Outcomes of Methylene Blue Single-Dye Sentinel Lymph Node Biopsy in Early Breast Cancer: A Retrospective Cohort Study from a Resource-Limited Tertiary Centre

Outcomes of Methylene Blue Single-Dye Sentinel Lymph Node Biopsy in Early Breast Cancer: A Retrospective Cohort Study from a Resource-Limited Tertiary Centre | 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 Outcomes of Methylene Blue Single-Dye Sentinel Lymph Node Biopsy in Early Breast Cancer: A Retrospective Cohort Study from a Resource-Limited Tertiary Centre Vathsal Bandaranayake, Amila Prathibha Nellihela, Jayanjana Asanthi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9328110/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 Background Sentinel lymph node biopsy (SLNB) is the standard method of axillary staging in clinically node-negative early breast cancer and is commonly performed using dual-tracer mapping with radioisotope and blue dye. However, access to nuclear medicine facilities is limited in many low- and middle-income countries (LMICs), restricting widespread implementation. This study evaluated the technical performance, oncologic safety, and economic implications of methylene blue (MB) single-dye SLNB in a resource-limited tertiary oncology centre. Methods A retrospective cohort study included 115 consecutive patients with clinically and radiologically node-negative early breast cancer who underwent MB single-dye SLNB between 2019 and 2023. Sentinel nodes were identified using subcutaneous injection of 2–3 mL of 1% methylene blue. Primary outcomes were sentinel node identification rate and axillary recurrence among SLNB-negative patients. The sentinel lymph node identification rate was defined as the proportion of procedures in which at least one sentinel lymph node was successfully identified. Secondary outcomes included nodal yield, additional nodal burden, recurrence-free survival, and tracer-related cost comparison with dual-tracer SLNB using published micro-costing data. Statistical analyses included Chi-square testing, independent t-tests or Mann–Whitney U tests, and Kaplan–Meier survival analysis. Results Sentinel nodes were successfully identified in 113 of 115 patients (98.3%). The mean number of sentinel lymph nodes retrieved was 3.25 ± 0.16 per patient. Nodal metastases were detected in 40 patients (34.8%). No isolated axillary recurrences were observed among SLNB-negative patients during follow-up (0/75; 95% CI 0–4.0%). Overall recurrence occurred in 5 patients (4.3%) over a mean follow-up of 36.3 months. No methylene blue-related adverse reactions were recorded. The estimated tracer cost per patient was approximately US $ 0.50 for MB single-dye mapping compared with US $ 135–221 for dual-tracer techniques, representing a reduction exceeding 99% in tracer-related costs. Conclusions MB single-dye SLNB achieved high identification rates with favourable short- to mid-term oncologic outcomes while providing substantial cost savings. These findings support MB single-dye SLNB as a safe, feasible, and economically sustainable approach to axillary staging in resource-limited settings where dual-tracer techniques are not readily available. Sentinel lymph node biopsy methylene blue single-dye technique breast neoplasms axillary staging low- and middle-income countries Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Sentinel lymph node biopsy (SLNB) has become the standard approach for axillary staging in clinically node-negative early breast cancer, replacing routine axillary lymph node dissection (ALND) in most patients [ 1 , 2 ]. By selectively identifying and excising the first draining lymph node(s), SLNB provides accurate nodal staging while significantly reducing surgical morbidity, including lymphedema and shoulder dysfunction. Landmark randomized trials, including those by Veronesi et al. and the ALMANAC study, demonstrated equivalent oncologic outcomes between SLNB and ALND in node-negative disease, with superior postoperative quality of life following SLNB [ 1 , 2 ]. In high-resource settings, SLNB is typically performed using a dual-tracer technique combining a radiolabeled colloid and a blue dye. Dual mapping improves sentinel node identification rates and reduces false-negative rates (FNRs) compared with either modality alone [ 3 , 4 ]. Meta-analyses report identification rates exceeding 95% and FNRs generally below 5–8% when dual tracers are used in experienced centres [ 3 , 4 ]. The most recent American Society of Clinical Oncology (ASCO) guideline update continues to recommend dual-tracer mapping where feasible and emphasizes adherence to established quality benchmarks to ensure oncologic reliability [ 5 ]. However, dual-tracer SLNB requires nuclear medicine infrastructure, radiopharmaceutical supply chains, gamma probes, and trained personnel. These requirements increase cost and logistical complexity and may not be available in many healthcare systems, particularly in low- and middle-income countries (LMICs) [ 6 ]. Regulatory constraints related to radioactive handling further limit widespread adoption. While blue dyes such as isosulfan blue are commonly used adjunctively, they carry a small but clinically relevant risk of anaphylaxis. In contrast, methylene blue (MB) is inexpensive, widely