The Study for Diagnostic Value and Safety of Transbronchial Mediastinal Cryobiopsy Combined With EBUS-TBNA in Mediastinal Lesions
Phase: NA
Conditions: Mediastinal Lymphadenopathy
Sponsor: Third Military Medical University

Date and version No: 8th February 2022 version 2.1
Chief Investigator Signature:
Confidentiality Statement
This document contains confidential information that must not be disclosed to
anyone other than the Sponsor, the Investigator Team, HRA, host organisation,
and members of the Research Ethics Committee and Regulatory Authorities
unless authorised to do so.
PROTOCOL
TITLE:
Transbronchial needle aspiration with
cryobiopsy in the diagnosis of
mediastinal disease: a randomised trial
VERSION NUMBER:
2.1
NCT NUMBER:
NCT04572984
Chief Investigator and trial leader:
Professor Felix JF, Department of
Pneumology and Critical Care Medicine,
Thoraxklinik, and Translational Lung
Research Center Heidelberg, University
of Heidelberg, Heidelberg, Germany
Co-Principal Investigator and Co-trial
leader：
Ye Fan, Department of Respiratory
Disease, Xinqiao Hospital, Third Military
Medical University, Chongqing, China
Sponsor:
University of Heidelberg, Heidelberg,
Germany
Xinqiao Hospital, Third Military Medical
University, Chongqing, China

Date and version No: 8th February 2022 version 2.1
TABLE OF CONTENTS
1. Background.......................................................................................................................... 1
2. Objective...............................................................................................................................1
3. Trial design and procedure................................................................................................ 1
3.1 Trial Participants........................................................................................................ 2
3.2 Sample size................................................................................................................3
3.3 Randomisation and blinding.................................................................................... 3
3.4 Patient withdrawal..................................................................................................... 4
4. Trial interventions................................................................................................................ 4
4.1 Sedation......................................................................................................................4
4.2 EBUS procedure........................................................................................................5
4.3 EBUS-TBNA...............................................................................................................6
4.4 Transbronchial mediastinal cryobiopsy after EBUS-TBNA.................................6
4.5 Postbiopsy.......................................................................................................7
4.6 Procedure interruption..............................................................................................7
4.7 Histological assessment...........................................................................................8
4.8 Patient follow-up........................................................................................................ 8
5. Study endpoints...................................................................................................................9
6. Data analysis........................................................................................................................9
7. Ethical considerations...................................................................................................... 10
8. Risks and benefits.............................................................................................................10
9. Privacy and personal information protection.................................................................11
10. Funding.............................................................................................................................11
11. Reference......................................................................................................................... 11

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1. Background
Mediastinal and hilar lymphadenopathy can be manifested in both malignant and benign
disorders. Accurate timely diagnosis of mediastinal disease is essential for choosing
appropriate treatment and for predicting prognosis, which usually requires sufficient samples
qualified for pathological and molecular assessment. Endobronchial ultrasound-guided
transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive and safe technique
that is widely employed for the sampling of mediastinal lesions1. Clinical guidelines
recommended that EBUS-TBNA is a preferred and initial modality for the invasive staging of
non-small cell lung cancer (NSCLC)2, 3. However, the relatively limited amount of intact tissue
obtained might restrict its diagnostic yield in mediastinal diseases of other etiologies4, 5.
We recently demonstrated that transbronchial mediastinal cryobiopsy provides larger
amounts of intact tissue and improves the overall diagnostic yield as compared to
EBUS-TBNA6, 7. The excellent performance of this approach, particularly in non-lung cancer
diseases, suggests a potentially additive value of mediastinal cryobiopsy to the standard
sampling strategy7.
2. Objective
We aim to prospectively evaluate the diagnostic accuracy and the safety of adding
transbronchial mediastinal cryobiopsy to standard sampling in mediastinal diseases.
3. Trial design and procedure
This is a prospective, mutlicentre, randomised, comparative, diagnostic accuracy study,
which is designed to evaluate diagnostic value and safety of combined application of
EBUS-TBNA and mediastinal cryobiopsy. The study is designed and reported following the
Consolidated Standards for Reporting Trials (CONSORT).

