Trimethylamine N-oxide as a potential biomarker for predicting axillary lymph node metastasis in breast cancer: a retrospective study.

Introduction
Breast cancer remains a leading global malignancy among female populations, characterized by elevated incidence and mortality rates [1]. Precise evaluation of axillary lymph node (ALN) metastasis status constitutes a critical determinant for both therapeutic strategy formulation and prognostic evaluation in breast cancer management [2, 3]. Since 2005, Sentinel Lymph Node Biopsy (SLNB) has become an essential part of the standard diagnostic procedure for breast cancer. It has remarkably enhanced the accuracy of assessing the axillary lymph node status and spared some patients from undergoing axillary lymph node dissection (ALND). [4, 5]. Nevertheless, SLNB retains inherent invasiveness, with potential complications including chronic lymphedema and sensory disturbances in the upper extremities [6]. These clinical challenges underscore the imperative for developing preoperative assessment modalities capable of reliably predicting ALN status in early-stage invasive breast carcinoma.
Trimethylamine N-oxide (TMAO), a key microbial-derived metabolite originating from gut microbiota, has garnered significant research interest due to its multifaceted involvement in disease pathogenesis. Substantial evidence delineates its pathophysiological associations with cardiovascular disorders (including atrial fibrillation, coronary atherosclerosis, and heart failure) and metabolic syndromes (encompassing chronic kidney disease and diabetes mellitus) [7, 8]. Emerging oncological investigations further identify TMAO as a critical modulator in tumorigenesis, influencing malignant initiation, progression, and metastatic dissemination [9, 10]. TMAO promotes the malignant phenotypes of colorectal cancer and hepatocellular carcinoma by modulating tumor cell metabolic reprogramming, oxidative stress responses, and the immune microenvironment [7, 10]. These collective insights position TMAO as both a promising diagnostic biomarker and a novel therapeutic target, offering potential translational opportunities for tumor early detection and prognostic stratification.
Building upon the emerging evidence of TMAO's pathophysiological significance, this investigation conducted a retrospective analysis of breast tissue specimens to quantitatively assess TMAO concentrations across benign lesions, malignant neoplasms, and distinct histopathological subtypes of breast carcinoma. The primary objective focused on delineating potential differential expression patterns of intra-tumoral TMAO. Furthermore, extending the investigation to clinicopathological correlations, we systematically evaluated the association between TMAO levels and axillary lymph node metastatic burden. These approaches aim to validate TMAO's biomarker potential for preoperative lymph node metastasis prediction in breast cancer management.

Material and methods

Study population and sample collection
To investigate inter-tissular TMAO variations, a pilot analysis was conducted on matched breast tissue specimens from three clinically defined cohorts: patients with benign breast nodules (BBN, n = 10), ductal carcinoma in situ (DCIS, n = 10), and invasive ductal carcinoma (IDC, n = 10). Subsequently, a retrospective cohort was identified through systematic review of surgical records from the Sixth Affiliated Hospital of Sun Yat-Sen University (July 2023-January 2025), ultimately enrolling 97 treatment-naive IDC patients stratified by nodal status: 26 node-positive (N +) and 71 node-negative (N0) cases. Finally, we collected plasma from 12 patients with positive lymph nodes and 20 patients with negative lymph nodes for further validation.
All excised tumor specimens underwent standardized processing: immediate post-resection rinsing with sterile normal saline followed by snap-freezing in liquid nitrogen within 5 min of resection. Cryopreserved samples were maintained at −80 °C in vapor-phase liquid nitrogen storage systems until biochemical analysis. Blood collection and preservation methods: 3 mL of venous blood was collected from the patient, centrifuged at 3000 r/min for 10 min, and the upper layer of serum was stored in liquid nitrogen.
This protocol received ethical approval from the Institutional Review Board of the Sixth Affiliated Hospital of Sun Yat-Sen University (Approval No: 2025ZSLYEC-373). Written informed consent was obtained from all participants prior to enrollment.

Clinicopathological characteristics
The analytical framework incorporated standardized tumor biomarkers and staging parameters: patient age at diagnosis, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) expression, Ki-67 proliferation index (%), histological grading, maximal tumor dimension (mm), TN staging (AJCC 8th edition), and lymphovascular invasion status.