accessible, and does not require specialized equipment, making it an attractive single mapping agent in resource-constrained environments [ 6 ]. Consequently, many centres in LMICs perform SLNB using 1% methylene blue alone as a pragmatic adaptation to limited infrastructure. This approach enables broader access to axillary staging while avoiding the morbidity associated with routine ALND. Nonetheless, concerns persist regarding the diagnostic accuracy of single-dye techniques. Earlier pooled analyses suggested that blue dye–only mapping achieved identification rates of approximately 85–91% and FNRs approaching 10–13%, exceeding commonly accepted surgical quality thresholds [ 3 , 4 ]. A systematic review of MB-only SLNB reported a pooled identification rate of 91% but highlighted variability in FNR across studies [ 4 ]. Despite these concerns, accumulating contemporary data suggest that outcomes with MB-only SLNB may improve with structured technique and surgical experience. A large retrospective series from China reported identification rates approaching 96% using methylene blue alone [ 8 ]. Studies from Nigeria, India, Malaysia, and other LMIC settings have demonstrated identification rates between 85% and 92%, with acceptable sensitivity for nodal metastasis detection [ 7 , 9 – 11 ]. Several of these reports describe improvements in performance metrics with increased operator experience and retrieval of multiple sentinel nodes, suggesting that technique standardization may narrow the performance gap between single- and dual-tracer methods [ 9 – 11 ]. Oncologic safety remains the critical benchmark. Long-term data from major SLNB trials indicate that axillary recurrence after a negative SLNB is uncommon, typically below 2%, and comparable to outcomes following ALND [ 1 , 2 , 12 ]. Although most of these data derive from dual-tracer cohorts, contemporary MB-only series have not demonstrated excess axillary relapse when appropriate patient selection and surgical protocols are applied [ 8 – 11 ]. These findings suggest that, in clinically node-negative early breast cancer, carefully executed single-dye SLNB may provide acceptable regional control. The relevance of this issue extends beyond technical considerations. Breast cancer incidence continues to rise across LMICs, where healthcare systems often face limited access to nuclear medicine services. In such contexts, omission of SLNB due to lack of radiotracers may lead either to unnecessary ALND with associated morbidity or to inadequate nodal staging. A validated, low-cost single-dye protocol could therefore facilitate guideline-concordant care while minimizing treatment-related harm. Nevertheless, high-quality real-world data from LMIC tertiary centres remain limited, and variability in technique, follow-up duration, and reporting standards complicates interpretation of existing literature. Few studies have evaluated identification rate, false-negative rate, nodal burden, clinicopathologic correlates, and recurrence outcomes within a standardized institutional protocol. In this context, we conducted a retrospective cohort study evaluating methylene blue–guided SLNB performed as the sole mapping technique in clinically and radiologically node-negative early breast cancer at a tertiary referral centre in Sri Lanka. We assessed sentinel node identification rate, nodal burden, clinicopathologic predictors of sentinel node positivity, and short- to mid-term oncologic outcomes, including axillary recurrence. By situating these findings within the broader global literature, we aim to clarify the feasibility and oncologic safety of MB-only SLNB in a resource-limited setting. Methods Study Design and Setting This retrospective cohort study was conducted at the Surgical Oncology Unit, Teaching Hospital Anuradhapura (THA), Sri Lanka, a tertiary referral centre serving a predominantly rural population. The study period extended from December 2019 to August 2023. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, and the Institutional Review Board of Teaching Hospital Anuradhapura (Ref. No. ERC/2024/22, dated July 15, 2024). As this study involved retrospective analysis of anonymized medical records without direct patient contact, the requirement for individual informed consent was formally waived. All eligible consecutive patients during the study period were included in order to minimise selection bias and to reflect real-world clinical practice. A summary of patient screening, exclusions, sentinel lymph node biopsy (SLNB) procedures, follow-up, and the final analysed cohort is presented in Fig. 1 . Flow diagram illustrating patient screening (n = 170), exclusions (n = 35), patients undergoing methylene blue–guided SLNB (n = 135), loss to follow-up (n = 20), and final analysed cohort (n = 115). Frozen section results and subsequent axillary management are also shown. Participants A total of 170 patients with invasive breast carcinoma were initially assessed for eligibility during the study period. Clinically node-negative (cN0) status was defined as: Absence of palpable axillary lymphadenopathy on clinical examination, and No suspicious axillary lymph nodes on preoperative ultrasonography. All patients underwent standard breast imaging with mammography and