Targeted Axillary Dissection Using Carbon Marking for Patients With Node-positive Breast Cancer Following Neoadjuvant Therapy (TADCOM): a Prospective, Multicenter, Randomized Controlled Trial
Phase: NA
Conditions: Breast Cancer, Surgery, Lymph Node Metastases
Sponsor: Second Affiliated Hospital, School of Medicine, Zhejiang University

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Targeted axillary dissection using carbon marking for 
patients with node-positive breast cancer following 
neoadjuvant therapy (TADCOM): study protocol for a 
prospective, multicenter, randomized controlled trial 
 
Version number 
1.2 
Date 
September 17, 2024 
Principal Investigator 
Jian Huang and Wuzhen Chen 
Study institution 
The Second Affiliated Hospital, Zhejiang University 
School of Medicine 
 
 
 
 

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I. Background 
Breast cancer is one of the most common cancers among women worldwide. 
Neoadjuvant chemotherapy (NAC) plays a pivotal role in the management of locally 
advanced cases, offering a pathway to pathological complete response (pCR) and 
reducing reliance on axillary lymph node dissection (ALND), which is often associated 
with complications like lymphedema and chronic pain. Approximately 21.1% of 
patients achieve pCR with NAC, with axillary pCR rates ranging from 23% to 74%. 
Such outcomes may allow some patients to avoid ALND, underscoring the clinical 
value of preserving axillary integrity. 
For patients with confirmed axillary metastasis before NAC, the use of tissue 
markers enables precise localization and removal of these nodes during sentinel lymph 
node biopsy (SLNB), known as targeted axillary dissection (TAD). This technique, 
which has been validated in studies like ACOSOG Z1071 and SenTa, shows a false-
negative rate (FNR) of 2% to 7%, reducing postoperative complications. 
Despite its benefits, TAD using traditional tissue markers faces challenges like 
high costs and potential marker loss. Recent advancements include the use of carbon 
nanoparticle suspension injection (CNSI) for lymph node mapping, known as carbon 
marking. CNSI's unique properties allow for enhanced visibility and prolonged stability 
within lymph nodes, making it a promising alternative to traditional methods.  
This research aims to compare various TAD techniques based on carbon marking 
against conventional methods, focusing on clinical feasibility, diagnostic accuracy, and 
the reduction of common issues such as marker loss. Additionally, the study explores 
the potential for CNSI in improving TAD outcomes in patients with pre-NAC cN2+ 
staging, seeking to develop a more effective, cost-efficient surgical approach. 
 
II. Objectives of the study 
The TADCOM trial is designed as a prospective, multicenter, randomized 
controlled trial (RCT) utilizing a non-inferiority framework. This study aims to 

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compare the feasibility and efficacy of CNSI for TAD against conventional tissue 
marker clip-based methods in patients with breast cancer following NAC. 
 
III. Study endpoint 
Primary Endpoints 
1. Marked lymph node retrieval rate: The proportion of successfully retrieved 
marked lymph nodes will be calculated and compared among the study groups to 
evaluate the effectiveness of each marking technique.  
2. Number of sentinel and marked lymph nodes: The mean, median, and range of 
the number of sentinel and marked lymph nodes harvested during surgery will be 
recorded and compared to assess the efficacy of the marking techniques in identifying 
lymph nodes of interest. 
3. Concordance between marked and sentinel lymph nodes: The consistency 
between marked lymph nodes and intraoperatively identified sentinel lymph nodes 
will be evaluated by calculating the percentage of marked nodes that are also sentinel 
nodes and vice versa to determine the accuracy of the marking techniques in 
identifying true sentinel lymph nodes. 
4. Complication rate: All surgery-related complications, including but not limited to 
hemorrhage, lymphedema, infection, pain, tissue damage, clip displacement, clip loss, 
absence of CNSI staining, and excessive CNSI staining, will be recorded and 
analyzed. The overall complication rate and rates for specific types of complications 
will be reported. The severity of complications will be assessed using the Clavien-
Dindo classification to provide a standardized evaluation of complication severity. 
 