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3.1 Trial Participants
Consecutive individuals who need bronchoscopic diagnosis based on suspicion of either
benign or malignant disease in mediastinal or hilar lymph nodes or masses, and who have
completed preoperative examinations, are prospectively enrolled by the team member from
each of the participating centres according to the inclusion criteria. Those individuals not
suitable for bronchoscopy or mediastinal biopsy or who meet the exclusive criteria are
excluded from study. The details of the inclusive and exclusive criteria are presented below.
3.1.1 Inclusive criteria
Aged ≥15 years old;
Patients with at least one mediastinal lesion with a short-axis ≥1 cm that is detected by
thoracic image;
Patients with recently discovered mediastinal lesions, clinical respiratory symptoms of
cough, expectoration, thoracalgia, apnea, or complicated lung lesions implicated by thoracic
image, which indicates a need for biopsy to identify the underlying etiology;
Patients should have undergone necessary preoperative laboratory examinations and
other examinations such as cardiac ultrasound or CTA when necessary, in order to exclude
potential contradictions;
Patient is willing and able to give informed consent for participation in the study.
3.1.2 Exclusive criteria
Patients with contradictions to endoscopic examination, such as severe cardiopulmonary
diseases,
coagulation
disorders,
intolerance
to
anesthesia
or
endoscopic
operation,
psychiatric disorders, or severe neurosis, and so on;
The EBUS procedure fails to detect the mediastinal lesion;
The mediastinal lesions are actually cysts or abscess;
Patients need for additional procedures other than EBUS examination (such as
endobronchial biopsy);

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Patients could not provide full informed consent;
Patients have been previously randomised to an arm of the present trial or involved in
other clinical trials in the recent 3 months;
Patients have any other conditions that are considered to be inappropriate to be involved
in this study.
3.2 Sample size
Sample size of this study is calculated by Power Analysis version 11.0 and Sample Size
version 11.0 software using two independent proportions analysis.
Sample size estimation is conducted according to the findings of our prior transbronchial
mediastinal biopsy report indicating the similarly low operational risks of EBUS-TBNA and
cryobiopsy (no serious adverse events have been observed in both groups), and thus the
number of the required cases is calculated mainly on the basis of potential diagnosis benefit7.
Assuming that the increase in the overall diagnostic yield by the combined approach is 13.5%
(a threshold of 79.9%), α=0.05, β=0.1, power is 0.9, N is calculated to be 130 for each group.
The result report is presented above.
3.3 Randomisation and blinding
Patients will be randomised using a computer-generated blocked randomisation scheme
(block size four based on a random table from an independent statistician who has no further

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involvement in the actual clinical study). The allocation is stratified at the centre level. Once
study participants are deemed eligible, a member of the trial team will randomise the
participant. The random allocation sequence will not be disclosed to patients and consenting
investigators until the interventions are assigned. Because of the nature of the intervention,
neither patients nor study personnel could be masked to group assignment. The radiologists
and the pathologists are unaware of the patient recruitment for a clinical trial. The
paper-based case forms are applied to record study data, and important information such as
patient-related information and the diagnostic results, are also recorded in an electronic
system. The data are further collected by an independent research assistant. Data monitoring
is conducted at each of the centres on the basis of the the local ethic committee
recommendation.
3.4 Patient withdrawal
All patients reserve the right to withdraw from the study at any time. For those who lack
appropriate decision-making ability, the expression of dissent in any form will be taken as an
indication that they do not wish to be included and these individuals are then withdrawn.
4. Trial interventions
All the procedures are performed by the same experienced bronchoscopist from each
centre.
4.1 Sedation
Patients are in supine position. The upper airway topical anesthesia is achieved by 2%
lidocaine, and conscious sedation is achieved by intravenous injection of midazolam and
fentanyl. Oxygen is initially administrated at 1-2 L/min and increased when oxygen saturation
is lower than 90%. Patients’ vital signs and pulse blood oxygen saturation are continuously
monitored.

