Dr. Samantha Herath, MPhil^1*, A/Prof Hema Mahajan, PhD², Dr. Elaine Yap, MBChB³, Prof. Alvin Ing, MD⁴

¹Department of Respiratory Medicine, Northern Beaches Hospital, 105, French’s Forest Road West, French’s Forest, NSW 2086, Sydney, Australia

²The University of Sydney, Sydney Medical School, Sydney, NSW, Australia.

³Middlemore Hospital, Auckland, New Zealand.

⁴Macquarie University Hospital, Macquarie Park, NSW, Australia

*Corresponding Author: Dr. Samantha Herath, MPhil, Department of Respiratory Medicine, Northern Beaches Hospital, 105, French’s Forest Road West, French’s Forest, NSW 2086, Sydney, Australia

Abstract
Introduction:
Radial EBUS (R-EBUS) biopsy for lung lesions has a marked safety profile with less than 1.5% pneumothorax in comparison to CT-guided transthoracic biopsy of 20 %. However, the diagnostic yield of R-EBUS remains suboptimal at 73 % in comparison to CT -TTB at 92 %. The size of the Guide Sheath (GS), the addition of aspiration needle biopsy and multi-modal sampling may improve the diagnostic yield.

Methodology:
This is a multi-center randomized controlled study. All patients referred to R-EBUS for a biopsy were offered the study and randomized to large or small GS arms. An aspiration needle is added to the large GS arm to assess the additional diagnostic yield. Each patient had 1-3 biopsies of forceps and brush (+ needle aspirate in large GS arm). All procedures were done under sedation. CXR has taken 1-hour post-procedure. The study was terminated due to slow recruitment at 42 patients planned interim analysis.

Results:
The large GS tended to provide a better diagnostic yield (72 %) in comparison with small GS (60 %) OR 1.77 (95 % CI 0.44- 7.17), this was improved further with an addition of aspiration needle (86 %) in the large GS arm. Aspiration needle was the sole diagnostic specimen 13 % and able to provide EGFR analysis similar to forceps biopsy. Multimodal biopsies seem to improve diagnostic yield. No pneumothorax or bleeding was recorded.

Conclusion:
Despite the type 2 error and small sample size, the large GS seem to provide better results. We suggest utilising an aspiration needle to improve the diagnostic yield. A multimodal biopsy is encouraging with better diagnostic yield, EGFR capability without additional side effects.

Keywords: Cryo-biopsy, radial EBUS, CT-Guided transthoracic biopsy, Diagnostic yield, EGFR, Bleeding, pneumothorax, Biopsy technique.

Introduction
Peripheral pulmonary Lesions (PPL) suspected of lung cancer requires a safe and effective biopsy. Radial Endobronchial ultrasound (R-EBUS) is a widely used guided bronchoscopy method [1,2] for diagnosis of PPL, with marked safety profile. In five meta-analyses of over 50 studies with 7000 patients on R-EBUS, the overall side effect rate was 1.5 % with 0.7 % requiring a chest drain insertion [1- 5]. This is in comparison to the “gold standard” CT-guided transthoracic (CT-TTB) biopsy, which has a pneumothorax rate of 20 % with a chest drain rate of 7.3 % in a meta-analysis of 48 papers consisting of 10.383  biopsies.[6].  Yet,  the diagnostic yield of   R-EBUS is suboptimal (69 %-73 %) [4,7-9], in comparison to the “gold standard”  CT-guided transthoracic biopsy yield of  92 % [10,11]. Despite being recommended by the American College of Chest Physicians (ACCP) clinical guidelines as a mainstream diagnostic method of PPL, this lower diagnostic yield hinders R - EBUS being a more widely used biopsy technique in PPL [12].

R-EBUS is a flexible wire-like ultrasound probe that gives a 360- degree radial view of the airway. It can be used via the working channel of the bronchoscope and advances to the bronchial subsegments beyond the bronchoscopy vision. This R-EBUS is introduced using a guide sheath (GS), which houses the ultrasound and has a snug fit to it. Once the PPL is located using the ultrasound, the ultrasound is withdrawn leaving the GS in situ which then acts as an extended working channel. Biopsy tools are introduced via this GS. There are two commercially available GS sizes. The small GS (K 201- Olympus) houses small instruments and accommodates biopsy equipment up to a diameter of 1.4 mm and the large GS (K203- Olympus) houses all conventional bronchoscopy biopsy equipment up to a diameter of 1.91 mm.