Secondary Endpoints 
1. Axillary and distant recurrence rates: During the 2-year and 5-year follow-up 
periods, the axillary and distant recurrence rates will be monitored and reported for 

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each study group to assess the long-term oncological outcomes of the different 
marking techniques. 
2. Overall survival (OS), and disease-free survival (DFS): OS and DFS will be 
monitored and reported for each study group at 2 years, 5 years, and other relevant 
time points. Kaplan-Meier analysis and Cox proportional hazards models will be used 
to estimate these endpoints and compare outcomes among the study groups. 
3. Surgical duration and learning curve: The total time from the start of the surgery 
to the removal of the last lymph node will be calculated to analyze the efficiency of 
different techniques in terms of surgical time. The learning curve associated with each 
marking technique will also be assessed to evaluate the impact on surgical duration 
and other outcomes over time. 
4. Postoperative complications: Complications such as lymphedema, infection, and 
pain will be assessed at specific time points (e.g., 1 month, 6 months, and 1 year post-
surgery) using validated tools or scales (e.g., Common Terminology Criteria for 
Adverse Events, Brief Pain Inventory) to determine the safety profile of each marking 
technique and its impact on patient morbidity. 
5. Quality of life: The European Organisation for Research and Treatment of Cancer 
Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) and the breast cancer-
specific module (EORTC QLQ-BR23) will be used to evaluate patients' quality of life 
before treatment, at 6 months, 1 year, and 2 years after treatment to assess the impact 
of the different marking techniques on patient-reported outcomes. 
6. Positive tumor margin rate and re-excision surgery: For patients undergoing 
breast-conserving surgery, the proportion of positive tumor margins, defined as tumor 
cells at the inked margin or within 1 mm from the margin, will be evaluated 
postoperatively. The rate of re-excision surgery due to positive margins will also be 
assessed to evaluate the impact of the marking techniques on the accuracy of surgical 
resection and the need for additional interventions. 
 
IV. Study subjects (national multi-center study in China) 

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4.1 Study population 
Patients with cT1-4N1-2aM0 breast cancer undergoing neoadjuvant chemotherapy and 
surgery (N = 126) 
4.2 Criteria for inclusion and exclusion 
Inclusion criteria: 
Participants in the study must meet the following criteria: 
1. Female patients aged 18 to 75 years are eligible. 
2. Participants must have a histologically confirmed diagnosis of breast cancer, 
classified as clinical stage T1-4N1-2aM0 according to the TNM classification system. 
3. Eastern Cooperative Oncology Group (ECOG) performance status must be 0 or 1. 
4. Clinical re-staging must indicate an axillary node status of ycN0 following NAC. 
5. Participants must provide written informed consent to partake in the trial, 
acknowledging understanding and agreement to the procedures and risks involved. 
Exclusion Criteria: 
Patients who met any of the following exclusion criteria were excluded from the study: 
1. Patients with metastatic breast cancer (Stage IV). 
2. Diagnosed with inflammatory breast cancer or bilateral breast cancer. 
3. History of axillary surgical procedures. 
4. Any medical, psychological, or social conditions that would prevent adherence to 
the study protocol or completion of the treatment or follow-up. 
5. Known allergy to carbon nanoparticles or presence of severe comorbid conditions 
or other serious underlying medical issues. 
6. Current or prior participation in another clinical trial that could interfere with the 
outcome of this study or affect the safety and well-being of the participants. 
4.3 Criteria for Termination 
1. If a participant exhibits local tumor progression (including the ipsilateral breast, 
chest wall, axillary, and supraclavicular or infraclavicular lymph nodes) or develop 

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distant metastases during NAC, the NAC will be discontinued, resulting in the 
termination of the participant’s involvement. 
2. Participants may choose to withdraw from the study voluntarily at any point. 
Such withdrawal will have no adverse effects on the participant’s future medical care 
or access to alternative treatment options. 
3. If continued participation is deemed to compromise the safety of the participant, 
as determined by the investigating team, the participant’s involvement will be 
terminated. 
4. Any serious breach of the study protocol by a participant, as identified by the 
investigators, will result in immediate termination of their participation. 
5. If a participant loses contact during the study and is unable to continue 
participating, they will be considered lost to follow-up and terminated from the study. 
 