TMAO quantification in breast tissue and plasma
Tissue processing was performed under cryopreserved conditions to minimize metabolite degradation. Precisely weighed 100 mg tissue aliquots were rapidly thawed in ice-cold phosphate-buffered saline (PBS, 4 °C) for blood contaminant removal. Following three wash cycles with chilled PBS, specimens were mechanically homogenized in 600 μL ice-cold PBS supplemented with protease inhibitors. The homogenization protocol comprised three 30-s pulses at 30 Hz with intermittent ice-bath cooling to maintain thermal stability.
Resulting homogenates underwent differential centrifugation: initial clarification at 3,000 × g for 10 min (4 °C) to remove cellular debris, followed by high-speed centrifugation at 12,000 × g for 15 min (4 °C) using a refrigerated centrifuge. The final supernatant was analyzed. The plasma was slowly thawed on ice, then centrifuged at 3000 r/min (4 °C) for 2 min, and the upper layer of plasma was taken for testing. Each sample undergoes triplicate testing.
TMAO concentrations were determined using a validated commercial enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer's protocol [11]. The TMAO kit was purchased from ELK Biotechnology (Catalog No.: ELK8356). Absorbance measurements at 450 nm were acquired with microplate reader. The standard curve formula for this test is: y = 40.626x2 + 336.55x—17.617; R2 = 0.991.

Statistical analysis
Continuous variables were analyzed by Mann–Whitney test. The comparison of categorical variables was performed by card placement test or Fisher exact test. Spearman correlation analysis was used to assess correlations between biomarker levels. The significance difference of this study is P < 0.05. All statistical tests were two-tailed tests. SPSS software, R software (version 3.6.1) and GraphPad Prism software were used for statistical analysis and plotting.

Results

TMAO expression in BBN, DICS, and IDC groups
Quantifiable TMAO levels were detected across all tissue specimens, including 10 BBN, 10 DCIS, and 10 IDC. Comparative analysis revealed significantly elevated TMAO concentrations in the IDC group (113.9 ± 16.36 ng/g) versus DCIS (82.29 ± 13.84 ng/g; p < 0.01) and BBN cohorts (76.09 ± 10.32 ng/g; p < 0.001). No significant intergroup difference was observed between DCIS and BBN specimens (p = 0.57) (Fig. 1).

Clinical features and TMAO expression of ALN + and ALN- groups
The study cohort comprised 97 eligible patients stratified by nodal status: 26 ALN-positive (ALN +) and 71 ALN-negative (ALN-) cases (Fig. 2). ALN + patients exhibited advanced T-stage distribution (p = 0.001) and higher lymphovascular invasion prevalence (42.3% vs 19.7%; p = 0.024) compared to ALN- counterparts. While imaging modalities (ultrasound, CT, MRI) demonstrated intergroup variability, no statistically significant differences emerged in hormone receptor profiles, HER2 status, Ki-67 indices, histological grade, or surgical approach (Table 1).
Postoperative tissue analysis revealed markedly elevated TMAO levels in ALN + specimens (210.92 ± 82.09 ng/g) versus ALN- controls (122.99 ± 48.23 ng/g; p < 0.001) (Fig. 3A). To determine whether the size of the tumor is related to the concentration of TMAO. Spearman correlation analysis indicated no significant association between TMAO concentrations and tumor diameter (R = 0.21) (Fig. 3B). Since the majority of the included patients were in T2-stage (63.9%), we conducted a subgroup analysis of the patients in T2-stage. Subgroup stratification by T-stage identified significantly higher TMAO expression in ALN + patients at T2-stage (219.89 ± 61.37 ng/g vs 122.56 ± 48.93 ng/g; p = 0.002) (Fig. 3C).
Since we only tested the TMAO concentration of the tumor, we further verified whether plasma TMAO is also related to axillary lymph node metastasis. We re-collected the plasma of 32 patients in a patient cohort(12 ALN + and 20 ALN-). Plasma analysis showed that the level of TMAO in ALN + was significantly higher than that in the ALN- control group (127.7 ± 16.58 ng/g vs 95.01 ± 19.18 ng/g, p < 0.001) (Fig. 3D).

Association of TMAO with lymph node metastasis in breast cancer
Univariable logistic regression identified significant predictors of lymph node metastasis, including elevated TMAO levels (OR = 1.022, 95% CI: 1.012–1.032), T-stage (T1 vs T2, OR = 1.236, 95% CI: 1.192–2.204), lymphovascular invasion (OR = 2.985, 95% CI: 1.428–7.874), and positive axillary imaging findings (ultrasound: OR = 10.638 (3.311–25.641); CT: OR = 6.098 (2.279–16.393); MRI: OR = 11.494 (4.032–33.333)). Estrogen/progesterone receptor status, HER2 expression, Ki-67 index, and histological grade showed no significant associations (p > 0.05).
Multivariable analysis confirmed TMAO (OR = 1.020, 95% CI: 1.009–1.031), T-stage (T1 vs T2, OR = 1.201, 95% CI: 1.019–2.169), and axillary ultrasound positivity (OR = 6.993, 95% CI: 1.099–45.455) as independent predictors (Table 2). In T2-stage subgroup analysis, TMAO retained independent predictive value (OR = 1.030, 95% CI: 1.013–1.046) after adjusting for imaging modalities and lymphovascular invasion (Table 3).