Methodology
This is a single-blind, randomized, multicentre, interventional exploratory study.
Ethics approval: Australia (HREC 2019/ETH02599) and New Zealand (15/STH/3).
Patients over 18y, referred for R-EBUS were recruited. Pregnant and lactating patients, patients on anti-coagulation that cannot be stopped for medical reasons were excluded. Aspirin was permitted. Informed consent was taken from all patients and randomized to the large-GS (K -203 Olympus-outer diameter 2.6mm and the largest diameter of biopsy tool that can be accommodated 1.91mm) or small-GS (K-201 Olympus-Outer diameter 2mm and the largest diameter of biopsy tool that can be accommodated 1.4mm) arms. A planning CT scan of the chest within one month of the procedure was needed with the slice thickness of at least 1mm for the bronchoscopist to perform a manual pathway to the segmental bronchi.

Patients in the large-GS arm was sampled with forceps, brush, and aspiration needle (either Exelon 21G;13 mm length or Medtronic’s 21G: 8mm length); while patients in the small-GS arm were sampled with forceps and brush only as a small-sized aspiration needle was not available. For each sampling method, 1-3 samples were taken at the discretion of the proceduralist. For each GS type, the sequence of the sampling method was randomized. The small USS probe 1.7mm Olympus UM-S20-17S was used for all procedures.

Results
42 patients from two tertiary hospitals were recruited from January 2015 to December 2017. 24 were assigned to the large GS and 18 to the small GS. There were no statistically significant differences between the two groups in any of these baseline parameters (Table 1).

Table 1: Comparing the patient demography and CT characteristics between the Large GS and small GS arms.

Two subjects in the small-GS arm and two in the large-GS arm had a diagnosis via Linear-EBUS prior to the R-EBUS procedure, performed as per institutional guidelines. One lesion in the thin-GS arm was not visible on ultrasound. After these three patients were excluded, a total of 22 patients from the large-GS group and 15 from the small GS group underwent R-EBUS. In the large-GS arm, 19/22 (86 %) had a diagnosis made on R-EBUS. (16 malignant, three benign). Of the three who were non-diagnostic, two proceeded with CT-guided biopsy which confirmed malignancy and one received radiotherapy based on high clinical probability of malignancy. In the small-GS arm, R-EBUS was diagnostic in 9/15 (60 %). (Five malignancies, four benign). Of the six who were non-diagnostic, four were confirmed malignancy (one lobectomy, three CT-guided biopsy), one was benign on follow-up, and one was lost to follow-up. The overall diagnostic yield, with only the forceps and brush, as sampling methods was 16/22 (72 %) in the large- GS arm and 9/15 (60 %) in the small-GS arm. OR 1.77 (95 % CI 0.44- 7.17). When the aspiration needle was added to the large-GS arm, the diagnostic yield improved to 19/22 (86 %) Vs 9/15 (60 %) OR 4.2 (95 CI 0.85-20), P =0.118. 3/22 (13.6 %) were diagnosed only from the aspiration needle. In the large-GS arm, a diagnosis was made solely from forceps in 18.1 %, needle aspirate in 13.6 % and brush in 4.5 %.

In terms of ability to perform EGFR testing,12/16 (75 %) had EGFR status diagnosed in the large-GS. The number of malignancies diagnosed and ability to perform EGFR were analysed: with the aspiration needle there were 13/16 (81 %) malignancies, with 62 % suitable for EGFR, Cytology brush had 11/16(69 %) malignancies with 54 % suitable for EGFR and Forceps had 9/16 (56 %) malignancies with 66 % suitable for EGFR.

In the small-GS, a diagnosis was made solely on forceps in 7 % and brush in 7 %. 40 % of the small-GS samples were suitable for EGFR. All patients had a chest-X ray one-hour post-procedure. No bleeding or pneumothorax was reported in both arms.

Discussion
Both large and small-GS arms had similar baseline patient demographics and lesion size.
In comparing the use of small biopsy forceps and brush through the small-GS, the larger biopsy forceps and brush, via the large-GS demonstrated a tendency for a better diagnostic yield and better suitability for EGFR testing. This may be due to the larger samples obtained from the large biopsy tools that would survive the processing better. It may also be possible that the large GS arm had more malignancies than the small GS producing better diagnostic yield. However, these results were not statistically significant in keeping with the only previously published study by Kurimato et al [19].

