Image-guided Ultrasound Robotic Intraoperative Evaluation of Lymph-nodes Status in Gynecological Malignancies
Phase: NA
Conditions: Lymph Node Metastasis, Gynecologic Cancer
Sponsor: Fondazione Policlinico Universitario Agostino Gemelli IRCCS

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
 
 
 
PROTOCOL TITLE 
 
Image-guided ultrasound robotic intraoperative evaluation of lymph-nodes 
status in gynecological malignancies  
 
(R-LYNUS prospective trial) 
 
 
 
 
 
 
 
 
Scientific Director 
Prof. Giovanni Scambia  
 
PI 
Prof.ssa Antonia Testa  
 
Sub -Investigator 
Prof. Denis Querleu  
 
 
PhD Candidate 
Dott. Matteo Pavone  
 
 
 
Collaborators  
Dott.ssa Elena Teodorico 
Dott. Nicolo’ Bizzarri  
Dott. Valerio Gallotta 
Dott.ssa Simona Fragomeni 
 
 
Statistician 
Prof.ssa Diana Giannarelli  
 
 
 
 
 
 
 
 
 
 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
Synopsis 
TITLE 
Image-guided ultrasound robotic intraoperative evaluation of lymph-nodes status in 
gynecological malignancies (R-LYNUS prospective trial) 
CENTERS 
Dipartimento per la salute della Donna e del Bambino e della Salute Pubblica, Fondazione 
Policlinico Universitario A. Gemelli, IRCCS, UOC Ginecologia Oncologica, Rome, Italy. 
 
BACKGROUND 
The assessment of lymph node status is of crucial importance in gynaecological malignancies. 
Indeed, the prognosis and adjuvant treatment regimens are strongly influenced by the presence 
of nodal involvement. Systematic extensive lymphadenectomies are often performed for 
staging, diagnosis of skip metastases and to define the radiation field when radiotherapy 
treatments are required. Nevertheless, these can lead to significant short-term and long-term 
lymphatic complications, which are difficult to justify if the lymph nodes are free from 
metastasis. To avoid unnecessary comprehensive procedures in early-stage cancers, evaluation 
of the sentinel lymph node has acquired a valuable role even if limitations are still present (rate 
of frozen section false negative, “empty packets” and mapping failure).  
The introduction of an intra-operative non-invasive imaging technique capable of describing 
the presence and characteristics of lymph nodes could help in (1) eliminating the risk of empty 
packet, (2) orienting the intraoperative decision while avoiding the drawbacks of frozen section 
(time and resources, partial destruction of the tissue material), (3) orientate the pathological 
section if frozen section is used. To date, technological advancements have paved the way for 
enhanced intra-operative assessment of cancerous organs and lesions. Over the past decade, the 
evolution of robotic surgery combined with advancements in image-guided surgery techniques 
has led to the introduction of ultrasound probes designed specifically for intraoperative 
ultrasound during robotic surgery (RIOUS). Apart from the conventional rigid laparoscopic 
probes, which can be inserted through an accessory trocar, there are robotic probes tailored to 
fit device arms, and drop-in flexible probes that are becoming increasingly relevant in the 
scientific panorama. Notably, the latter drop-in probes feature a rigid segment designed for 
compatibility with robotic graspers, leveraging the dexterity and rotational manoeuvrability 
inherent to robotic surgery. Such probes, already proven effective in evaluating liver and kidney 
lesions as well as tumour margins, hold promise for intraoperative lymph node assessments due 
to the possibility of reaching difficult anatomical spaces thanks to the robotic-assisted 
movements. 
 
PRIMARY 
ENDPOINT 
The aim of this prospective trial is to report the sensibility of RIUS in the metastasis detection 
(macro, micro and ITCs) from fresh, unstained in vivo lymph node samples.   
 