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4.2 EBUS procedure
In
our
procedure,
rigid
bronchoscopy
is
not
used.
An
EBUS
bronchoscope
(BF-UC260F-OL8 or BF-UC260F, Olympus, Tokyo, Japan) is inserted into the trachea through
the nose (if the nasal airway is too narrow for the EBUS bronchoscope to pass through or
there are other contradictions, the oral way can be the alternative option). As the EBUS
bronchoscope has been inserted in, the glottis, the trachea, the bronchuses are probed in turn.
After the examination of the airway, the EBUS bronchoscope is then contacted with the airway
wall, and a systematic examination of all mediastinal and hilar lymph node stations is
sequentially performed according to the Mountain-Dressler lymph node map. For each lesion,
the blood supply is identified by the Doppler Ultrasound and the size is measured by its long
axis and short axis.The most suspicious target (lesion with disease characteristics implicated
by imaging data [such as significantly enlarged size, contrast enchantment on CT imaging,
high FDG uptake on PET/CT imaging, et al.]) with relatively less biopsy risk is chosen for
biopsy sampling. The ultrasonic detection duration is defined as the time interval from the
insertion of the bronchoscope into the nose or mouth to the moment when the mediastinal
lesion is detected.
After the target is localized, EBUS-TBNA (step 4.3) with or without transbronchial
mediastinal cryobiopsy (step 4.4) is performed according to the randomised group. To avoid
the risk of severe bleeding, the vascularization status of the lesion is assessed by
Doppler-mode blood flow imaging as previously described by Nakakima and coworkers8, 9.
Lesions are classified as follows: grade 0: no blood flow or small amounts of flow; grade I: a
few main vessels running toward the center of the lesion; grade II: a few punctiform or
rod-shaped flow signals, a few small vessels found as a long strip of a curve; and grade III:
rich flow, more than four vessels found with different diameters and a twist or helical-flow
signal. If the lesion is too abundant in blood supply (Grade III), too close to large vessels, or
other conditions detected by EBUS that indicates high risk of biopsy, another appropriate
lesion would be considered for sampling.

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4.3 EBUS-TBNA
4.3.1 As the target lesion has been visualized by ultrasound, a dedicated TBNA needle
(21-gauge or 22-gauge, NA-201SX-4021 or NA-201SX-4022, Olympus, Tokyo, Japan, which
are the standard biopsy instruments for EBUS-TBNA as recommended by the current
guideline) is passed through the working channel of the EBUS bronchoscope, and advanced
through the tracheobronchial wall into the lesion under real-time ultrasound visualization,
avoiding areas with abundant blood supply or massive necrosis10. After the central stylet has
been removed, the suction is applied using a syringe while the needle is manipulated back
and forth for 25 times within the lesion. The specimen collected in the lumen of the needle is
pushed out using the central stylet and then blown by air with a syringe onto a glass slide. The
specimen on the glass slide is smeared and ﬁxed in 95% alcohol for cytologic examination.
The visible tissue fragment on the glass slide and the residual specimen in the lumen of the
needle are collected and transferred into the containers ﬁlled with formalin for cell block
analysis. Both cell block analyses and glass slide-based cytology analyses are performed.
4.3.2 Repeat 4.3.1 for 4 times. The operation duration for TBNA is recorded from the
insertion of the needle into the bronchoscope to its final exit from the working channel.
Patients assigned to the combined group will receive one-time mediastinal cryobiopsy after
the completion of EBUS-TBNA, which is specified as below.
4.4 Transbronchial mediastinal cryobiopsy after EBUS-TBNA
4.4.1 Opening of a window on the airway wall
The target lesion is supervised by ultrasound, and a high-frequency electric needle knife
(Olympus KD-31C-1, Olympus, Tokyo, Japan) is inserted through the working channel of the
EBUS bronchoscope, and an incision is then made (about 2 - 3 mm) on the airway wall
adjacent to the target lesions. The knife is then advanced into the lesion under real-time
ultrasound visualization, avoiding areas with abundant blood flow or massive necrosis. After