The addition of an aspiration needle improved the diagnostic yield of the large GS further. This may be due to the ability of the aspiration needle to sample PPL placed adjacent to bronchi as well as inside the bronchi. 13.6 % of patients had a diagnosis made solely from the aspiration needle. There is limited retrospective analysis published by Chen et al that had compared the aspiration needle against forceps biopsy with R-EBUS and demonstrated 80 % diagnostic yield with aspiration needle in comparison to 77 % with forceps biopsy [8]. Our data is also supported by the ACQUIRE registry data that demonstrate aspiration needle was able to provide a diagnosis 9.5 % of the time when forceps biopsy was negative [21]. The aspiration needle increased the diagnostic yield as the sole diagnostic sample and also complement the procedure by improving the overall diagnostic yield in our study and reported by previous limited studies as well [24-26]. The ability of aspiration needle sample to be examined at ROSE for immediate results and manoeuvre the GS to a better location, if needed, and the ability to perform the procedure under sedation are additional advantages [27].

Whilst the ability to perform EGFR had been reported to be 80 % for forceps biopsy with R-EBUS [22], studies have demonstrated cytology brush to be able to provide EGFR as well but less (66 %) than that of forceps biopsy [23], however in cytology samples with malignant cells, a 100 % ability for the cytology brush to provide EGFR analyse on the malignant samples had been demonstrated [28]. Studies have not prospectively compared the ability to perform EGFR in aspiration needles nor compared it with forceps biopsy in radial EBUS. In this study, aspiration needle was suitable for EGFR analysis similar to the forceps biopsy; adding further value to the already improved diagnostic yield.

The addition of the aspiration needle to the large-GS, improved the diagnostic yield further, without an increase in complications, especially pneumothorax, this has been confirmed by one other study demonstrating no increase in adverse events with the use of needles aspirate even when the PPL was not visible on ultrasound [24].

Multi-modal biopsy techniques have not been well considered as a measure of improving diagnostic yield. The tendency has been on high reliance on forceps biopsy alone and addition of cytology brush on a few studies with a very limited combination of aspiration needle as a multi-modal biopsy tool². Although studies in R-EBUS had demonstrated that the addition of cytology brush had improved the diagnostic yield compared to forceps alone, the concept of multimodal biopsy was not well explored. In this study, using multi-modal sampling (forceps, brush +/- aspiration needle) has resulted in an improved overall diagnostic yield in both arms.

With the multimodal biopsy techniques, the patients in the large GS arm received a maximal of 3 types (Forceps biopsy, cytology brush and aspiration needle) of biopsies with a maximum of three passes in each biopsy leading to nine biopsies per patient, yet there were no reported pneumothorax and bleeding, and all patients were discharged home as planned. Whilst this is in keeping with the overall safety of the R-EBUS procedure, as well as the haemostasis achieved by using a GS, this may also be due to the larger (mean 3.7-4.2cm) PPL size in this cohort as well. Multi-modal biopsy needed to be performed in smaller PPL with further studies to confirm safety. Using the GS consistently may have led to better haemostasis and higher diagnosis in both arms as acknowledged by the previous meta-analysis. [3].

Limitations
The main limitation of this study is the early termination due to lack of recruitment. These results are not statistically significant due to the small sample size with the resultant type 2 error. In this study all procedures were performed using the GS therefore, appreciating that some centres do not use the GS due to the extra expense or the inability to reach certain locations, the result of this study is not applicable when a GS is not used.

We were not able to perform a needle aspirate in the small GS arm due to the lack of availability of a small needle which is a future development requirement. We are unable to provide guidance on the best needle aspirate to use as there was no randomisation of the different types of needle aspirate and further studies are needed.

Conclusion
We suggest including an aspiration needle and using multi-modal biopsy tools with R-EBUS via a large GS to improve the diagnostic yield of R-EBUS. The ability to perform EGFR testing with aspiration needle samples further strengthens the argument. We noted utilising a GS consistently improved the side effect profile and the diagnostic yield as noted in the previous meta-analysis.

Multi-modal sampling is able to provide better diagnostic results and the tools used need to be tailored to PPL characteristics for the optimum results. Despite the higher number of biopsies performed the safety profile of R-EBUS was well preserved.

The R-EBUS procedure can be performed under sedation and doesn’t require fluoroscopy. If biopsy tools can be improved to provide better diagnostic yield, this extremely safe method would be widely utilised and form an integral pathway of a lung cancer diagnosis.

Conflicts of interest statement: All Authors listed here do not have any conflicts of interest to declare.

