/li\u003e\n\u003cli\u003eBotana M, A et al. Rendimiento diagn\u0026oacute;stico de la toracoscopia semirr\u0026iacute;gida para la caracterizaci\u0026oacute;n molecular de derrames pleurales malignos de origen pulmonar. Arch Bronconeumol 2018;54:489-91\u003c/li\u003e\n\u003cli\u003eBansal S, Mittal S, Tiwari P, Jain D, Arava S, Hadda V, et al. Rigid Mini-Thoracoscopy Versus Semirigid Thoracoscopy in Undiagnosed Exudative Pleural Effusion. The MINT Randomized Controlled Trial. J Bronchology Interv Pulmonol 2020;27:163-171.\u003c/li\u003e\n\u003cli\u003eWurps H, Sch\u0026ouml;nfeld N, Bauer TT, Bock M, Duve C, Sauer R, Mairinger T, Griff S. Intra-patient comparison of parietal pleural biopsies by rigid forceps, flexible forceps and cryoprobe obtained during medical thoracoscopy: a prospective series of 80 cases with pleural effusion. BMC Pulm Med 2016 Jul;16:98. \u003c/li\u003e\n\u003cli\u003eUdagawa H, Kirita K, Naito T, Nomura S, Ishibashi M, et al. (2020) Feasibility and utility of transbronchial cryobiopsy in precisi\u0026oacute;n medicine for lung cancer: prospective single-arm study. Cancer Sci 111:2488- 2498. \u003c/li\u003e\n\u003cli\u003eArimura K, Tagaya E, Akagawa H, Nagashima Y, Shimizu S, et al. (2019) Cryobiopsy with endobronchial ultrasonography using a guide sheath for peripheral pulmonary lesions and DNA analysis by next generation sequencing and rapi don-site evaluation. Respir Investig 57:150-156.\u003c/li\u003e\n\u003cli\u003eAlexander M, Kim SY, Cheng H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020;198:897-907. \u003c/li\u003e\n\u003cli\u003eAlduais Y, Zhang H, Fan F, Chen J, Chen B. Non-small cell lung cancer (NSCLC): A review of risk factors, diagnosis, and treatment. Medicine (Baltimore) 2023;102:e32899.\u003c/li\u003e\n\u003cli\u003eSafety and accuracy of semirigid pleuroscopy performed by pulmonary fellows at a major university hospital: our initial experience. Adil Shujaat , Abubakr A Bajwa, Faisal Usman, Lisa Jones, James D Cury. J Bronchology Interv Pulmonol 2013;20:213-23.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"biomarker, diagnostic, cryobiopsy, semirigid pleuroscopy, histology, pathological finding","lastPublishedDoi":"10.21203/rs.3.rs-7103432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7103432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCryobiopsy has emerged in recent years as a tool of growing interest in the diagnosis of non-small cell lung cancer (NSCLC). We conducted the first study with the primary objective of analyzing the techniques of biopsy during semirigid pleuroscopy to compare the quality of cryobiopsies versus conventional forceps biopsies, including expected tissue outcomes and the feasibility of histological characterization and molecular testing, of malignant pleural effusion (MPE). Prospective study including 14 caucasian patients with MPE due to NSCLC who underwent semirigid pleuroscopy with cryobiopsies. The median biopsy size for conventional flexible forceps and cryoprobe was 2.5 mm (1.5\u0026ndash;3.2 mm) and 5.5 mm (3.8\u0026ndash;7.6 mm), respectively (p\u0026thinsp;=\u0026thinsp;0.07). The number of biopsies also differed: flexible forceps: 5 (4-6.25) biopsies vs cryoprobe: 3 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) biopsies(p\u0026thinsp;=\u0026thinsp;0.01). The tumor/non-tumor ratio in the conventional forceps sample was 2.4 (1.2\u0026ndash;5.9), while in the cryoprobe sample, it was 3.6 (1.2\u0026ndash;10) (p\u0026thinsp;=\u0026thinsp;0.09). Only in one case, the samples obtained during semirigid pleuroscopy were insufficient for molecular diagnosis. The incorporation of cryobiopsy into semirigid pleuroscopy could reduce the number of biopsies required, the sample size was significantly larger, as was the tumor/non-tumor ratio. This technique could shortening procedure time and facilitating tissue collection without increasing procedural risks.\u003c/p\u003e","manuscriptTitle":"Optimizing Tissue Sampling During Medical Pleuroscopy for Diagnosis of Malignant Pleural Effusion Due to Lung Cancer: Study Criopleura","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 18:21:22","doi":"10.21203/rs.3.rs-7103432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T08:07:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T12:19:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T12:31:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194046931683269606962030804339845255299","date":"2025-08-02T11:19:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T10:28:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101410605491524884495641929012228562401","date":"2025-08-02T09:30:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12559135863224523871599360459929466243","date":"2025-07-30T13:55:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T13:37:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T13:34:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-22T14:07:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-16T18:25:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-16T18:22:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3330a456-7778-4a99-b5f4-5b74e5071fed","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52596888,"name":"Biological sciences/Cancer"},{"id":52596889,"name":"Health sciences/Medical research"},{"id":52596890,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-11-03T16:00:20+00:00","versionOfRecord":{"articleIdentity":"rs-7103432","link":"https://doi.org/10.1038/s41598-025-21170-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-27 15:57:23","publishedOnDateReadable":"October 27th, 2025"},"versionCreatedAt":"2025-08-04 18:21:22","video":"","vorDoi":"10.1038/s41598-025-21170-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-21170-8","workflowStages":[]},"version":"v1","identity":"rs-7103432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7103432","identity":"rs-7103432","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}