4.4 Criteria for ultrasound diagnosis of suspicious lymph nodes 
Ultrasound Scoring Criteria for Suspicious Axillary Lymph Nodes 
Ultrasound 
Characteristics 
Scoring Criteria 
Points 
Shape 
Fusiform or elongated (ratio of long to 
short axis ≥2) 
0 
Round or oval (ratio of long to short 
axis <2) 
1 
Border 
Smooth, regular 
0 
Irregular, lobulated 
1 
Hilum 
Intact, central 
0 
Absent or eccentric 
2 
Cortical thickness 
Uniform, ≤3mm 
0 
Focal thickening (>3mm) or diffuse 
thickening 
2 

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Calcification 
No calcification 
0 
Presence of microcalcifications 
1 
Necrosis 
No necrotic area 
0 
Presence of necrotic area 
1 
Vascular pattern 
Predominantly 
central 
hilar 
vascularity, 
no 
significant 
peripheral 
vascularity 
0 
Predominantly 
chaotic, 
irregular 
peripheral vascularity 
1 
*A total score of ≥3 points indicates a suspicious lymph node, and a fine-needle 
aspiration (FNA) or core-needle biopsy (CNB) is recommended to obtain a pathological 
diagnosis 
 
V. Study Methodology 
5.1 Subjects included 
A prospective, multicenter, randomized controlled trial (RCT) utilizing a non-
inferiority framework was conducted in 126 breast cancer patients who met the criteria 
of inclusion and exclusion. The relevant data were recorded according to the CRF form 
issued by the applicants. 
 
5.2 Interventions 
Group 1: Clip-Guided Targeted Axillary Dissection (CG-TAD) 
Participants will undergo an ultrasound-guided placement of tissue marker clips 
into clinically suspicious axillary lymph nodes before NAC. Following the completion 
of NAC, methylene blue dye will be used to facilitate the mapping of sentinel lymph 
nodes. Subsequently, TAD will be performed to excise both the sentinel and previously 
clipped lymph nodes. This group serves as the control arm, employing standard 

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methodologies consistent with those reported in existing TAD literature (25). 
Group 2: CNSI Lymph Node Marking (CN-LNM) 
participants will receive injections of carbon nanoparticle suspension injection 
(CNSI) under ultrasound guidance to mark clinically suspicious axillary lymph nodes 
before NAC. Post-NAC, methylene blue dye will also be used to map the sentinel 
lymph nodes. The TAD procedure will then be conducted to remove both the sentinel 
lymph nodes and the lymph nodes marked with CNSI. 
Group 3: Peritumoral CNSI Mapping (PCN-MAP) 
Participants will be administered CNSI injections around the primary breast tumor 
under ultrasound guidance before NAC, allowing for natural lymphatic drainage to 
transport the carbon nanoparticles to both sentinel and potentially involved axillary 
lymph nodes. If a lymph node is confirmed as metastatic through pathology, an 
additional tissue marker clip will be placed under ultrasound guidance before NAC. 
After NAC, the sentinel lymph nodes will be mapped using methylene blue, followed 
by TAD to excise the sentinel lymph nodes, CNSI-marked nodes, and any additional 
clipped nodes. 
 
5.3 Neoadjuvant chemotherapy 
NAC regimens for breast cancer will be customized based on the patient’s ECOG 
performance status, molecular subtype, and relevant biomarkers such as HER2 
overexpression. Treatment durations range from 3 to 6 months, following the latest 
evidence-based guidelines. Anti-HER2 therapies are added for cases with HER2 
overexpression to enhance efficacy. Throughout NAC, patient response is closely 
monitored via ultrasound, mammography, and MRI before and after chemotherapy 
cycles, adjusting treatments based on individual tolerance and effectiveness. This 
personalized approach ensures optimal safety and outcomes, incorporating patient and 
clinical input in decision-making. 
 

