Carbon-tattooed target lymph nodes improve detection of low-volume residual axillary disease after neoadjuvant chemotherapy

Carbon-tattooed target lymph nodes improve detection of low-volume residual axillary disease after neoadjuvant chemotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Carbon-tattooed target lymph nodes improve detection of low-volume residual axillary disease after neoadjuvant chemotherapy Pelin Basım, Emine Yıldırım, Neşe Uçar, Perya Abbasoglu, Nilay Bakoglu Malinowski, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9171161/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Purpose Accurate axillary staging after neoadjuvant chemotherapy (NAC) remains essential in breast cancer surgery. Low-volume residual axillary disease is frequently missed after neoadjuvant chemotherapy (NAC), raising concerns about the reliability of sentinel lymph node biopsy (SLNB). This study evaluated the detection performance of carbon-tattooed target lymph nodes (TLNs) compared with conventional blue dye (BD) mapping during axillary staging, particularly in the context of low-volume metastatic disease. Methods Eighty-three breast cancer (BC) patients undergoing SLNB after preoperative ultrasound-guided carbon injection of suspicious axillary lymph nodes were prospectively analyzed. Detection rates of TLNs and BD were assessed across metastasis categories including macrometastasis (MacroM), micrometastasis (MicroM), and isolated tumor cells (ITCs). Paired comparisons were performed using the McNemar exact test. Results TLNs were identified in all patients, achieving a 100% detection rate. BD detected sentinel lymph nodes in 62 patients (74.7%), failed in 14 (16.9%), and required ALND-only identification in 7 (8.4%). TLNs detected all metastatic nodes, including MacroM (n = 42), MicroM (n = 30), and ITCs (n = 41). In contrast, BD detected 64.3% of MacroM, 60.0% of MicroM, and 34.1% of ITCs. McNemar analysis demonstrated significantly higher detection with TLNs for MacroM (p = 0.000061), MicroM (p = 0.000488), and ITCs (p < 0.00000001). Conclusion Carbon-tattooed TLN mapping showed superior detection of metastatic lymph nodes compared with BD, particularly in low-volume disease after NAC. Carbon tattooing Blue Dye Targeted axillary dissection Macrometastasis Introduction In breast cancer (BC) surgery, the approach to the axilla is progressively evolving, emphasizing both the prevention of local recurrences and the determination of prognosis, while also prioritizing procedures that minimize morbidity. Although axillary lymph node dissection (ALND) has been used for a long time in traditional approaches to treatment, sentinel lymph node biopsy (SLNB), developed by Giuliano and Krag, has become the standard for early-stage clinically node-negative breast cancer [ 1 – 3 ]. In patients with initially node-positive disease (cN+), several studies are being conducted on axillary restaging and treatment following neoadjuvant chemotherapy (NAC) aimed at reducing tumor burden, with efforts to establish an optimal and standardized approach that could be universally applied across all patient groups. However, a critical limitation remains: SLNB may fail to detect low-volume residual axillary disease after NAC, particularly micrometastases (MicroM) and isolated tumor cells (ITC). Following NAC, the significant increase in axillary complete response rates, reaching up to 70%, has prompted investigations into the suitability of SLNB for locally advanced disease, as it allows for the avoidance of ALND and associated morbidities without negatively impacting survival or local recurrence when SLNB is negative. The selection of an axillary staging method that ensures high oncological safety while maintaining arm function and quality of life is still a subject of intense debate [ 4 – 6 ]. Subsequent studies have shown that in patients initially presenting with axillary lymph node metastasis (ALNM), SLNB can be safely performed in a selected group after NAC [ 7 , 8 ]. In the American College of Surgeons Oncology Group (ACOSOG) Z1071 trial, patients with ALNM confirmed by biopsy were marked with clips prior to NAC, and in the group where the clipped lymph node (cLN) was included in SLNB, the false negative rate (FNR) was found to be 6.8%. The same study reported that in patients whose cLN was not identified during SLNB and was instead included in ALND, the false negative rate increased to 19% [ 9 ]. In this context, marking the metastatic LN prior to NAC and including it in the SLNB specimen becomes crucial. Targeted axillary dissection (TAD) is a procedure that involves SLNB, in which all sentinel lymph nodes (SLN) are removed along with the metastatic LN that was clipped prior to treatment. Numerous clinical studies have shown that TAD is a viable option to reduce false negative ALND in breast cancer patients following NAC (10). During TAD, the localization of the cLN prior to treatment is determined either by placing a guidewire under preoperative ultrasound (US) guidance or by implanting a radioactive seed [ 10 – 12 ]. In cases where an iodine-125 seed is implanted in the cLN, intraoperative localization is achieved using a radioactive probe. The cLN, along with all SLN, is subsequently excised as part of the TAD procedure [ 12 ]. At present, the implementation of these procedures is constrained by factors such as high costs and the absence of the requisite equipment and nuclear medicine units in many centers. As a result, alternative methods that are more accessible and feasible are being explored for evaluating axillary response following NAC. Carbon nanoparticles, owing to their molecular dimensions, are particulate substances that can traverse lymphatic channels without penetrating blood vessels, and are utilized for lymph node visualization in thyroid surgery [ 13 ]. Similarly, sterile carbon particle suspension has long been used for the endoscopic marking of colonic lesions [ 14 ]. Numerous studies on the use of the tattooing technique in BC patients, particularly those with suspected metastasis undergoing primary surgical treatment or in known metastatic LNs after NAC, report favorable opinions regarding the effectiveness of this method [ 15 – 18 ]. In our study, patients with biopsy-proven 1–3 metastatic axillary lymph nodes (N1) before NAC, who had metallic clip placement, underwent concurrent lymph node marking by injection of SPOT® Ex Endoscopic Tattoo into the LNs cortex. The primary objective of this study was to assess the correlation between tattooed lymph nodes (TLNs), labeled with carbon suspension, and SLNs as well as to evaluate the detection rate of TLNs during surgery. The secondary objective was to evaluate the impact of TLNs on surgical outcomes, including evaluating the efficacy of TLN in detecting axillary macrometastases (MacroM), MicroM, and ITC through frozen section examination and final pathological analysis. In other words, we evaluated post-NAC concordance between the SLNs and the previously marked metastatic LNs. Additionally, metastatic burden was categorized and analyzed based on MacroM, MicroM and ITC. Thus, the reliability and safety of carbon dye for permanent axillary marking as an alternative to blue dye (BD) in axillary staging were assessed. Materials and Methods Study Design: This prospective, two-center study was carried out between February 2023 and June 2025 and conducted to evaluate the feasibility of using carbon dye as an alternative to clips for marking metastatic lymph nodes prior to NAC in patients with locally advanced breast cancer and histopathologically confirmed axillary metastasis. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (Ethics Committee) of Istanbul Medipol University (approval number: E-10840098-202.3.02-1418; 19 February 2026) Written informed consent was obtained from all patients before participation in the study, in accordance with institutional and ethical guidelines. Patients were recruited and clinical data were collected at Istanbul Medipol University Hospital, Istanbul, Türkiye. Patients: A total of 83 female patients aged ≥ 18 years with BC and biopsy-proven ALNM (confirmed by fine-needle aspiration (FNA) or core needle biopsy (CNB) who received NAC as initial treatment for clinical stage T1–3, N1, M0 disease were included in the study, regardless of breast cancer subtype or histopathological characteristics. Exclusion criteria included refusal to receive or complete NAC, disease progression during the ongoing treatment, a prior history of breast cancer or axillary surgery, as well as the presence of distant metastasis, clinical N2 and N3 disease, inflammatory breast cancer, pregnancy, and lactation. Radiological Evaluation: All patients were evaluated prior to the NAC regimen, using mammography (MG), breast and axillary US, and magnetic resonance imaging (MRI). The US evaluations were performed using a Toshiba Aplio 500 ultrasound device with a 5–14 MHz linear array probe and Toshiba software version 6.0 (Toshiba Corporation, Tokyo, Japan). LNs with lobulated contours, diffuse or asymmetric focal cortical thickness (> 3mm), anechoic/hypoechoic cortex compared to the subcutaneous tissue or obliterated cortex, and distorted fatty hilum or poorly visualized hilum were considered suspicious or malignant. For lymph nodes suspicious or appearing malignant in terms of metastasis, either an ultrasound-guided FNA or CNB was performed, depending on the size of the LNs. Multiple insertions were made into the thickest or focal thickened area of the LN cortex, and sufficient tissue samples were obtained. Pre-NAC Marking of Lymph Node and Tattoo Technique: LNs found to be metastatic histopathologically underwent a second session for clip placement and carbon dye application. A marking procedure was performed on one to three LNs per patient, corresponding to the number of biopsy-proven metastatic nodes. In other words, all LNs diagnosed as metastatic, ranging from one to three in number, were marked with both clip placement and carbon tattoo technique prior to NAC. Clip placement was performed according to the number of pathologically confirmed metastatic lymph nodes using a Geotek titanium tissue-marking clip (Geotek Medical, Ankara, Turkey), which is routinely used in clinical practice in Türkiye. The same LNs were injected with a highly purified carbon suspension, carefully deposited into the cortical area of LN and the adjacent soft tissues, with the total volume injected being documented. If feasible, physicians had the option to record the distinct volumes injected into