SECONDARY 
ENDPOINT 
The secondary endpoint is to assess the lowest size detectable with the adopted drop-in robotic 
ultrasound probe. 
TYPE OF STUDY 
Interventional, prospective monocentric clinical trial 
INCLUSION 
CRITERIA 
- 
Women undergoing robotic surgery for gynecological malignancies (ovarian, 
endometrial, cervical cencers) 
- 
Need for nodal excision (staging or cytoreductive reasons) 
- 
18-99 years old 
- 
Absence of contemporary lymphatic diseases 
- 
Absence of previous oncological disease in the last 5 years 
- 
Willingness to participate in the study and to provide informed consent 
EXCLUSION 
CRITERIA 
- 
Previous radiotherapy treatments in the area of analysed lymph nodes 
- 
Previous chemotherapy treatments 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
MATERIAL 
AND 
METHODS 
Women undergoing radical surgery for gynecological cancers who meet the inclusion criteria 
in absence of exclusion criteria will be considered candidate for the study. Intraoperative 
robotic ultrasound will be performed on in vivo lymph nodal samples. Lymph nodes 
morphology and imaging characteristics will be evaluated. The gold standard will be definitive 
pathology. 
SAMPLE SIZE 
The primary endpoint of this study is the sensitivity of RIOUS in detecting metastases. We will 
consider the number of nodes rather than the number of patients; the sample size is based on a 
10% risk of metastasis. A total of 351 nodes will ensure to estimate a sensitivity of about 90% 
with a 95% confidence interval semi-width of 10% and will allow to obtain a precision of 4% 
for the specificity.  Considering that an average of 2-3 nodes is sampled by patients, the number 
of patients can be estimated to 160. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
 
 
 