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being confirmed within the lesion and the measurement of its insertion depth, the needle knife
is then withdrawn. The time from the insertion of the needle knife into the bronchoscope to its
removal from the work channel is recorded as the window-opening duration.
4.4.2 Cryobiopsy
An Erbe frozen probe (Erbe 20402-401, 1.1 mm, Erbe, Tübingen, Germany) is then
passed through the working channel of the EBUS bronchoscope, and advanced through the
window on the airway wall into the lesion under real-time ultrasound visualization, avoiding
areas with abundant blood flow or massive necrosis. After measuring the insertion depth of
the cryoprobe, the probe is activated to cool down with nitrous oxide for around 7 seconds.
Then the bronchoscopist gently extracts the cryoprobe tip with the rapidly frozen biopsy
specimen attached to it, and then withdraws the bronchoscope and the cryoprobe. The frozen
specimen is released from the cryoprobe by thawing in saline and it is then fixed in formalin.
4.4.3 One-time cryobiopsy is performed. Cryobiopsy time is recorded from the insertion
of the cryoprobe into the bronchoscope to its final exit from the working channel. The size of
each specimen is measured and recorded by its long axis and area.
4.5 Postbiopsy
Hemorrhage within the airway is assessed through the bronchoscope, especially in the
biopsy
site.
The
procoagulants
are
locally
used
when
necessary.
Record
all
the
procedure-associated adverse events during the procedure, including bleeding, severe
hypoxemia, cardiac arrhythmia, hypotension, etc. The overall procedure duration is measured
from the insertion of bronchoscope to its removal from the patient’s nose or mouth.
4.6 Procedure interruption
The EBUS procedure will be interrupted accordingly in the following situations:
1) Patients are intolerant to the bronchoscopic procedure;

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2) The ultrasound fails to detect the lesion within 20 minutes;
3) The blood supply is so abundant in the lesion that is considered inappropriate to
continue biopsy due to the high risk;
4) The ultrasound identifies that the lesion is a cyst or abscess;
5) Severe adverse events occur during operation, such as severe bleeding. Bleeding is
classiﬁed as follows: grade 0, traces of blood not requiring suctioning; grade 1, bleeding only
requiring suctioning and hemostatic wedging for up to 2 minutes (two 1-minute cycles); grade
2, bleeding requiring hemostatic wedging for 3 minutes or more; grade 3, bleeding requiring
topical instillation of epinephrine or ice cold saline; and grade 4, bleeding requiring
hemodynamic support, transfusion of blood products, selective mainstem intubation,
bronchial blocker, hospital admission, or surgical intervention11, 12.
4.7 Histological assessment
All the specimens obtained from patients are sent to the Department of Pathology and
appropriately processed. The pathologic evaluation is made by the pathologists at each of the
centres, who are unaware of the patient recruitment for a clinical trial. In our study, the
pathological diagnosis is performed following the local workflow. All specimens are examined
by two pathologists.
4.8 Patient follow-up
Immediately after the operation, the patients are inquired about whether there are any
discomforts, and it is recorded if there are any. Chest X-ray or other imaging examinations are
performed within 24 hours to detect whether there is pneumothorax, pneumomediastinum, or
mediastinitis. Twenty-four hours after operation, a follow-up is conducted and the symptoms
are recorded, including fever, coughing, hemoptysis, chest pain, dyspnea, and so on.
Perioperative severe adverse events include moderate-to-severe bleeding (grade 3 or 4),
oversedation requiring ventilatory support or sedation reversal, pneumothorax with persistent
air leakage (> 5 days), unplanned hospital readmission, and death. ICU transfer within 48