the LN cortex, the surrounding soft tissue, and the path extending from the node to the skin. The creation of a carbon suspension track from the LN to the skin surface, as well as the intentional tattooing of the overlying dermis, were optional steps. Spot® Ex Endoscopic Tattoo (GI supply,Mechanicsburg,USA) was utilized for this procedure. Both clip placement and carbon tattooing were initially performed in the same lymph nodes due to concerns regarding potential fading or intraoperative non-visualization of carbon during the prolonged NAC interval. However, as carbon staining was consistently identified in all clipped nodes, demonstrating complete concordance, subsequent analyses were based on carbon-tattooed target lymph nodes. Wire localization of marker-clipped nodes: The targeted clipped axillary LNs were localized using a flexible marking wire (20-G, 10-cm breast localization needle; Geotek Medical, Ankara, Turkey) under ultrasound or mammographic guidance, depending on clip visibility, 30 minutes to 1 hour before surgery. In cases where the clipped LNs were adjacent to each other, the use of a single wire was deemed appropriate. To facilitate easy identification of the wire-marked LNs and shorten the duration of the surgical procedure, mapping was performed by a radiologist with over 10 years of experience in breast radiology. The vertical and horizontal distances from the wire insertion point to the LNs were mapped using ultrasonography, and the closest potential incision line was marked on the skin using a marker pen. Axillary surgery: In all patients exhibiting either a partial or complete clinical axillary response, the SLN was mapped conventionally by using BD inoculated in the periareolar region. During surgery, BD LNs and TLNs were identified by visual examination, subsequently confirmed by pathological examination; on the other hand, the guidance of hook wire marking was utilized to identify clipped LNs. The time taken for the identification of three distinct groups of LNs, each marked using different methods, in the axillary region was recorded separately for each group as well as cumulatively. All blue-dyed, carbon-labeled-clipped LNs identified during surgery were sent to the pathology unit for frozen section analysis. Subsequently, all patients with macrometastasis (MacroM) detected underwent ALND. All excised LNs were classified based on the size of the metastatic foci as 2 mm (MacroM). Database setting: Clinical and pathological data of the patients were retrieved from their medical records of the concerned hospitals. The variables of interest included age, body mass index (BMI), smoking status, menopause state, tumor size, greatest LN diameter, number of suspicious LNs, clinical T and N stage, radiological findings (MG, US and MRI), tumor type and subtype, presence of lymphvascular invasion, type of breast and axillary surgery, identification of blue-dyed, carbon-dyed and clipped LNs, intraoperative concordance of markings and axillary pathological response based on the presence and size of the metastatic foci with the marking technique used to identify it. Statistical Analysis: Statistical analyses were performed using IBM SPSS Statistics software (version 23; IBM Corp., Armonk, NY, USA). Paired comparisons between TLNs and blue dye detection rates were conducted using the McNemar exact test. A p-value of < 0.05 was considered statistically significant. Results 3.1. Patient Characteristics A total of 83 patients were included in the analysis. Among the study cohort, 48 patients (57.9%) were non-smokers and 35 (42.2%) were current smokers. Fifty patients (60.2%) were postmenopausal, while 33 (39.8%) were premenopausal. On preoperative axillary US, one suspicious LN was identified in 12 patients (14.5%), two suspicious LNs in 34 patients (41.0%), and ≥ 3 suspicious LNs in 37 patients (44.6%) (Table 1). BMI was comparable between patients with negative SLNB findings (25.70 ± 2.94) and those with metastatic SLNB involvement (25.97 ± 1.90). The mean age at surgery was 48.0 ± 10.08 years in the SLNB-negative group and 50.0 ± 11.48 years in the SLNB-positive group. Primary tumor diameter measured 27.0 ± 13.51 mm in patients without SLNB metastasis and 30.0 ± 14.28 mm in those with metastatic involvement. The greatest LN diameter on imaging was also similar between groups (19.0 ± 4.82 mm vs. 18.0 ± 6.59 mm) (Table 2). 3.2. Sentinel Lymph Node Identification BD successfully identified lymph nodes during SLNB in 62 patients (74.7%). In 7 patients (8.4%), LNs were identified only during subsequent ALND. In the remaining 14 patients (16.9%), BD failed to identify any lymph nodes during either SLNB or ALND. In contrast, TLNs were identified in all patients, achieving a 100% detection rate with complete one-to-one correspondence between injected and detected nodes. When the entire cohort was evaluated regardless of metastatic status, intraoperative concordance between TLNs and BD was observed in 47 patients (56.6%), whereas no concordance was observed in 36 patients (43.4%) (Table 1). 3.3. SLNB Metastatic Findings SLNB demonstrated metastatic involvement in 42 patients (50.6%), while no metastasis was detected in 41 patients (49.4%). MacroM was absent in 41 patients (49.4%). Among patients with MacroM (n = 42), detection was achieved exclusively with TLNs in 15 patients (35.7%), while combined detection with both TLNs and BD was observed in 27 patients (64.3%). MicroM was absent in 53 patients (63.9%). Among MicroM-positive patients (n = 30), 12 cases (40.0%) were detected exclusively with TLNs, whereas 18 cases (60.0%) were detected by both TLNs and BD. ITCs were absent in 42 patients (50.6%). Among ITC-positive patients (n = 41), detection was achieved solely by TLNs in 27 cases (65.9%), while combined TLN and BD detection was observed in 14 cases (34.1%) (Table 3). 3.4. Detection Rates According to Metastatic Burden TLNs demonstrated complete detection of metastatic lymph nodes across all pathological subtypes. All MacroM (n = 42), MicroM (n = 30), and ITC-positive LNs (n = 41) were successfully identified using TLNs, corresponding to a detection rate of 100% in each metastasis category. In contrast, BD identified a lower proportion of metastatic disease. BD positivity was observed in 27 MacroM (64.3%), 18 MicroM (60.0%), and 14 ITCs (34.1%). Detection rates decreased with lower-volume metastatic burden, with the lowest detection observed in LNs containing isolated tumor cells (Table 3). 3.5. Paired Comparison of TLNs and BD Detection A paired comparison of TLN and BD detection rates was performed using the McNemar exact test (Table 4). For macrometastasis, TLNs identified 15 metastatic nodes not detected by BD, while no nodes were detected exclusively by BD, resulting in a statistically significant difference (p = 0.000061). Similarly, for micrometastasis, TLNs detected 12 nodes that were BD-negative, with no cases of BD-only detection (p = 0.000488). For LNs containing ITCs, TLNs identified 27 additional metastatic nodes not detected by BD, again with no BD-only detection, yielding a highly significant difference favoring TLNs (p < 0.00000001). Importantly, no metastatic LN was detected by BD alone. Discussion Axillary lymph node involvement remains one of the most significant prognostic indicators in BC and plays a crucial role in determining both locoregional management and systemic therapeutic approach. In patients with node-positive disease, neoadjuvant chemotherapy is commonly employed and has been shown to induce notably encouraging nodal pCR rates, ranging from 40% to 75%. TAD, first described by Caudle et al., is an innovative approach based on marking LNs with biopsy-proven metastasis prior to systemic therapy and subsequently removing these targeted nodes in combination with conventional SLNB during surgery. This strategy enables the retrieval of originally pre-treatment metastatic nodes, thereby allowing a more accurate assessment of axillary tumor burden, reducing false-negative rates, and optimizing pathological axillary staging [ 11 , 19 ]. The findings of the present study suggest that, following NAC, TLNs may demonstrate a very high sensitivity for the detection of residual metastatic disease. In our patient cohort, regardless of metastatic burden, all metastatic LNs were successfully identified through TLNs across all pathological subgroups, including MacroM, MicroM and ITCs. In contrast, using the blue dye technique, only 64.3% of MacroM, 60.0% of MicroM, and 34.1% of LNs containing ITCs were detected. Notably, in 35.7% of patients with MacroM and in 40.0% of those with MicroM, metastatic LNs were identified exclusively by TLNs and were not visualized using blue dye. Collectively, these findings suggest that permanent LN marking with carbon may represent a feasible and potentially promising approach to reliably localize target LNs and enhance the accuracy of axillary staging after NAC. The ACOSOG Z1071 trial evaluated the reliability of SLNB after NAC in patients with biopsy-proven node-positive BC and demonstrated that false-negative rates remained clinically relevant despite the use of different SLNB techniques [ 8 ]. Secondary analyses of the same trial further showed that inclusion of the pre-treatment clipped LN along with the conventional sentinel LN specimen significantly reduced the false-negative rate, whereas failure to retrieve the targeted metastatic LN was associated with a substantially increased risk of false-negative results [ 9 ]. These findings underscore that a key determinant of post-NAC axillary evaluation is whether the LN known to be metastatic at diagnosis is successfully removed. Similarly, the SENTINA study reported that in patients initially presenting with node-positive disease, the performance of SLNB after NAC was highly dependent on technical factors and that false-negative rates remained clinically significant [ 7 ]. The SN FNAC trial also demonstrated that reliable removal of biopsy-proven metastatic LNs improved the accuracy of axillary staging; however, these LNs did not consistently correspond to the conventional sentinel LN basin, limiting the reliability of standard SLNB approaches [ 20 ]. These observations have been supported by several meta-analyses and systematic reviews, which emphasized that SLNB after NAC in initially node-positive patients is associated with variable and, at times, unacceptably high false-negative rates [ 21 , 23 ]. Moreover, multiple studies addressing on selective evaluation of clipped or marked LNs have consistently shown that successful