1. Background:  
 
The assessment of lymph node status is of crucial importance in gynaecological malignancies (ovarian, 
endometrial, cervical and vulvar cancers) (1,2). The rate of positive lymph nodes  in apparently  early 
stage cancers is far to be low (14.2% ovarian, 10% endometrial, cervical 15% and vulvar cancers 10%) 
(3–6). Indeed, the prognosis and adjuvant treatment regimens are strongly influenced by the presence of 
nodal involvement (7). Systematic extensive lymphadenectomies are often performed for staging, 
diagnosis of skip metastases and to define the radiation field when adjuvant radiotherapy treatments are 
required. Nevertheless, pelvic and/or para-aortic lymphadenectomy can lead to significant short-term 
and long-term complications. The main reported postoperative complications include lymphoceles in up 
to 38% of cases and the development of lymphedemas in the lower limbs, which greatly diminish the 
quality of life for patients (8–10). In vulvar cancer surgery, inguinofemoral lymphadenectomy carries a 
risk of wound infection or breakdown in 20% to 40% of cases and a risk of lymphedema in 13% to 48% 
of patients (11). Despite the high morbidity associated with these procedures, systematic 
lymphadenectomies have been performed for many years because accurately determining lymph node 
involvement is crucial for care assessing. In cases of endometrial cancer, the recurrence-free survival 
rate drops from 87% in patients without lymph node involvement to 71% and 36% in women with pelvic 
and aortic node involvement, respectively (12). Similarly, in cervical cancer, lymph node invasion has 
been recognized as a critical prognostic factor, as reflected in the latest FIGO classification (13). 
Lymphatic spread is a characteristic feature also of epithelial ovarian cancer (EOC) even at early stages 
(14).  Studies aiming to assess nodal involvement in all EOC stages, by performing systematic 
lymphadenectomy, have reported up to 55% rates of pelvic and para-aortic nodal metastases in patients 
with stage III and IV disease (15). 
However, what makes the burden of surgical morbidity even more difficult to bear is that in most cases, 
regardless of the type of pelvic malignancy, the lymph nodes are free from  metastasis (16,17). This 
means that the majority of patients undergo an unnecessary, risky, and burdensome procedure that has 
no proven impact on their survival. To solve this issue in early stages cancers, evaluation of the sentinel 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
lymph node (SLN) has acquired a valuable role allowing node-negative patients to be spared from the 
surgical comorbidities associated with total lymphadenectomy (18–21). Sentinel node frozen section 
analysis tough is not routinely performed also due to  its disadvantage in terms of time consumption and 
not accurate results for micro-metastasis detection (2,22).  Furthermore, in some case it  is reported the 
inadvertent failure to harvest nodal tissue (it can reach the 8% in obese patients with endometrial cancer)  
with "empty packets" that may lead to overtreatment for patient safety (23). Additionally, sometimes 
SLN mapping may fail, leading the surgeon to the need to extend the lymphadenectomy or making the 
SLN technique unuseful and the patient unstaged.  Even if efforts are focused on tailoring the lymph 
nodes removal to avoid the complications of extensive lymphadenectomies, the detection of pathological 
lymph nodes with micro or macro metastases is still an open challenge for pre-operative imaging 
techniques (24). While FDG PET/CT is the most accurate imaging examination for lymph node 
evaluation, it nevertheless results in false negative diagnoses (25,26). As an example, a study in 60 
patients with stage IB2 to IVA cervical cancer  found that 12 % of those with no finding of positive 
paraaortic nodes on PET/CT had pathologically positive paraaortic nodes (25). 
The introduction of an intra-operative imaging technique capable of describing the presence and 
characteristics of lymph nodes could help in the identification of the diseased ones tailoring surgical 
procedures and decreasing the risks associated with unuseful extensive lymphadenectomies (27).  
To date, advances in imaging guided surgery and artificial intelligence software are offering alternative 
solutions in the intraoperative diagnosis of nodal involvement (27). 
Among the novel intraoperative bedside imaging guided surgery techniques Full-Field Optical 
Coherence Tomography (FFOCT) (27),  High-Frequency Ultrasound (HFUS) (28,29) and Intraoperative 
Ultrasound (IOUS) (30) appear as the most promising in the scientific panorama.  The ultrasound lymph 
nodal morphology in gynecological malignancies has been extensively described. However, the intra-
operative assessment of lymph nodal status is far to be fully achieved.  
Intraoperative ultrasound (IOUS) is commonly utilized during open surgery with linear or finger probes, 
particularly in the hepato-biliary (HPB) and urological fields (31,32). Laparoscopically (LIOUS), 
ultrasound probes for guidance in MIS are more challenging to handle (33). To overcome this limitation, 
innovative approaches for robotic platforms integrate ultrasound imaging to facilitate its use in MIS 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
(34). Image-guided robotic approaches, that can integrate also  three-dimensional (3D) imaging, 
augmented reality (AR), and machine learning algorithms, offer advantages in the era of digital surgery 
(35). Real-time, non-invasive, cost-effective and dynamic intraoperative imaging of complex anatomy 
is the ultimate goal in computer-assisted surgery to attain unparalleled precision. In this landscape, IOUS 
has become the imaging modality of choice with the introduction of articulated robotic ultrasound probes 
(36). Image augmentation and fusion of imaging modalities is especially beneficial to delineate healthy 
and neoplastic tissue in oncological surgery (37). 
The navigation of ultrasound probes manoeuvred by articulated robotic graspers, can give access to 
anatomical spaces and angles that are inconvenient for rigid laparoscopic probes. First reports of 
applications of intraoperative ultrasound during robotic surgery (RIOUS) have been published in similar 
fields as open surgery with encouraging results (31,38).  
RIOUS has demonstrated superior performance compared to conventional LIOUS in liver surface 
exploration and tool manipulation. RIOUS’ success rate exceeded the one of LIOUS in liver surface 
exploration (85% vs. 73%, P = .030) and in tool manipulation (79% vs. 57%, P = .028)(33). Facilitating 
probe positioning in RIOUS results in enhanced precision while reducing the physical strain on surgeons 
during complex procedures (31) opportunity to identify otherwise undetected lesions, such as in 
pancreatic lesions (39). 
Similarly, rectal tumours were successfully detected using RIOUS, a finding that highlights its 
effectiveness in determining the optimal transection line for rectal surgeries, particularly in tumours too 
high for transanal palpation (40). A remarkable 100% success rate was demonstrated in identifying 
kidney lesions with RIOUS (41) to optimise tumour identification, thereby enhancing renal tissue 
preservation via partial nephrectomy, and without compromising oncological safety (34). In transoral 
robotic tongue base resection for obstructive sleep apnoea RIOUS has emerged as an invaluable tool. to 
locate the lingual artery and assess laryngeal tissues. The integration of RIOUS significantly enhanced 
efficiency by substantially reducing the risk of detrimental intraoperative bleeding complications (42). 
There are no studies in the literature reporting the use of RIOUS in lymph nodal assessment.  
The RIOUS image rendering, could be an useful tool  in the definition of in vivo lymph nodes from 
gynecological malignancies (43,44).  

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
 
2. Purposes and objective of the clinical trial 
The identification of an image-guided surgery technique capable of intraoperatively detecting the 
presence of lymph node metastases would overcome the disadvantages and complications of extensive 
staging lymphadenectomies. 
 
3. Experimental design  
 
Primary endpoint 
 
The aim of this prospective trial is to report the sensibility of RIOUS in the metastasis detection 
(macro, micro and ITCs) from fresh, unstained in vivo lymph node samples.   
Secondary endpoint 
 
The secondary endpoint is to assess the lowest size detectable with the adopted imaging technique.  
 
