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

A Prospective Multi-center Cohort Study Based on Deep Learning-based cfDNA Fragment Omics to Verify the TuFEst Model for Axillary Lymph Node Status Assessment in Breast Cancer Patients Undergoing Primary Surgery or Neoadjuvant Therapy

Through the research of this project, we expect to validate the clinical utility of the TuFEst-LN model in assessing axillary lymph node status in breast cancer patients. Specifically, we aim to prospectively validate its ability to identify pathologically node-negative patients among clinically and radiologically assessed cN0 patients undergoing upfront surgery ，and explore the predictive value of the TuFEst-LN model combined with preoperative MRI for ypN status assessment in initially node-positive patients following neoadjuvant therapy.

Study Overview

• Status: Recruiting
• Conditions: Breast Cancer; Axillary Lymph Node Metastasis
• Intervention / Treatment: Other: No Intervention: Observational Cohort

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 400

Contacts and Locations

• Study Contact: Name: Chao Ni; Phone Number: +8613989463951; Email: drnichao@zju.edu.cn

Study Locations

• China
  • Zhejiang
    • Hangzhou, Zhejiang, China
      • Recruiting
      • Second affiliated hospital of Medical school, Zhejiang university
      • Contact: Yao Dr; Phone Number: +8613989463951; Email: drnichao@zju.edu.cn

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Breast cancer patients receiving care across the six participating centers who fulfill the eligibility criteria for either the cN0 cohort undergoing upfront surgery or the initially cN+ cohort after neoadjuvant systemic therapy.

Description

Cohort 1-Specific Inclusion Criteria

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

1. Histologically confirmed invasive breast cancer.
2. Clinical stage cT1-3, cN0, M0 disease at enrollment.
3. No clinically palpable suspicious metastatic axillary lymph nodes identified by physical examination.
4. 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.
5. No prior neoadjuvant therapy, including chemotherapy, targeted therapy, immunotherapy, endocrine therapy, or breast/axillary radiotherapy before enrollment.
6. Planned to undergo definitive breast surgery with sentinel lymph node biopsy (SLNB) and/or axillary lymph node dissection (ALND).
7. 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:

1. Histologically confirmed invasive breast cancer.
2. Ipsilateral axillary lymph node metastasis confirmed by fine-needle aspiration or core needle biopsy at initial diagnosis.
3. No evidence of distant metastasis (M0), and planned to receive standard neoadjuvant systemic therapy followed by definitive breast and axillary surgery.
4. Completion of the planned neoadjuvant therapy, or premature discontinuation due to clinical reasons while remaining eligible for subsequent surgery.
5. 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).
6. 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.
7. 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.
8. Availability of complete postoperative breast and axillary pathological evaluation results.

Exclusion Criteria

Participants meeting any of the following criteria will be excluded:

1. Pregnancy or breastfeeding.
2. Prior surgical removal of the primary breast lesion before enrollment, resulting in inability to obtain the required preoperative blood samples.
3. Presence of confirmed distant metastasis.
4. Presence of supraclavicular, internal mammary, or other lymph node lesions that cannot be adequately assessed by planned surgery and pathological evaluation.
5. 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.
6. Receipt of whole blood, plasma, or other blood product transfusion within 30 days prior to enrollment.
7. Insufficient plasma sample volume, severe hemolysis, or failure to meet cfDNA sequencing quality control requirements determined by the central laboratory.
8. Absence of evaluable axillary surgical pathological results.
9. Any other condition considered by the investigator to make the patient unsuitable for participation in this study.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
1; Invasive breast cancer patients with cT1-3N0M0 disease who have received no prior neoadjuvant therapy or radiotherapy and undergo SLNB and/or ALND for axillary evaluation. A total of 300 participants are planned to be enrolled in Cohort 1	Other: No Intervention: Observational Cohort; No Intervention: Observational Cohort
2; Patients with newly diagnosed invasive breast cancer and biopsy-confirmed axillary lymph node metastasis without distant metastasis, who are scheduled to receive standard neoadjuvant systemic therapy followed by breast and axillary surgery . Patients who complete or prematurely discontinue neoadjuvant therapy for clinical reasons but proceed to surgery are eligible.At least 100 participants are planned to be enrolled in Cohort 2.	Other: No Intervention: Observational Cohort; No Intervention: Observational Cohort