retrieval of the pre-treatment targeted LN improves axillary staging accuracy and significantly reduces false-negative rates [ 22 , 23 , 24 , 27 ]. In this context, the finding in the present study that all metastatic LNs, across all metastatic burden subgroups, were identified through TLNs suggests that this approach may represent a practical alternative for reliable localization of target LNs after NAC. The lack of requirement for radioactive seeds or specialized nuclear medicine infrastructure may further enhance its clinical applicability; however, its impact on false-negative rates and oncologic outcomes should be validated in future comparative studies. From a broader perspective, pre-treatment carbon tattooing may not only aim to maintain oncologic safety but could also be regarded as a low-cost adaptation of the targeted axillary approach; in this regard, it raises the possibility of a cost-effectiveness advantage, particularly in resource-limited healthcare settings. In the post-neoadjuvant chemotherapy (NAC) setting, the reliability of conventional sentinel lymph node biopsy (SLNB), and particularly of surgical approaches based solely on blue dye mapping, remains controversial for both physiological and technical reasons. After NAC, lymphatic drainage pathways may be altered by fibrosis, obliteration, and tumor regression, potentially resulting in failure of the true metastatic lymph node to exhibit sentinel behavior; moreover, the possibility of skip metastases should not be overlooked [ 28 , 29 ]. Indeed, large prospective series and meta-analyses have demonstrated that in patients initially presenting with node-positive disease, false-negative rates of SLNB after NAC are variable and at times exceed clinically acceptable thresholds [ 8 , 9 , 20 , 30 ]. The SENTINA and SN FNAC trials further emphasized that sentinel lymph nodes do not always reliably represent residual metastatic disease and that, despite technical success, the biologically relevant “true target node” may be missed [ 7 , 22 ]. In the present study, blue dye identified only 64.3% of MacroM, 60.0% of MicroM, and 34.1% of ITCs, whereas all metastatic LNs across all subgroups were identified through TLNs. Moreover, in 35.7% of patients with MacroM and in 40.0% of those with MicroM, metastatic LN were detected exclusively via TLNs, suggesting a clinically meaningful discordance between sentinel negativity and true axillary response. These findings support the need to question the biological validity of the “sentinel” concept after NAC and underscore the importance of approaches based on direct evaluation of the target LN [ 8 , 9 , 21 , 24 ]. In this context, whether an SLNB that does not include the target node can truly provide biologically accurate axillary staging after NAC warrants critical consideration. Accordingly, the post-NAC axillary approach invites renewed debate as to whether technical success in sentinel node identification or biological representation of the initially involved LN should be regarded as the principal determinant of accurate staging. The clinical and biological significance of residual low-volume axillary disease (MicroM and ITCs) detected during surgery after NAC remains controversial and is often regarded by many authors as “incidental”; nevertheless, these findings continue to have implications for the definition of pCR, axillary staging, adjuvant treatment decisions, and regional radiotherapy planning [ 31 , 34 ]. Although the impact of MicroMs—and particularly ITCs—on long-term survival has not yet been clearly established, several high-quality studies suggest that this disease burden may reflect residual tumor biology and may carry prognostic relevance in selected subgroups [ 35 , 36 ]. Accurate detection of low-volume axillary disease is therefore of particular importance, especially for the appropriate definition pCR after NAC. However, conventional SLNB has demonstrated notable limitations in identifying low-volume disease, with ITCs and MicroM representing the most frequently missed forms of residual nodal metastasis in the post-NAC setting [ 9 , 20 , 30 ]. In the present study, blue dye mapping identified only 34.1% of ITCs and 60.0% of MicroM, consistent with the existing literature; in contrast, carbon-tattooed lymph nodes enabled detection of both MicroM and ITCs in 100% of cases, a finding that is particularly noteworthy. It should be acknowledged that restriction of the study population to patients with N1 disease may have contributed to these results. Nevertheless, these findings raise the possibility that failure to detect low-volume residual disease could lead to excessive axillary downstaging and false-negative interpretations of pCR. At a time when treatment paradigms are increasingly shifting toward biologically driven decision-making and de-escalation strategies, the clinical relevance of accurately identifying low-volume disease is likely to become even more pronounced [ 37 , 38 ]. In this context, the potential role of TLNs in reliably detecting low-volume metastatic disease and contributing to future residual disease–based risk stratification warrants further investigation. TLN marking relies on a labeling technique characterized by favorable physicochemical properties and biological compatibility, permitting long-term stability within the lymphatic system without significant migration. This characteristic allows reliable visual identification of the target LN during surgery even after months-long NAC protocols, with a low risk of fading or displacement, as may occasionally occur with metallic clips. The low cost of carbon tattooing, the absence of radiation exposure, the lack of requirement for specialized detection devices or nuclear medicine infrastructure, and the fact that no additional operating room equipment is needed constitute major advantages that enhance its feasibility and accessibility, particularly in developing countries and in healthcare systems with economic constraints. Compared with other targeting techniques based on metallic clip placement, carbon marking may also be less invasive and more cost-effective, potentially facilitating broader clinical implementation. The literature indicates that carbon particles have been safely used for LNs mapping in thyroid surgery, have long been established as a standard technique for endoscopic marking of colorectal lesions, and that an increasing number of studies with relatively large series support their feasibility in breast surgery, both in the primary setting and for post-NAC axillary evaluation [ 39 , 42 ]. These studies have demonstrated that carbon can persist in local tissues for extended periods without inducing significant inflammatory reactions and can facilitate intraoperative visual localization. The findings of the present study suggest that these technical advantages may translate into clinically meaningful benefits; in particular, the reliable detection of TLNs in patients with one to three suspicious or biopsy-proven metastatic nodes indicates that this approach may contribute not only to theoretical feasibility but also to practical and effective localization of target LNs, including the reliable identification of low-burden axillary metastatic disease, namely MicroM and ITC. In this context, carbon tattooing may be considered a potential tool to enhance the applicability of target-node–based axillary approaches, especially in centers with limited access to nuclear medicine facilities. Nevertheless, randomized controlled trials stratified according to clinical axillary stage are required to further clarify the role of this method within clinical algorithms. The findings of the present study prompt reconsideration of whether post-NAC axillary surgery should be driven primarily by technical success in sentinel LN identification or by reliable removal of the biologically relevant target LN. When the target node can be consistently localized, the possibility of more minimal, more targeted, and potentially less morbid axillary surgical strategies after NAC warrants discussion. Nevertheless, several limitations of this study must be acknowledged. The relatively small sample size, the two-center, non-randomized design, and the restriction to patients with clinical N1 disease (1–3 suspicious or biopsy-proven lymph nodes) may limit the generalizability of our findings. Furthermore, the lack of direct comparison with other target-node localization techniques, such as radioactive seeds, magnetic markers, or radioisotope-guided approaches, precludes conclusions regarding superiority or equivalence. Our analysis focused on short-term surgical and pathological outcomes; long-term oncologic endpoints, including axillary recurrence, disease-free survival, overall survival, and functional outcomes, were not assessed. In addition, the injection technique, ultrasonographic assessment, surgical exploration, and pathological evaluation are operator-dependent processes that may influence reproducibility across different centers. Despite these limitations, our results suggest that carbon-targeted LNs may provide a biologically meaningful representation of residual axillary disease after NAC and support the hypothesis that target-node–based, de-escalation-oriented surgical algorithms merit further investigation, potentially offering a simplified, low-cost and widely accessible alternative to conventional localization techniques. To define the role of this approach within clinical pathways, prospective, multicenter, comparative studies incorporating different axillary stages and long-term outcomes are warranted. Conclusions In conclusion, carbon-tattooed TLNs demonstrated superior detection of residual axillary disease compared with BD, particularly in low-volume disease. These findings suggest that reliance on SLNB alone after NAC may lead to clinically relevant underestimation of residual disease. Target-node–based approaches may provide a more accurate and biologically relevant strategy for post-NAC axillary staging. Declarations Conflicts of Interest: The authors declare that they have no competing interests. Informed Consent Statement Written informed consent has been obtained from the patient(s) to publish this paper. Funding: No funding was received for this study. Author Contribution Conceptualization, P.B. and E.Y.; methodology, P.B., A.Ç. and E.Y.; software, P.B.; formal analysis, P.B. and P.A.; investigation, P.B., N.U., N.B.M. and A.Ç.; resources, P.B. and A.Ç.; data curation, P.B., N.U., N.B.M. and A.