Funding: This study did not receive any funding

Prior abstract publication:
Herath, S, Yap, E, Low, I, Mahajan, H, & Ing, A. (2019, March). Multi-centre a randomized controlled trial with Radial EBUS (R-EBUS) comparing thin vs thick guide sheath (GS) with the addition of an aspiration needle biopsy, in the diagnosis of peripheral lung lesions (REBUST 2). Respirology supplement 2019 (vol. 24, pp. 91-91). 111 River St, Hoboken 07030-5774, NJ USA: Wiley.

Abbreviations List:
R- EBUS: Radial Endobronchial Ultrasound-guided biopsy CT: Computed Tomography

EGFR: Epidermal Growth Factor Receptor GS: Guide Sheath

Guarantor: Dr. Samantha Herath is the Guarantor of the content of the manuscript including the data and analysis.

Author contribution

1. Dr. Samantha Herath (Respiratory Physician) is the principal investigator and designed the study protocol, designed consent forms, and data entry forms, obtained ethics approval for ethics committees in Australia and New Zealand. Dr. Herath applied and secured grant funding and equipment funding for the Cryo-Radial procedure. Dr. Herath performed the Cryo-Radial procedure and collected data. She was responsible for patient follow-up, data entry, and data analysis as well as the write-up of this paper.
2. Dr. Alvin Ing (Respiratory Physician) is a co-investigator and assisted with designing the study protocol, performed the Cryo- Radial procedure, and collected data. He was responsible for patient follow-up and the write-up of this paper.
3. Dr. Elaine Yap (Respiratory Physician) assisted with designing the study protocol, assisted in obtaining ethics approval for ethics committees in New Zealand. She performed the Cryo-Radial procedure and collected the data. She was responsible for patient follow-up, data entry, and analysis. She assisted in the write-up of this paper.
4. Dr. Hema Mahajan (Pathologist) is a co-investigator. She assisted in the design of the study protocol and assisted in the analysis of pathological specimens.