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Pathological axillary lymph node positivity rate among TuFEst-LN-negative patients	Defined as the proportion of patients with pathological axillary lymph node positivity (pN1mi or higher) among patients predicted as negative by the locked TuFEst-LN model in Cohort 1. FOR = FN/(TN+FN) = 1-NPV.	up to 2 weeks

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Negative Predictive Value (NPV) of the TuFEst-LN Model	The proportion of patients with negative pathological axillary lymph node status (pN0 or pN0(i+)) among patients classified as negative by the locked TuFEst-LN model.	2 weeks
Sensitivity and False Negative Rate (FNR) for Detecting Pathological Axillary Lymph Node Positivity	The ability of the locked TuFEst-LN model to identify patients with pathological axillary lymph node metastasis (pN1mi or higher). FNR is defined as 1-sensitivity.	2 weeks
Specificity of the TuFEst-LN Model	The proportion of patients with true pathological node-negative status (pN0 or pN0(i+)) who are correctly classified as negative by the locked TuFEst-LN model.	2 weeks
Positive Predictive Value (PPV), Overall Accuracy, and Discrimination Performance	Evaluation of the predictive performance of the locked TuFEst-LN model, including PPV, overall accuracy, area under the receiver operating characteristic curve (ROC-AUC), area under the precision-recall curve (PR-AUC), and likelihood ratios.	2 weeks
Proportion of Patients Classified as Negative by the TuFEst-LN Model	The proportion of evaluable patients in Cohort 1 who are classified as negative by the locked TuFEst-LN model, used to estimate the potential rate of sentinel lymph node biopsy (SLNB) omission.	2 weeks
Calibration and Net Clinical Benefit of the TuFEst-LN Model	Assessment of model calibration and clinical utility using Brier score, calibration intercept, calibration slope, calibration curve, and decision curve analysis.	2 weeks
Pathological Burden of Missed Positive Cases	Evaluation of pathological characteristics among false-negative patients, including the number of micrometastatic and macrometastatic lymph node cases, extranodal extension, and other adverse pathological features.	2 weeks

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Predictive Performance of the TuFEst-LN Model for ypN Status	Using final surgical pathology as the reference standard, the predictive performance of the TuFEst-LN model for post-neoadjuvant pathological axillary lymph node status (ypN) will be evaluated, including area under the receiver operating characteristic curve (ROC-AUC), sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and calibration performance.	2 weeks
Predictive Performance of Preoperative MRI for ypN Status	Evaluation of the ability of preoperative magnetic resonance imaging (MRI) clinical and quantitative imaging features after neoadjuvant therapy to predict residual pathological axillary lymph node status (ypN0 versus ypN-positive).	2 weeks
Performance of Combined cfDNA Fragmentomics and MRI Model	Evaluation of the incremental value of integrating TuFEst-LN cfDNA fragmentomic features with MRI-based prediction models by comparing changes in ROC-AUC, Brier score, calibration performance, and decision curve analysis.	2 weeks
Changes in cfDNA Fragmentomic Features From T0 to T1	Exploration of the association between longitudinal changes in cfDNA fragmentomic scores or features before and after neoadjuvant therapy and pathological outcomes, including ypN status, breast pathological complete response (pCR), residual cancer burden (RCB), and residual tumor burden.	2 weeks
Exploratory Subgroup Analysis	Exploration of the predictive performance of the TuFEst-LN model across clinically relevant subgroups, including molecular subtype, treatment regimen, and baseline lymph node burden.	2 weeks