Ç.; writing—original draft preparation, P.B. and P.A.; writing—review and editing, P.B. and P.A.; visualization, P.A.; supervision, P.B. and A.Ç.; project administration, P.B. and E.Y. All authors have read and agreed to the published version of the manuscript. Acknowledgments: The authors would like to thank Istanbul Medipol University for providing the necessary facilities to conduct this study and for supporting the data collection process. The authors also extend their gratitude to the staff of the general surgery outpatient clinic for their assistance with patient coordination and administrative support throughout the study. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare no financial relationships or conflicts of interest related to this study. All authors confirm that they have no affiliations or financial involvement with any organization or entity that has a financial interest in or conflict with the subject matter discussed in this manuscript. Derived data supporting the findings of this study are available from the corresponding author PB on request. 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JAMA 310(14):1455–1461. 10.1001/jama.2013.278932 Boughey JC, Ballman KV, Le-Petross HT, McCall LM, Mittendorf EA, Ahrendt GM, Wilke LG, Taback B, Feliberti EC, Hunt KK (2016) Identification and Resection of Clipped Node Decreases the False-negative Rate of Sentinel Lymph Node Surgery in Patients Presenting With Node-positive Breast Cancer (T0-T4, N1-N2) Who Receive Neoadjuvant Chemotherapy: Results From ACOSOG Z1071 (Alliance). Ann Surg 263(4):802–807. 10.1097/SLA.0000000000001375 Caudle AS, Yang WT, Krishnamurthy S, Mittendorf EA, Black DM, Gilcrease MZ, Bedrosian I, Hobbs BP, DeSnyder SM, Hwang RF, Adrada BE, Shaitelman SF, Chavez-MacGregor M, Smith BD, Candelaria RP, Babiera GV, Dogan BE, Santiago L, Hunt KK, Kuerer HM (2016) Improved Axillary Evaluation Following Neoadjuvant Therapy for Patients With Node-Positive Breast Cancer Using Selective Evaluation of Clipped Nodes: Implementation of Targeted Axillary Dissection. J Clin Oncol 34(10):1072–1078. 10.1200/JCO.2015.64.0094 Kim EY, Byon WS, Lee KH, Yun JS, Park YL, Park CH, Youn IY, Choi SH, Choi YJ, Kook SH, Do SI (2018) Feasibility of Preoperative Axillary Lymph Node Marking with a Clip in Breast Cancer Patients Before Neoadjuvant Chemotherapy: A Preliminary Study. World J Surg 42(2):582–589. 10.1007/s00268-017-4171-8 Mittendorf EA, Caudle AS, Yang W, Krishnamurthy S, Shaitelman S, Chavez-MacGregor M, Woodward WA, Bedrosian I, Kuerer HM, Hunt KK (2014) Implementation of the american college of surgeons oncology group z1071 trial data in clinical practice: is there a way forward for sentinel lymph node dissection in clinically node-positive breast cancer patients treated with neoadjuvant chemotherapy? Ann Surg Oncol 21(8):2468–2473. 10.1245/s10434-014-3775-6 Zhu Y, Chen X, Zhang H, Chen L, Zhou S, Wu K, Wang Z, Kong L, Zhuang H (2016) Carbon nanoparticle-guided central lymph node dissection in clinically node-negative patients with papillary thyroid carcinoma. Head Neck 38(6):840–845. 10.1002/hed.24060 Milone M, Vignali A, Manigrasso M, Velotti N, Sarnelli G, Aprea G, De Simone G, Maione F, Gennarelli N, Elmore U, De Palma GD (2019) Sterile carbon particle suspension vs India ink for endoscopic tattooing of colonic lesions: a randomized controlled trial. Tech Coloproctol 23(11):1073–1078. 10.1007/s10151-019-02101-y Choy, N., Lipson, J., Porter, C., Ozawa, M., Kieryn, A., Pal, S., … Wapnir, I. (2015).Initial results with preoperative tattooing of biopsied axillary lymph nodes and correlation to sentinel lymph nodes in breast cancer patients. Annals of surgical oncology, 22, 377–382. doi.org/10.1245/s10434-014-4034-6 Hartmann S, Kühn T, de Boniface J, Stachs A, Winckelmann A, Frisell J, Wiklander-Bråkenhielm I, Stubert J, Gerber B, Reimer T (March 2021) Carbon tattooing for targeted lymph node biopsy after primary systemic therapy in breast cancer: prospective multicentre TATTOO trial. Br J Surg 108:302–307. https://doi.org/10.1093/bjs/znaa083 Natsiopoulos I, Intzes S, Liappis T, Zarampoukas K, Zarampoukas T, Zacharopoulou V, Papazisis K (2019) Axillary lymph node tattooing and targeted axillary dissection in breast cancer patients who presented as cN+ before neoadjuvant chemotherapy and became cN0 after treatment. Clin Breast Cancer 19(3):208–215. doi.org/10.1016/j.clbc.2019.01.013 Kim, W. H., Kim, H. J., Kim, S. H., Jung, J. H., Park, H. Y., Lee, J., … Lee, S. J.(2019). Ultrasound-guided dual-localization for axillary nodes before and after neoadjuvant chemotherapy with clip and activated charcoal in breast cancer patients: a feasibility study. BMC cancer, 19, 1–8. doi.org/10.1186/s12885-019-6095-1 Kuemmel, S., Heil, J., Rueland, A., Seiberling, C., Harrach, H., Schindowski, D.,… Reinisch, M. (2022). A prospective, multicenter registry study to evaluate the clinical feasibility of targeted axillary dissection (TAD) in node-positive breast cancer patients.Annals of surgery, 276(5), e553-e562. doi.org/10.1097/SLA.0000000000004572 Boileau, J. F., Poirier, B., Basik, M., Holloway, C. M., Gaboury, L., Sideris, L.,… Robidoux, A. (2015). Sentinel node biopsy after neoadjuvant chemotherapy in biopsy-proven node-positive breast cancer: the SN FNAC study. Journal of Clinical Oncology, 33(3), 258–264. doi.org/10.1200/JCO.2014.55.7827 Simons JM, van Nijnatten TJA, van der Pol CC et al (2019) Diagnostic accuracy of different surgical procedures for axillary staging after neoadjuvant systemic therapy in node-positive breast cancer: a systematic review and meta-analysis. Ann Surg 269(3):432–442. doi.org/10.1097/SLA.0000000000003075 Chehade HEH, Headon H, El Tokhy O, Heeney J, Kasem A, Mokbel K (2016) Is sentinel lymph node biopsy a viable alternative to complete axillary dissection following neoadjuvant chemotherapy in women with node-positive breast cancer at diagnosis? An updated meta-analysis involving 3,398 patients. Am J Surg 212(5):969–981 Pilewskie M, Morrow M (2017) Axillary nodal management following neoadjuvant chemotherapy: a review. JAMA Oncol 3(4):549–555. 10.1001/jamaoncol.2016.4163 Kuemmel, S., Heil, J., Bruzas, S., Breit, E., Schindowski, D., Harrach, H., … Reinisch,M. (2023). Safety of targeted axillary dissection after neoadjuvant therapy in patients with node-positive breast cancer. JAMA surgery, 158(8), 807–815. Kanesalingam, K., Sriram, N., Heilat, G., Ng, E. E., Meybodi, F., Elder, E., … French,J. (2020). Targeted axillary dissection after neoadjuvant systemic therapy in patients with node-positive breast cancer. ANZ journal of surgery, 90(3), 332–338. Pelizzari, G., Gerratana, L., Basile, D., Fanotto, V., Bartoletti, M., Liguori, A.,… Puglisi, F. (2019). Post-neoadjuvant strategies in breast cancer: From risk assessment to treatment escalation. Cancer Treatment Reviews, 72, 7–14. Yildirim E, Basim P, Ucar N, Bektas S, Iscen K, Karci E, Ozdemir AA (2024) Management of the Axilla After Neoadjuvant Chemotherapy: Can Axillary Needle Biopsy Replace Sentinel Node Biopsy? in vivo , 38 (5), 2523–2530 van Deurzen, C. H., Vriens, B. E., Tjan-Heijnen, V. C., van der Wall, E., Albregts,M., van Hilligersberg, R., … van Diest, P. J. (2009). Accuracy of sentinel node biopsy after neoadjuvant chemotherapy in breast cancer patients: a systematic review. European journal of cancer, 45(18), 3124–3130. Classe, J. M., Bordes, V., Campion, L., Mignotte, H., Dravet, F., Leveque, J., … Giard,S. (2009). Sentinel lymph node biopsy after neoadjuvant chemotherapy for advanced breast cancer: results of Ganglion Sentinelle et Chimiotherapie Neoadjuvante, a French prospective multicentric study. Journal of clinical oncology, 27(5), 726–732. Xing Y, Foy M, Cox DD, Kuerer HM, Hunt KK, Cormier JN (2006) Meta-analysis of sentinel lymph node biopsy after preoperative chemotherapy in patients with breast cancer. J Br Surg 93(5):539–546 Giuliano, A. E., Hunt, K. K., Ballman, K. V., Beitsch, P. D., Whitworth, P. W., Blumencranz,P. W., … Morrow, M. (2011). Axillary dissection vs no axillary dissection in women with invasive breast cancer and sentinel node metastasis: a randomized clinical trial.Jama, 305(6), 569–575. Galimberti, V., Cole, B. F., Zurrida, S., Viale, G., Luini, A., Veronesi, P., … Veronesi,U. (2013). Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23–01): a phase 3 randomised controlled trial. The lancet oncology, 14(4), 297–305. Giuliano, A. E., Ballman, K., McCall, L., Beitsch, P., Whitworth, P. W., Blumencranz,P., … Hunt, K. K. (2016). Locoregional recurrence after sentinel lymph node dissection with or without axillary dissection in patients with sentinel lymph node metastases:long-term follow-up from the American College of Surgeons Oncology Group (Alliance)ACOSOG Z0011 randomized trial. Annals of surgery, 264(3), 413–420. Galimberti, V., Cole, B. F., Viale, G., Veronesi, P., Vicini, E., Intra, M., … Forbes,J. (2018). Axillary dissection versus no axillary dissection in patients with breast cancer and sentinel-node micrometastases (IBCSG 23 – 01): 10-year follow-up of a randomised,controlled phase 3 trial. The Lancet Oncology, 19(10), 1385–1393. De Boer, M., Van Deurzen, C. H., Van Dijck, J. A., Borm, G. F., Van Diest, P. J.,Adang, E. M., … Tjan-Heijnen, V. C. (2009). Micrometastases or isolated tumor cells and the outcome of breast cancer. New England Journal of Medicine, 361(7), 653–663. Weaver, D. L., Ashikaga, T., Krag, D. N., Skelly, J. M., Anderson, S. J., Harlow,S. P., … Wolmark, N. (2011). Effect of occult metastases on survival in node-negative breast cancer. New England Journal of Medicine, 364(5), 412–421. Cortazar, P., Zhang, L., Untch, M., Mehta, K., Costantino, J. P., Wolmark, N., … Von Minckwitz, G. (2014). Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis. The Lancet, 384(9938), 164–172. Symmans, W. F., Wei, C., Gould, R., Yu, X., Zhang, Y., Liu, M., … Hortobagyi, G. N.(2017). Long-term prognostic risk after neoadjuvant chemotherapy associated with residual cancer burden and breast cancer subtype. Journal of Clinical Oncology, 35(10), 1049–1060. Xu S, Li Z, Xu M, Peng H (2020) The role of carbon nanoparticle in lymph node detection and parathyroid gland protection during thyroidectomy for non-anaplastic thyroid carcinoma-a meta-analysis. PLoS ONE, 15(11), e0223627 Wang L, Yang D, Lv JY, Yu D, Xin SJ (2017) Application of carbon nanoparticles in lymph node dissection and parathyroid protection during thyroid cancer surgeries: a systematic review and meta-analysis. OncoTargets therapy, 1247–1260 Nowak N, Dziedzic J, Nurczyk K, Zakoscielny A, Bury P, Zgodzinski W, Zinkiewicz K (2020) Application of endoscopic tattooing in intraoperative localization of colon tumours and sentinel lymph nodes. J Pre-Clinical Clin Res, 14 (4) Park, S., Koo, J. S., Kim, G. M., Sohn, J., Kim, S. I., Cho, Y. U., … Kim, E. K. (2018).Feasibility of charcoal tattooing of cytology-proven metastatic axillary lymph node at diagnosis and sentinel lymph node biopsy after neoadjuvant chemotherapy in breast cancer patients. Cancer research and treatment: official journal of Korean Cancer Association, 50(3), 801–812. Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Table4.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 21 Mar, 2026 Submission checks completed at journal 21 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9171161","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627448096,"identity":"c9aa8481-655d-4bf5-9420-c23cc92ad39c","order_by":0,"name":"Pelin Basım","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYFAC5oYDDAwJCQwSDIwPgFwePsJaGOFamA1AWtiI0cIA1cImAeIT1GLO3th4uKAmLY9/dvOzyq85djJsDMwPH93Ao8Wy52DD4RnHcool7hwzuy27LRnoMDZj4xw8WgxuJDYc5mGrSGy4kWB2W3IbM1ALD5s0Xi33HwK1/KtInH8j/Vux5LZ6IrTcYGw4zNuWk7jhRo4Z48dthwlrsewBOoy3L63Y8M6ZYmnGbcd52JgJ+MWc/fDhzzzfkvPkbrdv/PhzW7U9P3vzw8d4HYbMYeYBk3iUY2hh/EFA9SgYBaNgFIxMAAD1nE2o52G2mwAAAABJRU5ErkJggg==","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":true,"prefix":"","firstName":"Pelin","middleName":"","lastName":"Basım","suffix":""},{"id":627448098,"identity":"9c157ded-7eef-4e73-a061-da3be05adba7","order_by":1,"name":"Emine Yıldırım","email":"","orcid":"","institution":"Istanbul Atlas 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03:17:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18298,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9171161/v1/c81fd4243e4993d2e3f0202e.docx"},{"id":107870663,"identity":"45742f77-b44b-4195-b326-86f5e316ca7b","added_by":"auto","created_at":"2026-04-27 07:40:18","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15849,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9171161/v1/399388bd57ef4b73453b3f45.docx"},{"id":107853777,"identity":"7201c5f7-a2ef-49c1-a21f-19fa0245a121","added_by":"auto","created_at":"2026-04-27 03:17:19","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15954,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9171161/v1/48c0e9fbdd5bfa2823a7231a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCarbon-tattooed target lymph nodes improve detection of low-volume residual axillary disease after neoadjuvant chemotherapy \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn breast cancer (BC) surgery, the approach to the axilla is progressively evolving, emphasizing both the prevention of local recurrences and the determination of prognosis, while also prioritizing procedures that minimize morbidity. Although axillary lymph node dissection (ALND) has been used for a long time in traditional approaches to treatment, sentinel lymph node biopsy (SLNB), developed by Giuliano and Krag, has become the standard for early-stage clinically node-negative breast cancer [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In patients with initially node-positive disease (cN+), several studies are being conducted on axillary restaging and treatment following neoadjuvant chemotherapy (NAC) aimed at reducing tumor burden, with efforts to establish an optimal and standardized approach that could be universally applied across all patient groups. However, a critical limitation remains: SLNB may fail to detect low-volume residual axillary disease after NAC, particularly micrometastases (MicroM) and isolated tumor cells (ITC). Following NAC, the significant increase in axillary complete response rates, reaching up to 70%, has prompted investigations into the suitability of SLNB for locally advanced disease, as it allows for the avoidance of ALND and associated morbidities without negatively impacting survival or local recurrence when SLNB is negative. The selection of an axillary staging method that ensures high oncological safety while maintaining arm function and quality of life is still a subject of intense debate [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Subsequent studies have shown that in patients initially presenting with axillary lymph node metastasis (ALNM), SLNB can be safely performed in a selected group after NAC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the American College of Surgeons Oncology Group (ACOSOG) Z1071 trial, patients with ALNM confirmed by biopsy were marked with clips prior to NAC, and in the group where the clipped lymph node (cLN) was included in SLNB, the false negative rate (FNR) was found to be 6.8%. The same study reported that in patients whose cLN was not identified during SLNB and was instead included in ALND, the false negative rate increased to 19% [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this context, marking the metastatic LN prior to NAC and including it in the SLNB specimen becomes crucial. Targeted axillary dissection (TAD) is a procedure that involves SLNB, in which all sentinel lymph nodes (SLN) are removed along with the metastatic LN that was clipped prior to treatment. Numerous clinical studies have shown that TAD is a viable option to reduce false negative ALND in breast cancer patients following NAC (10). During TAD, the localization of the cLN prior to treatment is determined either by placing a guidewire under preoperative ultrasound (US) guidance or by implanting a radioactive seed [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In cases where an iodine-125 seed is implanted in the cLN, intraoperative localization is achieved using a radioactive probe. The cLN, along with all SLN, is subsequently excised as part of the TAD procedure [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. At present, the implementation of these procedures is constrained by factors such as high costs and the absence of the requisite equipment and nuclear medicine units in many centers. As a result, alternative methods that are more accessible and feasible are being explored for evaluating axillary response following NAC. Carbon nanoparticles, owing to their molecular dimensions, are particulate substances that can traverse lymphatic channels without penetrating blood vessels, and are utilized for lymph node visualization in thyroid surgery [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, sterile carbon particle suspension has long been used for the endoscopic marking of colonic lesions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Numerous studies on the use of the tattooing technique in BC patients, particularly those with suspected metastasis undergoing primary surgical treatment or in known metastatic LNs after NAC, report favorable opinions regarding the effectiveness of this method [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, patients with biopsy-proven 1\u0026ndash;3 metastatic axillary lymph nodes (N1) before NAC, who had metallic clip placement, underwent concurrent lymph node marking by injection of SPOT\u0026reg; Ex Endoscopic Tattoo into the LNs cortex. The primary objective of this study was to assess the correlation between tattooed lymph nodes (TLNs), labeled with carbon suspension, and SLNs as well as to evaluate the detection rate of TLNs during surgery. The secondary objective was to evaluate the impact of TLNs on surgical outcomes, including evaluating the efficacy of TLN in detecting axillary macrometastases (MacroM), MicroM, and ITC through frozen section examination and final pathological analysis. In other words, we evaluated post-NAC concordance between the SLNs and the previously marked metastatic LNs. Additionally, metastatic burden was categorized and analyzed based on MacroM, MicroM and ITC. Thus, the reliability and safety of carbon dye for permanent axillary marking as an alternative to blue dye (BD) in axillary staging were assessed.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design:\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e This prospective, two-center study was carried out between February 2023 and June 2025 and conducted to evaluate the feasibility of using carbon dye as an alternative to clips for marking metastatic lymph nodes prior to NAC in patients with locally advanced breast cancer and histopathologically confirmed axillary metastasis. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (Ethics Committee) of Istanbul Medipol University (approval number: E-10840098-202.3.02-1418; 19 February 2026) Written informed consent was obtained from all patients before participation in the study, in accordance with institutional and ethical guidelines. Patients were recruited and clinical data were collected at Istanbul Medipol University Hospital, Istanbul, T\u0026uuml;rkiye.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatients:\u003c/h3\u003e\n\u003cp\u003eA total of 83 female patients aged\u0026thinsp;\u0026ge;\u0026thinsp;18 years with BC and biopsy-proven ALNM (confirmed by fine-needle aspiration (FNA) or core needle biopsy (CNB) who received NAC as initial treatment for clinical stage T1\u0026ndash;3, N1, M0 disease were included in the study, regardless of breast cancer subtype or histopathological characteristics.\u003c/p\u003e \u003cp\u003eExclusion criteria included refusal to receive or complete NAC, disease progression during the ongoing treatment, a prior history of breast cancer or axillary surgery, as well as the presence of distant metastasis, clinical N2 and N3 disease, inflammatory breast cancer, pregnancy, and lactation.\u003c/p\u003e\n\u003ch3\u003eRadiological Evaluation:\u003c/h3\u003e\n\u003cp\u003eAll patients were evaluated prior to the NAC regimen, using mammography (MG), breast and axillary US, and magnetic resonance imaging (MRI). The US evaluations were performed using a Toshiba Aplio 500 ultrasound device with a 5\u0026ndash;14 MHz linear array probe and Toshiba software version 6.0 (Toshiba Corporation, Tokyo, Japan). LNs with lobulated contours, diffuse or asymmetric focal cortical thickness (\u0026gt;\u0026thinsp;3mm), anechoic/hypoechoic cortex compared to the subcutaneous tissue or obliterated cortex, and distorted fatty hilum or poorly visualized hilum were considered suspicious or malignant. For lymph nodes suspicious or appearing malignant in terms of metastasis, either an ultrasound-guided FNA or CNB was performed, depending on the size of the LNs. Multiple insertions were made into the thickest or focal thickened area of the LN cortex, and sufficient tissue samples were obtained.