A Prospective Multicenter Study of the TuFEst-LN Model for Axillary Lymph Node Status Assessment in Breast Cancer

Through the research of this project, we aim to prospectively validate the locked TuFEst-LN model, a cfDNA fragmentomics-based liquid biopsy model, for axillary lymph node status assessment in patients with breast cancer. This study will evaluate the ability of the TuFEst-LN model to identify pathologically node-negative patients among clinically and radiologically assessed cN0 patients with cT1-3 invasive breast cancer undergoing upfront surgery without neoadjuvant therapy. In addition, this study will explore the predictive value of the TuFEst-LN model combined with preoperative magnetic resonance imaging (MRI) for post-neoadjuvant pathological axillary lymph node status (ypN) in initially node-positive breast cancer patients. Peripheral blood samples and clinical data will be prospectively collected from multiple centers, and model predictions will be compared with final surgical pathology as the reference standard. This study aims to validate the clinical utility of cfDNA fragmentomics-based liquid biopsy for noninvasive axillary lymph node assessment and provide evidence for individualized axillary management in breast cancer.

Cohort 1-Specific Inclusion Criteria

Participants in Cohort 1 must meet all of the following criteria:

• Histologically confirmed invasive breast cancer.
• Clinical stage cT1-3, cN0, M0 disease at enrollment.
• No clinically palpable suspicious metastatic axillary lymph nodes identified by physical examination.
• No radiologically suspicious metastatic axillary lymph nodes identified by preoperative axillary imaging (at least ultrasound examination). Patients with suspicious lymph nodes must have negative cytological or histological findings confirmed by fine-needle aspiration or core needle biopsy.
• No prior neoadjuvant therapy, including chemotherapy, targeted therapy, immunotherapy, endocrine therapy, or breast/axillary radiotherapy before enrollment.
• Planned to undergo definitive breast surgery with sentinel lymph node biopsy (SLNB) and/or axillary lymph node dissection (ALND).
• Ability to provide qualified preoperative plasma samples for cfDNA analysis.

Cohort 2-Specific Inclusion Criteria

Participants in Cohort 2 must meet all of the following criteria:

• Histologically confirmed invasive breast cancer.
• Ipsilateral axillary lymph node metastasis confirmed by fine-needle aspiration or core needle biopsy at initial diagnosis.
• No evidence of distant metastasis (M0), and planned to receive standard neoadjuvant systemic therapy followed by definitive breast and axillary surgery.
• Completion of the planned neoadjuvant therapy, or premature discontinuation due to clinical reasons while remaining eligible for subsequent surgery.
• Ability to provide a baseline plasma sample (T0) within 24-72 hours before initiation of the first systemic treatment (including chemotherapy, immunotherapy, or targeted therapy).
• Availability of preoperative breast and axillary magnetic resonance imaging (MRI) after completion of neoadjuvant therapy. Patients unable to undergo MRI due to contraindications or other reasons may be included in the cfDNA-only analysis set but will not be included in the complete-case analysis of the combined cfDNA-MRI model.
• Ability to provide a second plasma sample (T1) within 24-72 hours before surgery after completion of neoadjuvant therapy. Patients without T0 samples may still be included in the exploratory analysis of T1 cfDNA combined with MRI for ypN prediction.
• Availability of complete postoperative breast and axillary pathological evaluation results.

Exclusion criteria

Participants meeting any of the following criteria will be excluded:

• Pregnancy or breastfeeding.
• Prior surgical removal of the primary breast lesion before enrollment, resulting in inability to obtain the required preoperative blood samples.
• Presence of confirmed distant metastasis.
• Presence of supraclavicular, internal mammary, or other lymph node lesions that cannot be adequately assessed by planned surgery and pathological evaluation.
• History of another active malignancy within the previous 5 years, except for cured non-melanoma skin cancer, cervical carcinoma in situ, or other malignancies considered by investigators unlikely to affect study outcomes.
• Receipt of whole blood, plasma, or other blood product transfusion within 30 days prior to enrollment.
• Insufficient plasma sample volume, severe hemolysis, or failure to meet cfDNA sequencing quality control requirements determined by the central laboratory.
• Absence of evaluable axillary surgical pathological results.
• Any other condition considered by the investigator to make the patient unsuitable for participation in this study.