Collaborators and Investigators

• Sponsor: Second Affiliated Hospital, Zhejiang University, School of Medicine
• Collaborators: First People's Hospital of Hangzhou; Ningbo Medical Center Lihuili Hospital; Shandong Cancer Hospital and Institute; Taizhou Hospital of Zhejiang Province affiliated to Wenzhou Medical University; Suzhou Municipal Hospital

Publications and helpful links

General Publications

• Gentilini OD, Botteri E, Sangalli C, Galimberti V, Porpiglia M, Agresti R, Luini A, Viale G, Cassano E, Peradze N, Toesca A, Massari G, Sacchini V, Munzone E, Leonardi MC, Cattadori F, Di Micco R, Esposito E, Sgarella A, Cattaneo S, Busani M, Dessena M, Bianchi A, Cretella E, Ripoll Orts F, Mueller M, Tinterri C, Chahuan Manzur BJ, Benedetto C, Veronesi P; SOUND Trial Group. Sentinel Lymph Node Biopsy vs No Axillary Surgery in Patients With Small Breast Cancer and Negative Results on Ultrasonography of Axillary Lymph Nodes: The SOUND Randomized Clinical Trial. JAMA Oncol. 2023 Nov 1;9(11):1557-1564. doi: 10.1001/jamaoncol.2023.3759.
• Bruhm DC, Vulpescu NA, Foda ZH, Phallen J, Scharpf RB, Velculescu VE. Genomic and fragmentomic landscapes of cell-free DNA for early cancer detection. Nat Rev Cancer. 2025 May;25(5):341-358. doi: 10.1038/s41568-025-00795-x. Epub 2025 Mar 4.
• Reimer T, Stachs A, Veselinovic K, Kuhn T, Heil J, Polata S, Marme F, Muller T, Hildebrandt G, Krug D, Ataseven B, Reitsamer R, Ruth S, Denkert C, Bekes I, Zahm DM, Thill M, Golatta M, Holtschmidt J, Knauer M, Nekljudova V, Loibl S, Gerber B. Axillary Surgery in Breast Cancer - Primary Results of the INSEMA Trial. N Engl J Med. 2025 Mar 13;392(11):1051-1064. doi: 10.1056/NEJMoa2412063. Epub 2024 Dec 12.
• Zhu Y, Zheng S, Shao Y, Zhou J, Gu X, Shen L, Li X, Liu W, Xue W, Lu H, Zhou J, Ding J, Deng H, Chen J, Yu Z, Yao Y, Xia W, Chen W, Sun S, Wang Z, Qian T, Yu X, Liu J, Chen Y, Lin Z, Huang J, Ni C. Fragmentomic liquid biopsy enables early breast cancer detection, molecular subtyping and lymph node assessment. Nat Commun. 2026 Mar 6;17(1):2276. doi: 10.1038/s41467-026-70204-w.

Study record dates

Study Major Dates

• Study Start (Actual): August 1, 2025
• Primary Completion (Estimated): December 31, 2027
• Study Completion (Estimated): December 31, 2027

Study Registration Dates

• First Submitted: November 19, 2025
• First Submitted That Met QC Criteria: December 11, 2025
• First Posted (Actual): December 26, 2025

Study Record Updates

• Last Update Posted (Actual): August 10, 2026
• Last Update Submitted That Met QC Criteria: August 5, 2026
• Last Verified: October 1, 2025

More Information

Terms related to this study

• Keywords: breast cancer; Magnetic resonance imaging; Neoadjuvant therapy; cfDNA; Diagnostic accuracy; Sentinel lymph node biopsy; Machine learning; Fragmentomics; Axillary lymph node; TuFEst model
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Skin Diseases; Breast Diseases; Skin and Connective Tissue Diseases; Breast Neoplasms
• Other Study ID Numbers: 2025-1028

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No