\u003c/p\u003e\n\u003ch3\u003ePre-NAC Marking of Lymph Node and Tattoo Technique:\u003c/h3\u003e\n\u003cp\u003eLNs found to be metastatic histopathologically underwent a second session for clip placement and carbon dye application. A marking procedure was performed on one to three LNs per patient, corresponding to the number of biopsy-proven metastatic nodes. In other words, all LNs diagnosed as metastatic, ranging from one to three in number, were marked with both clip placement and carbon tattoo technique prior to NAC. Clip placement was performed according to the number of pathologically confirmed metastatic lymph nodes using a Geotek titanium tissue-marking clip (Geotek Medical, Ankara, Turkey), which is routinely used in clinical practice in T\u0026uuml;rkiye. The same LNs were injected with a highly purified carbon suspension, carefully deposited into the cortical area of LN and the adjacent soft tissues, with the total volume injected being documented. If feasible, physicians had the option to record the distinct volumes injected into the LN cortex, the surrounding soft tissue, and the path extending from the node to the skin. The creation of a carbon suspension track from the LN to the skin surface, as well as the intentional tattooing of the overlying dermis, were optional steps. Spot\u0026reg; Ex Endoscopic Tattoo (GI supply,Mechanicsburg,USA) was utilized for this procedure.\u003c/p\u003e \u003cp\u003eBoth clip placement and carbon tattooing were initially performed in the same lymph nodes due to concerns regarding potential fading or intraoperative non-visualization of carbon during the prolonged NAC interval. However, as carbon staining was consistently identified in all clipped nodes, demonstrating complete concordance, subsequent analyses were based on carbon-tattooed target lymph nodes.\u003c/p\u003e\n\u003ch3\u003eWire localization of marker-clipped nodes:\u003c/h3\u003e\n\u003cp\u003eThe targeted clipped axillary LNs were localized using a flexible marking wire (20-G, 10-cm breast localization needle; Geotek Medical, Ankara, Turkey) under ultrasound or mammographic guidance, depending on clip visibility, 30 minutes to 1 hour before surgery. In cases where the clipped LNs were adjacent to each other, the use of a single wire was deemed appropriate. To facilitate easy identification of the wire-marked LNs and shorten the duration of the surgical procedure, mapping was performed by a radiologist with over 10 years of experience in breast radiology. The vertical and horizontal distances from the wire insertion point to the LNs were mapped using ultrasonography, and the closest potential incision line was marked on the skin using a marker pen.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAxillary surgery:\u003c/h2\u003e \u003cp\u003eIn all patients exhibiting either a partial or complete clinical axillary response, the SLN was mapped conventionally by using BD inoculated in the periareolar region. During surgery, BD LNs and TLNs were identified by visual examination, subsequently confirmed by pathological examination; on the other hand, the guidance of hook wire marking was utilized to identify clipped LNs. The time taken for the identification of three distinct groups of LNs, each marked using different methods, in the axillary region was recorded separately for each group as well as cumulatively. All blue-dyed, carbon-labeled-clipped LNs identified during surgery were sent to the pathology unit for frozen section analysis. Subsequently, all patients with macrometastasis (MacroM) detected underwent ALND. All excised LNs were classified based on the size of the metastatic foci as \u0026lt;\u0026thinsp;0.2 mm (isolated tumor cells, ITC), 0.2\u0026ndash;2 mm (micrometastasis-MicroM), and \u0026gt;\u0026thinsp;2 mm (MacroM).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDatabase setting:\u003c/h3\u003e\n\u003cp\u003eClinical and pathological data of the patients were retrieved from their medical records of the concerned hospitals. The variables of interest included age, body mass index (BMI), smoking status, menopause state, tumor size, greatest LN diameter, number of suspicious LNs, clinical T and N stage, radiological findings (MG, US and MRI), tumor type and subtype, presence of lymphvascular invasion, type of breast and axillary surgery, identification of blue-dyed, carbon-dyed and clipped LNs, intraoperative concordance of markings and axillary pathological response based on the presence and size of the metastatic foci with the marking technique used to identify it.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using IBM SPSS Statistics software (version 23; IBM Corp., Armonk, NY, USA). Paired comparisons between TLNs and blue dye detection rates were conducted using the McNemar exact test. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e3.1. Patient Characteristics\u003c/p\u003e \u003cp\u003eA total of 83 patients were included in the analysis. Among the study cohort, 48 patients (57.9%) were non-smokers and 35 (42.2%) were current smokers. Fifty patients (60.2%) were postmenopausal, while 33 (39.8%) were premenopausal.\u003c/p\u003e \u003cp\u003eOn preoperative axillary US, one suspicious LN was identified in 12 patients (14.5%), two suspicious LNs in 34 patients (41.0%), and \u0026ge;\u0026thinsp;3 suspicious LNs in 37 patients (44.6%) (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eBMI was comparable between patients with negative SLNB findings (25.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94) and those with metastatic SLNB involvement (25.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90). The mean age at surgery was 48.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.08 years in the SLNB-negative group and 50.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.48 years in the SLNB-positive group.\u003c/p\u003e \u003cp\u003ePrimary tumor diameter measured 27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;13.51 mm in patients without SLNB metastasis and 30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.28 mm in those with metastatic involvement. The greatest LN diameter on imaging was also similar between groups (19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82 mm vs. 18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.59 mm) (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e3.2. Sentinel Lymph Node Identification\u003c/p\u003e \u003cp\u003eBD successfully identified lymph nodes during SLNB in 62 patients (74.7%). In 7 patients (8.4%), LNs were identified only during subsequent ALND. In the remaining 14 patients (16.9%), BD failed to identify any lymph nodes during either SLNB or ALND.\u003c/p\u003e \u003cp\u003eIn contrast, TLNs were identified in all patients, achieving a 100% detection rate with complete one-to-one correspondence between injected and detected nodes.\u003c/p\u003e \u003cp\u003eWhen the entire cohort was evaluated regardless of metastatic status, intraoperative concordance between TLNs and BD was observed in 47 patients (56.6%), whereas no concordance was observed in 36 patients (43.4%) (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e3.3. SLNB Metastatic Findings\u003c/p\u003e \u003cp\u003eSLNB demonstrated metastatic involvement in 42 patients (50.6%), while no metastasis was detected in 41 patients (49.4%).\u003c/p\u003e \u003cp\u003eMacroM was absent in 41 patients (49.4%). Among patients with MacroM (n\u0026thinsp;=\u0026thinsp;42), detection was achieved exclusively with TLNs in 15 patients (35.7%), while combined detection with both TLNs and BD was observed in 27 patients (64.3%).\u003c/p\u003e \u003cp\u003eMicroM was absent in 53 patients (63.9%). Among MicroM-positive patients (n\u0026thinsp;=\u0026thinsp;30), 12 cases (40.0%) were detected exclusively with TLNs, whereas 18 cases (60.0%) were detected by both TLNs and BD.\u003c/p\u003e \u003cp\u003eITCs were absent in 42 patients (50.6%). Among ITC-positive patients (n\u0026thinsp;=\u0026thinsp;41), detection was achieved solely by TLNs in 27 cases (65.9%), while combined TLN and BD detection was observed in 14 cases (34.1%) (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e3.4. Detection Rates According to Metastatic Burden\u003c/p\u003e \u003cp\u003eTLNs demonstrated complete detection of metastatic lymph nodes across all pathological subtypes. All MacroM (n\u0026thinsp;=\u0026thinsp;42), MicroM (n\u0026thinsp;=\u0026thinsp;30), and ITC-positive LNs (n\u0026thinsp;=\u0026thinsp;41) were successfully identified using TLNs, corresponding to a detection rate of 100% in each metastasis category.\u003c/p\u003e \u003cp\u003eIn contrast, BD identified a lower proportion of metastatic disease. BD positivity was observed in 27 MacroM (64.3%), 18 MicroM (60.0%), and 14 ITCs (34.1%). Detection rates decreased with lower-volume metastatic burden, with the lowest detection observed in LNs containing isolated tumor cells (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e3.5. Paired Comparison of TLNs and BD Detection\u003c/p\u003e \u003cp\u003eA paired comparison of TLN and BD detection rates was performed using the McNemar exact test (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eFor macrometastasis, TLNs identified 15 metastatic nodes not detected by BD, while no nodes were detected exclusively by BD, resulting in a statistically significant difference (p\u0026thinsp;=\u0026thinsp;0.000061).\u003c/p\u003e \u003cp\u003eSimilarly, for micrometastasis, TLNs detected 12 nodes that were BD-negative, with no cases of BD-only detection (p\u0026thinsp;=\u0026thinsp;0.000488).\u003c/p\u003e \u003cp\u003eFor LNs containing ITCs, TLNs identified 27 additional metastatic nodes not detected by BD, again with no BD-only detection, yielding a highly significant difference favoring TLNs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00000001).\u003c/p\u003e \u003cp\u003eImportantly, no metastatic LN was detected by BD alone.