Diagnostic performance of combined TMAO-imaging model
Based on multivariable logistic regression outcomes, receiver operating characteristic (ROC) curve analysis was conducted for TMAO levels, T-stage classification, and axillary ultrasound findings. As illustrated in Fig. 4A, the individual diagnostic performance metrics were as follows:TMAO demonstrated an AUC of 0.836 (95% CI: 0.730–0.943);T-stage showed an AUC of 0.681 (95% CI: 0.568–0.793);Axillary ultrasound yielded an AUC of 0.748 (95% CI: 0.631–0.865). A composite diagnostic model incorporating all three parameters was subsequently developed. This integrated model exhibited enhanced predictive capability with a combined AUC of 0.915 (95% CI: 0.838–0.992) for discriminating lymph node metastasis status, demonstrating 88.5% sensitivity and 90.1% specificity at the optimal cutoff value. The calibration curve shows that the consistency between the predicted probability of the model and the actual observed probability is good, as shown in Fig. 4B.

Discussion
The status of axillary lymph nodes is closely related to the survival rate of breast cancer patients, and the accurate evaluation of axillary lymph node staging is crucial for the formulation of breast cancer treatment plan and prognosis judgment [12]. Currently, the evaluation methods of breast cancer lymph node metastasis include clinical examination, imaging examination and invasive examination. Clinical examination mainly includes physical examination and palpation of lymph nodes, which can provide preliminary diagnostic information [13]. Imaging tests, such as ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET-CT), can provide more accurate information about lymph node metastasis [14]. Invasive tests, such as lymph node biopsies and lymphadenectomy, can directly obtain a sample of lymph node tissue, which may help confirm the diagnosis and staging [15]. However, although these assessment methods are widely used in breast cancer patients, they all have limitations. Clinical examination is subject to the doctor's experience and palpation techniques, and lymph nodes may not always be accurately determined to be involved. Although imaging studies can provide detailed structural information, there are limitations in detecting small metastases or assessing the extent of metastases [16]. Invasive tests, while providing clear results, are somewhat limited by their invasiveness and associated risks. [17]. Therefore, in order to more accurately assess lymph node metastasis in breast cancer patients, the development of new and more accurate assessment methods is of great clinical significance. This study demonstrated a significant dose-dependent relationship between TMAO concentration in tumors and lymph node metastasis in breast cancer (OR = 1.020, 95%CI 1.009–1.031). At the same time, TMAO combined with tumor T staging and ultrasonography can predict the occurrence of lymph node metastasis. The results show that the combined multi-parameter predictive model exhibits excellent performance (AUC 0.915). If confirmed in larger, prospective cohorts, combining TMAO with imaging might reduce—but not replace—the need for surgical axillary staging.
Metabolic abnormalities are common in disease and can lead to dysfunction of metabolic pathways and abnormal accumulation or deficiency of metabolites. Metabolites in human tissues provide promising biomarkers for clinical diagnosis, classification and prognosis prediction [18]. Identifying alterations in metabolites, or metabolic pathways, can not only provide valuable insights into the pathophysiology of the disease, but also improve the accuracy of patient diagnosis and risk prediction, and help identify potential therapeutic targets [19, 20]. TMAO is a compound produced from dietary nutrients by the gut microbiome, which is increasingly recognized as a potential risk factor for various diseases, including cancer [7]. Although our study showed no differences between the two groups in cardiovascular disease, chronic kidney disease, diabetes, or recent antibiotic use, larger prospective cohorts or propensity-score analyses are still needed to rule out subtle imbalances in cardiovascular or renal risk factors. A meta-analysis examining the relationship between TMAO concentrations and cancer risk showed that high TMAO levels were positively associated with an increased risk of colorectal, prostate, primary liver, and pancreatic cancers [21]. Previous studies have shown through metabolomics analysis that the level of TMAO in plasma samples of breast cancer patients is higher than that of healthy controls, suggesting that TMAO is closely related to breast cancer [22]. In this study, it was found that the TMAO content in IDC group (113.9 ± 16.36 ng/g) was significantly higher than that in DCIS group (82.29 ± 13.84 ng/g) and BBN group (76.09 ± 10.32 ng/g), while no significant difference was found between DCIS group and BBN group. This suggests that not only is TMAO in plasma strongly associated with breast cancer, but TMAO in breast tissue is also associated with breast cancer progression.