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hours after the procedure is also considered a severe adverse event. One month later, a
second follow-up is conducted, and examinations such as X-ray or CT scan would be
arranged according to patient symptom. The complications relative to the operation are
recorded. Patients with an indefinite diagnosis (especially for benign disorders) are followed
up continuously or receive surgical procedures (such as mediastinoscopy or surgical lymph
node dissection), and the final diagnosis should be revised at any time if more reliable
evidence supports new diagnosis. Six months later, imaging reexaminations are required for
patients with an indefinite diagnosis (especially for benign disorders).
5. Study endpoints
The co-primary outcomes are the diagnostic yield and safety of the EBUS procedure. The
diagnostic yield of the EBUS procedure is defined as the percentage of patients for whom the
EBUS procedures provided a definite diagnosis. Suspicious findings from the biopsy
procedures are regarded as negative cases. Safety is evaluated with regard to the prevalence
of the procedure-related adverse events.
Secondary
end-points
include
the
diagnostic
yield
of
transbronchial
mediastinal
cryobiopsy, specimen adequacy and size, suitability of samples for molecular genetic assay,
and duration of the bronchoscopic procedure.
6. Data analysis
The full analysis set, including all the patients who do not violate the inclusion criteria and
have undergone the biopsy, is used for demographic summaries and safety analyses.
Inter-individual diagnostic analyses are performed based on the full analysis set, and the
intra-individual diagnostic analyses include subjects receiving both EBUS-TBNA and
mediastinal cryobiopsy, which are performed in concern of the potential intergroup
differences13. Subgroup analyses are performed on disease etiology. Analyses of the

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secondary outcomes are done in the combined biopsy population, except the analysis of
procedural duration that is based on the full analysis set.
Statistical analyses are performed using IBM SPSS 21.0 software (IBM Corp., Armonk,
NY, USA). Categorical variables are reported as counts and percentages, and continuous
variables as means and standard deviation. Pearson’s Chi-squared or Fisher’s exact test is
used
to
compare
proportions,
as
appropriate.
For
continuous
data,
between-group
comparisons are performed by Student´s t test or the Mann-Whitney U-test for parametric or
non-parametric data, respectively. A P<0.05 is considered to denote statistical significance.
Because of discussions within the research and statistical team about the need for more
details on how analyses are to be carried out, amendments to the data analysis section of the
protocol are made on Feb 8th 2022 before our analyses are started. The changes made are
that more specified information on the populations for the primary and subgroup analyses are
provided in this part.
7. Ethical considerations
The research group will ensure that this study is conducted in accordance with the
principles of the Declaration of Helsinki and Good Clinical Practice. All participants will be
asked to provide informed consent.
8. Risks and benefits
Similar to the application of standard needle aspiration biopsy, bleeding may occur during
the combined mediastinal sampling. In most situations, it requires no additional interventions.
Other adverse events include pneumothorax, infection, and hematoma or emphysema in
mediastinum; however, these occurrences are much less frequent7. The bronchoscopist will
try the best to operate carefully to avoid the damage to blood vessels and other important
tissues and organs. Patients will be reexamined by Chest X-ray or other imaging

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examinations within 24 hours to detect whether there is pneumothorax, or emphysema,
hematoma, or infection in mediastinum.
Compared with the routine application of EBUS-TBNA alone, the operative duration of
the combined sampling may be prolonged by approximately 5 minutes (the duration required
for cryobiopsy), which may increase the discomfort of the patients. During the procedure,
conscious sedation, local anesthesia, and continuous vital signs and oxygen saturation
monitoring will be used to minimize the discomfort and ensure the safety of the patients.
Compared with the traditional EBUS-TBNA, the operation scheme of the combined
approach helps to obtain more intact tissues and improve diagnostic utilities. This lessened
the need for more invasive examinations and surgical procedures.
9. Privacy and personal information protection
The research group hereby promises to protect the privacy and personal information of
the subjects that participate in the study. The security measures for protecting the privacy and
confidentiality of personal information include hiding information that can identify the subjects
during data reports, restricting access to such information, data anonymity, and so on.
Legally, there are exceptions for researchers to protect the privacy and personal
information of subjects when inspections are required by administrative authorities, or ethics
committee, etc.
10. Funding
The study is funded by National Natural Science Foundation of China.
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