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAxillary lymph node involvement remains one of the most significant prognostic indicators in BC and plays a crucial role in determining both locoregional management and systemic therapeutic approach. In patients with node-positive disease, neoadjuvant chemotherapy is commonly employed and has been shown to induce notably encouraging nodal pCR rates, ranging from 40% to 75%. TAD, first described by Caudle et al., is an innovative approach based on marking LNs with biopsy-proven metastasis prior to systemic therapy and subsequently removing these targeted nodes in combination with conventional SLNB during surgery. This strategy enables the retrieval of originally pre-treatment metastatic nodes, thereby allowing a more accurate assessment of axillary tumor burden, reducing false-negative rates, and optimizing pathological axillary staging [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The findings of the present study suggest that, following NAC, TLNs may demonstrate a very high sensitivity for the detection of residual metastatic disease. In our patient cohort, regardless of metastatic burden, all metastatic LNs were successfully identified through TLNs across all pathological subgroups, including MacroM, MicroM and ITCs. In contrast, using the blue dye technique, only 64.3% of MacroM, 60.0% of MicroM, and 34.1% of LNs containing ITCs were detected. Notably, in 35.7% of patients with MacroM and in 40.0% of those with MicroM, metastatic LNs were identified exclusively by TLNs and were not visualized using blue dye. Collectively, these findings suggest that permanent LN marking with carbon may represent a feasible and potentially promising approach to reliably localize target LNs and enhance the accuracy of axillary staging after NAC.\u003c/p\u003e \u003cp\u003eThe ACOSOG Z1071 trial evaluated the reliability of SLNB after NAC in patients with biopsy-proven node-positive BC and demonstrated that false-negative rates remained clinically relevant despite the use of different SLNB techniques [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Secondary analyses of the same trial further showed that inclusion of the pre-treatment clipped LN along with the conventional sentinel LN specimen significantly reduced the false-negative rate, whereas failure to retrieve the targeted metastatic LN was associated with a substantially increased risk of false-negative results [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings underscore that a key determinant of post-NAC axillary evaluation is whether the LN known to be metastatic at diagnosis is successfully removed. Similarly, the SENTINA study reported that in patients initially presenting with node-positive disease, the performance of SLNB after NAC was highly dependent on technical factors and that false-negative rates remained clinically significant [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The SN FNAC trial also demonstrated that reliable removal of biopsy-proven metastatic LNs improved the accuracy of axillary staging; however, these LNs did not consistently correspond to the conventional sentinel LN basin, limiting the reliability of standard SLNB approaches [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These observations have been supported by several meta-analyses and systematic reviews, which emphasized that SLNB after NAC in initially node-positive patients is associated with variable and, at times, unacceptably high false-negative rates [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, multiple studies addressing on selective evaluation of clipped or marked LNs have consistently shown that successful retrieval of the pre-treatment targeted LN improves axillary staging accuracy and significantly reduces false-negative rates [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this context, the finding in the present study that all metastatic LNs, across all metastatic burden subgroups, were identified through TLNs suggests that this approach may represent a practical alternative for reliable localization of target LNs after NAC. The lack of requirement for radioactive seeds or specialized nuclear medicine infrastructure may further enhance its clinical applicability; however, its impact on false-negative rates and oncologic outcomes should be validated in future comparative studies. From a broader perspective, pre-treatment carbon tattooing may not only aim to maintain oncologic safety but could also be regarded as a low-cost adaptation of the targeted axillary approach; in this regard, it raises the possibility of a cost-effectiveness advantage, particularly in resource-limited healthcare settings.\u003c/p\u003e \u003cp\u003eIn the post-neoadjuvant chemotherapy (NAC) setting, the reliability of conventional sentinel lymph node biopsy (SLNB), and particularly of surgical approaches based solely on blue dye mapping, remains controversial for both physiological and technical reasons. After NAC, lymphatic drainage pathways may be altered by fibrosis, obliteration, and tumor regression, potentially resulting in failure of the true metastatic lymph node to exhibit sentinel behavior; moreover, the possibility of skip metastases should not be overlooked [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Indeed, large prospective series and meta-analyses have demonstrated that in patients initially presenting with node-positive disease, false-negative rates of SLNB after NAC are variable and at times exceed clinically acceptable thresholds [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The SENTINA and SN FNAC trials further emphasized that sentinel lymph nodes do not always reliably represent residual metastatic disease and that, despite technical success, the biologically relevant \u0026ldquo;true target node\u0026rdquo; may be missed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, blue dye identified only 64.3% of MacroM, 60.0% of MicroM, and 34.1% of ITCs, whereas all metastatic LNs across all subgroups were identified through TLNs. Moreover, in 35.7% of patients with MacroM and in 40.0% of those with MicroM, metastatic LN were detected exclusively via TLNs, suggesting a clinically meaningful discordance between sentinel negativity and true axillary response. These findings support the need to question the biological validity of the \u0026ldquo;sentinel\u0026rdquo; concept after NAC and underscore the importance of approaches based on direct evaluation of the target LN [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this context, whether an SLNB that does not include the target node can truly provide biologically accurate axillary staging after NAC warrants critical consideration. Accordingly, the post-NAC axillary approach invites renewed debate as to whether technical success in sentinel node identification or biological representation of the initially involved LN should be regarded as the principal determinant of accurate staging.\u003c/p\u003e \u003cp\u003eThe clinical and biological significance of residual low-volume axillary disease (MicroM and ITCs) detected during surgery after NAC remains controversial and is often regarded by many authors as \u0026ldquo;incidental\u0026rdquo;; nevertheless, these findings continue to have implications for the definition of pCR, axillary staging, adjuvant treatment decisions, and regional radiotherapy planning [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although the impact of MicroMs\u0026mdash;and particularly ITCs\u0026mdash;on long-term survival has not yet been clearly established, several high-quality studies suggest that this disease burden may reflect residual tumor biology and may carry prognostic relevance in selected subgroups [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Accurate detection of low-volume axillary disease is therefore of particular importance, especially for the appropriate definition pCR after NAC. However, conventional SLNB has demonstrated notable limitations in identifying low-volume disease, with ITCs and MicroM representing the most frequently missed forms of residual nodal metastasis in the post-NAC setting [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the present study, blue dye mapping identified only 34.1% of ITCs and 60.0% of MicroM, consistent with the existing literature; in contrast, carbon-tattooed lymph nodes enabled detection of both MicroM and ITCs in 100% of cases, a finding that is particularly noteworthy. It should be acknowledged that restriction of the study population to patients with N1 disease may have contributed to these results. Nevertheless, these findings raise the possibility that failure to detect low-volume residual disease could lead to excessive axillary downstaging and false-negative interpretations of pCR. At a time when treatment paradigms are increasingly shifting toward biologically driven decision-making and de-escalation strategies, the clinical relevance of accurately identifying low-volume disease is likely to become even more pronounced [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this context, the potential role of TLNs in reliably detecting low-volume metastatic disease and contributing to future residual disease\u0026ndash;based risk stratification warrants further investigation.\u003c/p\u003e \u003cp\u003eTLN marking relies on a labeling technique characterized by favorable physicochemical properties and biological compatibility, permitting long-term stability within the lymphatic system without significant migration. This characteristic allows reliable visual identification of the target LN during surgery even after months-long NAC protocols, with a low risk of fading or displacement, as may occasionally occur with metallic clips. The low cost of carbon tattooing, the absence of radiation exposure, the lack of requirement for specialized detection devices or nuclear medicine infrastructure, and the fact that no additional operating room equipment is needed constitute major advantages that enhance its feasibility and accessibility, particularly in developing countries and in healthcare systems with economic constraints. Compared with other targeting techniques based on metallic clip placement, carbon marking may also be less invasive and more cost-effective, potentially facilitating broader clinical implementation. The literature indicates that carbon particles have been safely used for LNs mapping in thyroid surgery, have long been established as a standard technique for endoscopic marking of colorectal lesions, and that an increasing number of studies with relatively large series support their feasibility in breast surgery, both in the primary setting and for post-NAC axillary evaluation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These studies have demonstrated that carbon can persist in local tissues for extended periods without inducing significant inflammatory reactions and can facilitate intraoperative visual localization. The findings of the present study suggest that these technical advantages may translate into clinically meaningful benefits; in particular, the reliable detection of TLNs in patients with one to three suspicious or biopsy-proven metastatic nodes indicates that this approach may contribute not only to theoretical feasibility but also to practical and effective localization of target LNs, including the reliable identification of low-burden axillary metastatic disease, namely MicroM and ITC. In this context, carbon tattooing may be considered a potential tool to enhance the applicability of target-node\u0026ndash;based axillary approaches, especially in centers with limited access to nuclear medicine facilities. Nevertheless, randomized controlled trials stratified according to clinical axillary stage are required to further clarify the role of this method within clinical algorithms.