In addition, the results of this study show that the concentration of TMAO in tumors is significantly dose-dependent with lymph node metastasis in breast cancer and can be used to distinguish patients with IDC with or without lymph node metastasis. However, Seo et al. found in thyroid cancer that the TMAO level in the lymph node metastasis group was lower than that in the non-metastatic group [23], and the potential role of targeted metabolites, including choline, in predicting lymph node metastasis in patients with thyroid cancer was investigated. To verify whether the concentration of TMAO in the patient's plasma is also related to the relevant axillary lymph node metastasis. We re-collected a batch of patients' plasma for testing, the results showed that Plasma analysis showed that the level of TMAO in ALN + was significantly higher than that in the ALN- group. However, the consistency between TMAO in patient tissues and plasma still requires further verification through prospective studies.
However, in triple-negative breast cancer (TNBC), recent studies have shown that TMAO can drive immune activation and promote anti-tumor immunity [24]. The study demonstrated that TMAO promotes CD8 + T cell-mediated TNBC immunotherapy via a PERK-ER stress-GSDME pyroptosis axis. However, it should be noted that this study was limited to patients with immunomodulatory (IM) subtype TNBC and anti-PD-1 therapy, and its conclusions should be further validated in other subtypes of TNBC and immunotherapy for other cancers. The different effects of TMAO in tumors may be attributed to complex interactions with the tumor microenvironment. On the other hand, TMAO can promote liver cancer progression by regulating cell proliferation, invasion, and angiogenesis [10]. This dual role highlights the complex and dynamic nature of TMAO in tumors and underscores the need for further research to fully understand its role in cancer biology.
Our research demonstrates that T stage is an independent prognostic factor for axillary lymph node metastasis in breast cancer. Prior studies have shown that a maximum tumor diameter exceeding 2 cm is an independent factor for axillary lymph node metastasis [25, 26]. The larger the tumor, the greater the extent of surrounding tissue invasion and lymphatic vessel involvement, thereby increasing the risk of lymph node metastasis. In breast ultrasound, a mass aspect ratio over 1 is commonly considered a characteristic of malignant tumors [27]. This vertical growth pattern may allow cancer tissue to more easily obtain nutrients from surrounding tissues, suggesting higher invasiveness and a potential link to axillary lymph node metastasis. However, as a retrospective analysis, our database lacks detailed aspect ratio data, so the correlation between mass aspect ratio and axillary lymph node status requires further study. Additionally, our study reveals a weak correlation between TMAO and tumor diameter, indicating that TMAO may not be closely related to tumor staging but may have a certain association with lymph node metastasis. Subgroup analysis indicates that in the T2 stage, TMAO remains an independent risk factor for lymph node metastasis.
Our study does not show a higher likelihood of axillary lymph node metastasis in HER-2-positive breast cancer. Existing research indicates that invasive breast cancer has a significantly higher risk of axillary lymph node metastasis than non-invasive breast cancer [28]. HER-2 positivity is typically associated with a higher risk of axillary lymph node metastasis in breast cancer patients. However, our study only included patients who underwent surgery directly after their initial diagnosis. Some HER-2-positive patients might have received neoadjuvant therapy and thus were not included in the study, which could explain why we did not observe a relationship between HER-2 positivity and axillary lymph node metastasis in breast cancer.
There are still some limitations in this study. First of all, this is a retrospective study. In the detection of TMAO concentrations, there was no one-to-one match between the patient's tumor tissue and plasma. We only conducted plasma tests on some patients. In subsequent prospective experiments, the concentrations of TMAO in both the plasma and tumor tissues of patients should be detected simultaneously. Secondly, this study relied on a commercial ELISA kit whose specificity is still inferior to mass-spectrometry methods. All measurements were performed on a single microplate reader platform in one laboratory, and no inter-batch or inter-laboratory variation was assessed. Finally, this study did not fully elucidate the mechanism of action of TMAO on tumors or determine whether it can be used as a potential target for cancer prevention and treatment.

Conclusions
Our study revealed differential levels of TMAO in breast tissue between benign and malignant lesions, as well as among different pathological subtypes of breast cancer. Notably, the TMAO concentration was significantly associated with lymph node metastasis, suggesting its potential utility as a novel biomarker for predicting axillary lymph node metastasis in breast cancer patients.

Supplementary Information