References

1. Steinfort DP, Khor YH, Manser RL, Irving LB (2011) Radial probe endobronchial ultrasound for the diagnosis of peripheral lung cancer: Systematic review and meta-analysis. Eur Respir J. 37(4): 902-10.
2. Ali MS, Trick W, Mba BI, Mohananey D, Sethi J, et al. (2017) Radial endobronchial ultrasound for the diagnosis of peripheral pulmonary lesions: A systematic review and meta-analysis. Respirology. 22(3): 443-453.
3. Memoli JSW NP, Silvestri GA (2012) Meta-analysis of Guided Bronchoscopy for the Evaluation of the Pulmonary Nodule. Chest. 142(2): 385-93.
4. McGuire AL, Myers R, Grant K, Lam S, Yee J (2020) The diagnostic accuracy and sensitivity for malignancy of radial- endobronchial ultrasound and electromagnetic navigation bronchoscopy for sampling of peripheral pulmonary lesions: Systematic review and meta-analysis. Journal of Bronchology and Interventional Pulmonology. 27(2): 106-121.
5. Sainz Zuñiga PV, Vakil E, Molina S, Bassett RL Jr, Ost DE (2020) Sensitivity of radial endobronchial ultrasound-guided bronchoscopy for lung cancer in patients with peripheral pulmonary lesions: an updated meta-analysis,. Chest. 157(4): 994–1011.
6. DiBardino DM, Yarmus LB, Semaan RW (2015) Transthoracic needle biopsy of the lung-Review Article on Percutaneous Needle Aspiration/Transthoracic Needle Aspiration (PCNA/TTNA). J Thoracic Dis. 7(S4): S304-S316.
7. Lee KH, Lim KY, Suh YJ, Hur J, Han DH, et al. (2019) Diagnostic accuracy of percutaneous transthoracic needle lung biopsies: A multicenter study. Korean J Radiol. 20(8): 1300-1310.
8. Chen A, Chenna P, Loiselle A, Massoni J, Mayse M, et al. (2014) Radial probe endobronchial ultrasound for peripheral pulmonary lesions: A 5-year institutional experience. Ann Am Thorac Soc. 11(4): 578-82.
9. Izumo T, Hayama M, Matsumoto Y, Hiraishi Y, Tsuchida T. Safety of endobronchial ultrasound with a guide sheath (EBUS- GS) for peripheral pulmonary lesions; More than 1,200 our consecutive cases. Respirology 2015; 3): 24.
10. DiBardino DM, Yarmus LB, Semaan RW (2015) Transthoracic needle biopsy of the lung. J Thorac Dis. 7: S304-16.
11. Fu YF, Zhang JH, Wang T, Shi YB (2021) Endobronchial ultrasound-guided versus computed tomography-guided biopsy for peripheral pulmonary lesions: A meta-analysis. Clinical Respiratory Journal. 15(1): 3-10.
12. Rivera MP, Mehta AC, Wahidi MM (2013) Establishing the diagnosis of lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians Evidence- Based Clinical Practice Guidelines. Chest. 143 (5 Suppl): e142S- e165S.
13. Herth FJF, Eberhardt R, Becker HD, Ernst A (2006) Endobronchial ultrasound-guided transbronchial lung biopsy in fluoroscopically invisible solitary pulmonary nodules: A prospective trial. Chest. 129(1): 147-50.
14. Asano F, Shinagawa N, Ishida T, Shindoh J, Anzai M, et al. (2013) Virtual bronchoscopic navigation combined with ultrathin bronchoscopy a randomized clinical trial. Am J Respir Crit Care Med. 188(3): 327-33.
15. Yamada N, Yamazaki K, Kurimoto N, Asahina H, Kikuchi E, et al. (2007) Factors related to diagnostic yield of transbronchial biopsy using endobronchial ultrasonography with a guide sheath in small peripheral pulmonary lesions. Chest. 132(2): 603-8.
16. Guvenc C, Yserbyt J, Testelmans D, Zanca F, Carbonez A, et al. (2015) Computed tomography characteristics predictive for radial EBUS-miniprobe-guided diagnosis of pulmonary lesions. Journal of Thoracic Oncology. 10(3): 472-8.
17. Ostendorf U, Scherff A, Khanavkar B, Ewig S, Hecker E (2011) Diagnosis of peripheral lung lesions by EBUS-guided TBB in routine practice. Pneumologie. 65(12): 730-5.
18. Kramer T, Manley CJ, Annema JT (2021) Robotic Bronchoscopy for Diagnosing Peripheral Lung Lesions: Are We There Yet? Chest. 160(3): E326-E327.
19. Kurimoto N, Osada H, Miyazu Y, Ishida A, Miyazawa M (2007) Endobronchial ultrasonography using two types of the guide sheath (ebus-gs) for peripheral pulmonary lesions. Chest. 132(4): 439a.
20. Zhang L, Wu H, Wang G (2017) Endobronchial ultrasonography using a guide sheath technique for diagnosis of peripheral pulmonary lesions. Endoscopic Ultrasound. 6(5): 292-299.
21. Ost DE, Ernst A, Lei X, Kovitz KL, Benzaquen S, et al. (2016) Diagnostic Yield and Complications of Bronchoscopy for Peripheral Lung Lesions Results of the AQuIRE Registry. American Journal of Respiratory and Critical Care Medicine. 193(1): 68-77.
22. Guisier F, Salaün M, Lachkar S, Lamy A, Piton N, et al. (2016) Molecular analysis of peripheral non-squamous non-small celllung cancer sampled by radial EBUS. Respirology. 21(4): 718-26.
23. Yu KL, Tsai TH, Ho CC, Liao WY, Lin CK, et al. (2018) The value of radial endobronchial ultrasound-guided bronchial brushing in peripheral non-squamous non-small cell lung cancer. Sci. 8(1): 5837.
24. Hayama M, Izumo T, Matsumoto Y, Chavez C, Tsuchida T, Sasada S. Tbna through a guide sheath (GS-TBNA) for peripheral pulmonary lesions that cannot be precisely detected by radial EBUS. Respirology 2014; 3): 80.
25. Hayama M, Izumo T, Chavez C, Matsumoto Y, Tsuchida T, et al. (2015) Additional transbronchial needle aspiration through a guide sheath for peripheral pulmonary lesions that cannot be detected by radial EBUS. Clin Respir J. 11(6): 757-764.
26. Chao TY, Chien MT, Lie CH, Chung YH, Wang JL, et al. (2009) Endobronchial ultrasonography-guided transbronchial needle aspiration increases the diagnostic yield of peripheral pulmonary lesions: A randomized trial. Chest. 136(1): 229-236.
27. Chhajed PN, Tamm M (2006) Bronchoscopy for small pulmonary nodules and mediastinal staging of lung cancer: just do it!. Am J Respir Crit Care Med. 174(9): 961-2.
28. Sánchez-Font A, Chalela R, Martín-Ontiyuelo C, Albero- González R, Dalmases A, et al. (2018) Molecular analysis of peripheral lung adenocarcinoma in brush cytology obtained by EBUS plus fluoroscopy-guided bronchoscopy. Cancer cytopathol. 126(10): 860-871.