\u003c/p\u003e \u003cp\u003eThe findings of the present study prompt reconsideration of whether post-NAC axillary surgery should be driven primarily by technical success in sentinel LN identification or by reliable removal of the biologically relevant target LN. When the target node can be consistently localized, the possibility of more minimal, more targeted, and potentially less morbid axillary surgical strategies after NAC warrants discussion. Nevertheless, several limitations of this study must be acknowledged. The relatively small sample size, the two-center, non-randomized design, and the restriction to patients with clinical N1 disease (1\u0026ndash;3 suspicious or biopsy-proven lymph nodes) may limit the generalizability of our findings. Furthermore, the lack of direct comparison with other target-node localization techniques, such as radioactive seeds, magnetic markers, or radioisotope-guided approaches, precludes conclusions regarding superiority or equivalence. Our analysis focused on short-term surgical and pathological outcomes; long-term oncologic endpoints, including axillary recurrence, disease-free survival, overall survival, and functional outcomes, were not assessed. In addition, the injection technique, ultrasonographic assessment, surgical exploration, and pathological evaluation are operator-dependent processes that may influence reproducibility across different centers. Despite these limitations, our results suggest that carbon-targeted LNs may provide a biologically meaningful representation of residual axillary disease after NAC and support the hypothesis that target-node\u0026ndash;based, de-escalation-oriented surgical algorithms merit further investigation, potentially offering a simplified, low-cost and widely accessible alternative to conventional localization techniques. To define the role of this approach within clinical pathways, prospective, multicenter, comparative studies incorporating different axillary stages and long-term outcomes are warranted.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, carbon-tattooed TLNs demonstrated superior detection of residual axillary disease compared with BD, particularly in low-volume disease. These findings suggest that reliance on SLNB alone after NAC may lead to clinically relevant underestimation of residual disease. Target-node\u0026ndash;based approaches may provide a more accurate and biologically relevant strategy for post-NAC axillary staging.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eInformed Consent Statement\u003c/h2\u003e \u003cp\u003e\u003cb\u003e\u003c/b\u003eWritten informed consent has been obtained from the patient(s) to publish this paper.\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eNo funding was received for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, P.B. and E.Y.; methodology, P.B., A.\u0026Ccedil;. and E.Y.; software, P.B.; formal analysis, P.B. and P.A.; investigation, P.B., N.U., N.B.M. and A.\u0026Ccedil;.; resources, P.B. and A.\u0026Ccedil;.; data curation, P.B., N.U., N.B.M. and A.\u0026Ccedil;.; writing\u0026mdash;original draft preparation, P.B. and P.A.; writing\u0026mdash;review and editing, P.B. and P.A.; visualization, P.A.; supervision, P.B. and A.\u0026Ccedil;.; project administration, P.B. and E.Y. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Istanbul Medipol University for providing the necessary facilities to conduct this study and for supporting the data collection process. The authors also extend their gratitude to the staff of the general surgery outpatient clinic for their assistance with patient coordination and administrative support throughout the study. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare no financial relationships or conflicts of interest related to this study. All authors confirm that they have no affiliations or financial involvement with any organization or entity that has a financial interest in or conflict with the subject matter discussed in this manuscript. Derived data supporting the findings of this study are available from the corresponding author PB on request.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data presented in this study are available from the corresponding author upon reasonable request. The data are not publicly available due to institutional ethical regulations and patient privacy considerations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiuliano AE, Kirgan DM, Guenther JM, Morton DL (1994) Lymphatic mapping and sentinel lymphadenectomy for breast cancer. 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Long-term prognostic risk after neoadjuvant chemotherapy associated with residual cancer burden and breast cancer subtype. Journal of Clinical Oncology, 35(10), 1049\u0026ndash;1060.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu S, Li Z, Xu M, Peng H (2020) The role of carbon nanoparticle in lymph node detection and parathyroid gland protection during thyroidectomy for non-anaplastic thyroid carcinoma-a meta-analysis. PLoS ONE, 15(11), e0223627\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Yang D, Lv JY, Yu D, Xin SJ (2017) Application of carbon nanoparticles in lymph node dissection and parathyroid protection during thyroid cancer surgeries: a systematic review and meta-analysis. OncoTargets therapy, 1247\u0026ndash;1260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNowak N, Dziedzic J, Nurczyk K, Zakoscielny A, Bury P, Zgodzinski W, Zinkiewicz K (2020) Application of endoscopic tattooing in intraoperative localization of colon tumours and sentinel lymph nodes. J Pre-Clinical Clin Res, \u003cem\u003e14\u003c/em\u003e(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, S., Koo, J. S., Kim, G. M., Sohn, J., Kim, S. I., Cho, Y. U., \u0026hellip; Kim, E. K. (2018).Feasibility of charcoal tattooing of cytology-proven metastatic axillary lymph node at diagnosis and sentinel lymph node biopsy after neoadjuvant chemotherapy in breast cancer patients. Cancer research and treatment: official journal of Korean Cancer Association, 50(3), 801\u0026ndash;812.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research-and-treatment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brea","sideBox":"Learn more about [Breast Cancer Research and Treatment](https://www.springer.com/journal/10549)","snPcode":"10549","submissionUrl":"https://submission.nature.com/new-submission/10549/3","title":"Breast Cancer Research and Treatment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carbon tattooing, Blue Dye, Targeted axillary dissection, Macrometastasis","lastPublishedDoi":"10.21203/rs.3.rs-9171161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9171161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eAccurate axillary staging after neoadjuvant chemotherapy (NAC) remains essential in breast cancer surgery. Low-volume residual axillary disease is frequently missed after neoadjuvant chemotherapy (NAC), raising concerns about the reliability of sentinel lymph node biopsy (SLNB). This study evaluated the detection performance of carbon-tattooed target lymph nodes (TLNs) compared with conventional blue dye (BD) mapping during axillary staging, particularly in the context of low-volume metastatic disease.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEighty-three breast cancer (BC) patients undergoing SLNB after preoperative ultrasound-guided carbon injection of suspicious axillary lymph nodes were prospectively analyzed. Detection rates of TLNs and BD were assessed across metastasis categories including macrometastasis (MacroM), micrometastasis (MicroM), and isolated tumor cells (ITCs). Paired comparisons were performed using the McNemar exact test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTLNs were identified in all patients, achieving a 100% detection rate. BD detected sentinel lymph nodes in 62 patients (74.7%), failed in 14 (16.9%), and required ALND-only identification in 7 (8.4%). TLNs detected all metastatic nodes, including MacroM (n\u0026thinsp;=\u0026thinsp;42), MicroM (n\u0026thinsp;=\u0026thinsp;30), and ITCs (n\u0026thinsp;=\u0026thinsp;41). In contrast, BD detected 64.3% of MacroM, 60.0% of MicroM, and 34.1% of ITCs. McNemar analysis demonstrated significantly higher detection with TLNs for MacroM (p\u0026thinsp;=\u0026thinsp;0.000061), MicroM (p\u0026thinsp;=\u0026thinsp;0.000488), and ITCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00000001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCarbon-tattooed TLN mapping showed superior detection of metastatic lymph nodes compared with BD, particularly in low-volume disease after NAC.\u003c/p\u003e","manuscriptTitle":"Carbon-tattooed target lymph nodes improve detection of low-volume residual axillary disease after neoadjuvant chemotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 03:17:11","doi":"10.21203/rs.3.rs-9171161/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-19T16:28:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-21T08:38:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-21T08:37:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer Research and Treatment","date":"2026-03-19T15:09:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research-and-treatment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brea","sideBox":"Learn more about [Breast Cancer Research and Treatment](https://www.springer.com/journal/10549)","snPcode":"10549","submissionUrl":"https://submission.nature.com/new-submission/10549/3","title":"Breast Cancer Research and Treatment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8e9c94b-4f24-4c6c-83d5-d126aa721e81","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T03:17:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 03:17:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9171161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9171161","identity":"rs-9171161","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}