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5.4 Procedures for labeling axillary lymph nodes 
For CNSI: 
Using sterile technique, prepare the CNSI for injection. 
Under continuous ultrasound guidance, inject a small volume of CNSI (specific volume 
as per protocol, e.g., 0.1 mL) directly into the identified suspicious lymph nodes. 
Confirm the dispersion of CNSI within the lymph node visually on ultrasound. 
For Tissue Clips: 
Prepare the clip applier loaded with a sterile tissue marker clip. 
Under ultrasound guidance, deploy the clip directly into or adjacent to the lymph node, 
ensuring the clip is securely placed within the node without displacement. 
5.5 Post-Labeling Monitoring 
1) Perform an immediate post-procedure ultrasound to ensure the integrity of the 
lymph node labeling. 
2) Document any immediate adverse reactions or complications. 
3) Schedule a follow-up ultrasound within a week to check the stability of the markers 
and the condition of the lymph nodes. 
5.6 Pathological evaluation of lymph nodes 
Following TAD, lymph nodes will undergo histological examination to determine 
the presence of disease. If the histological findings are positive, an extensive dissection 
of levels I-II axillary lymph nodes will be required. For enhanced accuracy in detecting 
axillary lymph node metastases, all specimens are processed using hematoxylin and 
eosin (H&E) staining, complemented by immunohistochemical (IHC) staining 
techniques. Following NAC, the detection of isolated tumor cells (ITCs) should be 
categorized as ypN0(i+), indicating non-invasive residual disease, while the 
identification of micro-metastases should be recorded as ypN1mi, denoting minimal 
residual disease. Considering the risk of residual disease, it is recommended that all 

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patients receive postoperative axillary regional nodal irradiation (RNI) to minimize the 
likelihood of local recurrence and enhance long-term disease management. 
 
VI. Record of patient information 
6.1 Basic patient information 
Demographic and Physical Data: 
• 
Age: Documented in years. 
• 
Weight: Measured in kilograms. 
• 
Body Mass Index (BMI): Calculated using the formula (weight in kg)/(height 
in m²). 
Clinical Assessment: 
• 
Clinical Staging: Includes documentation of tumor size, site, and lymph node 
status according to the TNM classification. 
• 
Neoadjuvant Therapy: Details of any chemotherapy, hormone therapy, or 
radiation therapy administered prior to surgery. 
Surgical Intervention: 
• 
Operation Mode: Specified as either breast-conserving surgery or radical 
mastectomy, based on the clinical judgment and patient consent. 
Postoperative Care: 
• 
Endocrine Therapy and Radiotherapy: Specifics of any hormonal treatments 
or radiation therapy administered post-surgery. 
Histopathological Evaluation: 
• 
Histopathology: Classification of the tumor as invasive carcinoma or 
carcinoma in situ. 
• 
Histologic Grading: Grading of the tumor based on cellular differentiation. 
• 
Hormone Receptor Status: Results for estrogen and progesterone receptors. 
• 
Ki-67: Proliferation index measured as a percentage. 
• 
HER-2 Status: Determined via immunohistochemistry or fluorescence in situ 

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hybridization (FISH). 
• 
Lymph Node Status: Documented findings from the pathological examination 
of excised lymph nodes. 
Additional Clinical Data: 
• 
Menopausal State: Classified as premenopausal, perimenopausal, or 
postmenopausal. 
• 
Family History: Detailed record of any familial incidence of breast cancer or 
related cancers. 
 
6.2 Placement information 
Brand and Model: Each CNSI clip used in the study is meticulously documented, 
noting the specific brand and model to ensure consistency and traceability in the 
research findings. 
Placement Position: The exact anatomical location where each clip is placed 
within the axillary region is recorded. This includes specifying the depth and proximity 
to critical structures to facilitate accurate replication and assessment of the clip’s 
effectiveness in marking the lymph nodes. 
Puncture Details: The condition of the puncture site (e.g., any immediate 
complications such as bleeding or infection) and the precise time of puncture are 
recorded to provide a comprehensive timeline and context for subsequent analyses. 
Operator Information: The identity of the operator performing the clip placement 
is logged to assess the impact of operator skill and experience on the success of the 
procedure. 
Follow-Up: Follow-up data on each clip are collected, focusing on whether any 
displacement or loss of the clip occurred post-placement. This is essential for evaluating 
the reliability and stability of the CNSI clips used in marking axillary lymph nodes. 
 