Setting 
 
Patients referred to the Gynecology Oncology Unit in Fondazione Policlinico A. Gemelli 
IRCCS, Italy, Rome with a diagnosis of gynecological malignancy requiring surgical lymph nodes 
harvest will be evaluated for the enrolment. 
Experimental plan 
 
Interventional, prospective monocentric clinical trial. 
 
4. Study enrolment 
 
Inclusion criteria 
 
- 
Women undergoing robotic surgery for gynecological malignancies (ovarian, endometrial, 
cervical cancer) 
- 
Need for nodal excision (staging or cytoreductive reasons) 
- 
18-99 years old 
- 
Absence of contemporary lymphatic diseases 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
- 
Absence of previous oncological disease in the last 5 years 
- 
Willingness to participate in the study and to provide informed consent 
 
Exclusion criteria 
 
- 
Previous radiotherapy treatments 
- 
Previous chemotherapy treatments 
- 
Recurrence of disease 
 
5. Treatments  
 
Robotic intraoperative ultrasound 
Women undergoing radical surgery for gynecological cancers who meet the inclusion criteria will be 
enrolled. RIOUS will be performed on in vivo nodal samples collected during surgeries at Department 
of Gynecology Oncology, Fondazione Policlinico A. Gemelli IRCCS. Lymph nodes morphology and 
imaging characteristics will be evaluated. Comparison with the definitive pathology will be made to 
accomplish the study endpoints. 
The robotic drop-in ARIETTA L51K probe (Hitachi, Tokio, Japan)  will be used to describe normal and 
pathological lymph node by a gynecologist expert in oncological ultrasound and surgery.  Lymph nodes 
images will be evaluated in order to investigate the presence or the absence of morphological parameters 
to be significant in the macroscopic disease detection according to the  VITA (Vulva International 
Tumor Analysis) group consensus (28,29): nodal shape; inhomogeneous echo structure; intranodal 
deposits (described as a hyperechoic or hypoechoic area detected within the node); hilum anomalies 
(absence, displacement or interface distortion); cortical thickening; and nodal grouping. Moreover, the 
following dimensional parameters will be recorded: long-axis (L) and short-axis (S); L/S ratio, 
considering as suspicious a value < 2; cortical (C) and medullar (M) thickness of the node; and C/M 
thickness ratio, considering as suspicious a value > 1. Three additional morphological parameters will 
be assessed, which comprised: presence of perinodal hyperechoic ring (as a sign of inflammatory 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
perinodal stroma); cortical interruption (as a sign of extracapsular tumor spread); and presence of rich 
vascularization. A microscopic evaluation will be then carried out. Observational parameters will be 
recorded and Doppler images of intra-nodal vascularization scored as 0-3. Detection of macro and micro 
metastases (< 2mm) or ITCs (< 0.2mm) will be recorded. After collecting these morphological and 
dimensional parameters, a morphometric ultrasound pattern (MUP) will be expressed. The classification 
system is based on the tool for breast imaging published by the American College of Radiology (ACR), 
the breast imaging-reporting and data system (BI-RADS®) provides the following categories for 
assessment: negative; benign; probably benign; suspicious; and highly suggestive of malignancy. Based 
on this model, we will classify the LN status into five groups, according to a subjective assessment: 
normal (U1); reactive-but-negative (U2); minimally suspicious/probably groups U3, U4 and U5 as 
positive. Finally, specimens will be given to the pathologist for the histological evaluation. 
 
6. Sample size  
 
The primary endpoint of this study is the sensitivity of RIOUS in detecting metastases. We will consider 
the number of nodes rather than the number of patients; the sample size is based on a 10% risk of 
metastasis. A total of 351 nodes will ensure to estimate a sensitivity of about 90% with a 95% confidence 
interval semi-width of 10% and will allow to obtain a precision of 4% for the specificity.  Considering 
that an average of 2-3 lymph nodes is sampled by patients, the number of patients can be estimated to 
160. 
 
7. Statistical analysis 
 
In general, data will be summarized using appropriate summary statistics: Categorical data will be 
described using absolute counts and percentages. Continuous variables will be evaluated using the 
following standard descriptive summary statistics: number of observations, arithmetic mean and 
standard deviation, median and interquartile range. Results will be reported at lymph nodes and patients 
level. Punctual estimates regarding primary and secondary endpoints will be presented with their 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
confidence intervals. Analysis and reporting will follow the Standard for Reporting Diagnostic Accuracy 
Studies (STARD) guidelines. 
The secondary endpoint will be evaluated with paired test either parametric or not according to the 
deviation form normality assumptions evaluated with the Kolmogorov-Smirnov test. 
 