6.3 Surgical pathology information 

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SLNB/TAD region 
Marked 
X
X 
Positive 
X 
pCR 
X 
Unmarked 
X
X 
Positive 
X 
 
 
ALND does not contain the SLNB/TAD region 
Marked 
X
X 
Positive 
X 
pCR 
X 
Unmarked 
X
X 
Positive 
X 
 
 
* reporting concerns about post-treatment responses 
 
6.4 Long-term Follow-up Information 
Survival/Death: The survival status of each participant will be tracked, recording 
the time from study entry until death or the last follow-up, whichever comes first. 
Recurrence: The occurrence and location of any cancer recurrence will be 
documented, along with the timing of such events relative to the initial treatment. 
Follow-up Treatment: Any subsequent treatments administered post-recurrence or 
as part of ongoing care will be systematically recorded, noting the type and timing of 
treatment. 
 
VII. Data Management 
1. Patient Identification: 
• 
Upon enrollment, each participant will be assigned a unique identification 
number (ID). This ID will be used to maintain participant confidentiality and ensure 
that all collected data can be accurately linked to the corresponding participant without 
revealing personal information. 

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2. Data Updating Protocol: 
• 
All study data will be updated on a quarterly basis to ensure that information is 
current and reflects any new developments or changes in participant status. This regular 
updating is crucial for maintaining the integrity of the data set and ensuring that 
analyses are based on the most recent information. 
3. Annual Data Analysis and Reporting: 
• 
Comprehensive data analysis will be conducted annually. The findings from 
these analyses will be compiled into progress reports that will be disseminated to all 
participating units. These reports will provide insights into the study’s progress, 
preliminary outcomes, and any significant findings or deviations from expected trends. 
 
VIII. Quality Control of clinical trials 
1. Ethics and Protocol Approval: 
• 
Prior to implementation, the pilot program of each multicenter clinical trial must 
be reviewed and approved by the Ethics Committee responsible for the respective unit. 
This ensures that all ethical guidelines and regulatory requirements are met. 
2. Participant and Researcher Consistency: 
• 
To maintain consistency in participant management across all centers, clinical 
trial participants should be relatively fixed. Key researchers must conduct technical 
training sessions before the trial commences. These sessions will focus on a thorough 
review of the clinical trial protocols and technical procedures to ensure uniformity in 
technology application, equipment use, data judgment standards, and documentation 
methods. 
3. Data Handling and CRF Compliance: 
• 
Researchers are required to fill out the Case Report Form (CRF) accurately, 
promptly, and in detail according to the instructions provided. Data should be uploaded 
in real time to maintain data integrity and facilitate timely analysis. 
4. Verification of Observations and Findings: 
• 
All observations and findings from the clinical trials must be rigorously verified. 

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This verification process ensures the reliability of the data and confirms that the trial 
conclusions are directly derived from the original data collected. 
5. Mid-Term Reviews and Data Verification: 
• 
Each responsible unit must organize regular mid-term seminars to discuss the 
ongoing progress of the trial, address any issues encountered, and verify the data 
collected from each center. These discussions are critical for assessing interim 
outcomes and making necessary adjustments to the trial protocol. 
6. Participant Retention Strategies: 
• 
Researchers must implement proactive strategies, such as regular notifications 
and follow-up appointments, to maintain a dropout rate below 30%. These measures 
are essential for minimizing bias and ensuring the statistical power of the trial results. 
7. Quality Control Systems: 
• 
Each test center must establish a stringent quality control system to oversee and 
ensure the quality of the trial execution. This system should include regular audits and 
reviews to monitor compliance with the trial protocols and maintain high standards of 
research integrity.