9. Administrative and ethical considerations 
 
Ethics considerations 
 
a) Risk-benefit considerations  
The RIOUS evaluation will be made during the scheduled surgeries. The patients will not have any 
additional discomfort due to the imaging exam. The result of the following study will in no way 
influence the patients' subsequent therapies or the usual standard of care. In this study we will not 
propose any different treatment compared to the standard of care for patients with gynecological 
malignancies. 
 
b) Data protection and privacy 
 
The data collection will be performed pseudonymously, and the patient’s name will not appear. All 
collected data will be kept confidential. This study is performed in accordance with the revision of the 
Declaration of Helsinki. 
 
c) Institutional review board / ethics committee 
 
Study protocol, patient information and informed consent will be submitted to the ethics committee of 
the FPG-IRCCS and collaborators centres for approval. The study will start after being approved by the 
ethics committee. The principal investigator will inform the ethics committee about any changes in the 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
study protocol which could interfere with patient’s safety. Furthermore, the committee will be informed 
of the planned or premature end of the study. The investigators are obliged to consult their responsible 
ethics committee and to wait for approval before including patients into the study, as well as to inform 
their ethics committee of changes in the protocol and end of study. 
 
d) Informed consent 
 
Before enrolment in the study, the treating investigator informed the patient about the nature of the 
procedure, its aims, expected advantages as well as possible risks. Each patient must consent in writing 
to participate in the study. The patient must be given enough time and opportunity to decide on 
participation and to clarify questions before inclusion in the trial. The informed consent will be signed 
by both patient and treating investigator. The original document is kept by the investigator. 
 
Publications 
 
The study’s results will be published irrespective of the nature of the results. The coordinating principal 
investigator, the other investigators, the statistician, the study coordinators and other authors will be 
included depending on recruitment numbers and absolute number of authors allowed for the respective 
journal. 
 
Adherence to protocol and protocol amendments 
 
The study protocol must be thoroughly adhered, and any deviation must be documented and justified by 
the investigator. Changes or supplements to the study protocol can only be decided on and authorised 
by the coordinating principal investigator (amendment), the study coordinators and the biometrician. 
 
8. Quality assurance/monitoring 
 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
According to the guidelines of Good Clinical Practice the study monitoring will be done by personal 
which is assigned by the coordinating principal investigator. The applicable directives for data protection 
law will be kept. It is the responsibility of the Clinical or Field Monitor to follow the study via telephone 
contact, written correspondence, and regular visits to the Investigator and study sites to review records. 
The Clinical or Field Monitor will maintain current, personal knowledge of the study through 
observation, review of the records, comparison with source documents, and discussion of the conduct 
of the study with the investigators. Within this trial a 100% SDV (Source Data Verification) will be 
performed for the inclusion and exclusion criteria, verification of treatment compliance by surgery 
reports, histopathological results, primary and secondary endpoints and complications. Other trial data 
will undergo 100% SDV for 20% of patients. The participation of the patient in the clinical trial has to 
be documented in the patient record. 
 
9. Data management 
 
Data will be prospectively collected by principal investigator or co-investigator, or by trainees under 
direct supervision.  Data from registration will be entered to an eCRF (electronic Case Report Form) for 
every patient. Study data will be managed using REDCap electronic data capture tools hosted at 
Fondazione Policlinico Universitario A. Gemellli, IRCCS (https://redcap-irccs.policlinicogemelli.it/). 
REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support 
data capture for research studies, providing 1) an intuitive interface for validated data entry; 2) audit 
trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless 
data downloads to common statistical packages; and 4) procedures for importing data from external 
sources (A metadata-driven methodology and workflow process for providing translational research 
informatics support . Research electronic data capture (REDCap). Only people officially registered as 
study investigators or data managers will receive a user login to access the REDCap web platform and 
enter/manage data. 
 
Storage of study documents 

  
 
Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
 
Originals of all central study documents will be kept in the study office for a period of 10 years after 
preparation of the final report. The investigator keeps accrued administrative documents 
(correspondence with ethics committee, surveillance authority, study coordinators, central study office), 
the patient identification list, signed informed consent, and general study documents (protocol, 
amendments) for the above-mentioned period. Original data of study patients (medical records) must be 
stored according to the archiving period valid at the respective study site, but not less than 10 years.  
 
 
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Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
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Protocol_Study_R-LYNUS Versione 1.0 del 07/11/2023                                                                                            
 
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