ultrasound. Breast MRI was performed selectively when clinically indicated due to resource limitations. Thirty-five patients were excluded for the following reasons: Biopsy-proven axillary nodal metastasis (cN1 or higher) Multifocal or multicentric invasive lobular carcinoma requiring separate mapping injections Locally advanced disease not suitable for SLNB Prior ipsilateral breast or axillary surgery Receipt of neoadjuvant chemotherapy or radiotherapy After exclusions, 135 consecutive eligible patients underwent primary breast surgery with sentinel lymph node biopsy (SLNB) using 1% methylene blue as the sole mapping agent. Of the 135 patients who underwent SLNB, 20 (14.8%) were excluded from final outcome analysis due to incomplete follow-up data, relocation, or failure to attend postoperative oncology surveillance clinics. No direct patient contact was attempted in accordance with ethics approval. The final study cohort consisted of 115 patients included in the outcome analysis (Fig. 1 ). Sample Size This study was designed as a retrospective consecutive cohort; therefore, no a priori sample size was specified. All eligible consecutive patients undergoing sentinel lymph node biopsy (SLNB) using methylene blue single-dye technique between December 2019 and August 2023 at Teaching Hospital Anuradhapura were included. The final analysed cohort comprised patients with complete clinical records and adequate follow-up data. The precision of key outcome estimates was assessed using 95% confidence intervals. SLNB Surgical Technique All procedures were performed under general anesthesia by consultant surgical oncologists specializing in breast surgery, using a standardized institutional protocol. After induction, 2–3 mL of 1% methylene blue was injected subcutaneously in the periareolar or peritumoral region, according to tumor location. The injection site was gently massaged for 2–5 minutes to facilitate lymphatic uptake as demonstrated in Fig. 2 . An axillary incision was then made, and the blue-stained lymphatic channels were traced toward the axilla. All blue-stained lymph nodes and any palpably suspicious nodes were excised. An intraoperative view of a blue-dyed sentinel lymph node during axillary dissection is presented in Fig. 3 , and the harvested sentinel lymph node with adjacent lymph nodes following methylene blue injection is illustrated in Fig. 4 . In cases where no blue-stained lymphatic channels or nodes were identified intraoperatively, sentinel node mapping was considered unsuccessful. In such instances, limited axillary sampling was performed at the discretion of the operating surgeon. Sentinel Lymph Node Pathology and Axillary Management Excised sentinel lymph nodes were immediately sent for intraoperative frozen section analysis. The histopathology laboratory and on-call pathologist were pre-notified before surgery to ensure availability. Sentinel lymph node biopsy was performed first, and while awaiting frozen section results definitive breast tumour resection was undertaken. If metastatic disease was identified on frozen section analysis, immediate completion axillary lymph node dissection (ALND) involving Levels I and II was performed during the same operation. Patients had been counselled preoperatively and consented for possible completion ALND if sentinel lymph node metastasis was detected. When frozen section analysis was negative, no additional axillary surgery was performed. Final paraffin-embedded histology confirmed sentinel lymph node status. Pathological Assessment Histopathologic evaluation was performed using hematoxylin–eosin staining, with immunohistochemistry of all tumour and axillary dissections including sentinel samples. Pathological staging followed the Tumour–Node–Metastasis (TNM) classification of the American Joint Committee on Cancer (AJCC), 8th edition [ 13 ]. Tumour grading was performed using the Nottingham histological grading system (Elston–Ellis grading system) [ 14 ]. Recorded clinicopathological variables included tumour size, histological subtype, tumour grade, lymphovascular invasion (LVI), extranodal extension, ooestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2) status, Ki-67 proliferation index, number and status of sentinel lymph nodes, axillary lymph node dissection findings when performed, and adjuvant treatment details. Outcomes and Definitions Primary outcomes were sentinel node identification rate and axillary recurrence among SLNB-negative patients. Sentinel lymph node identification rate was defined as the proportion of patients in whom at least one sentinel lymph node was successfully identified. Axillary safety was assessed using the axillary recurrence rate among sentinel lymph node–negative patients during follow-up. Because completion axillary lymph node dissection was performed only in patients with positive intraoperative sentinel lymph node findings, a classical false-negative rate defined against axillary dissection as a reference standard could not be calculated. Axillary safety was therefore assessed clinically using the axillary recurrence rate among sentinel lymph node–negative patients. Secondary outcomes included nodal yield, additional nodal burden, recurrence-free survival, and tracer-related cost comparison with dual-tracer SLNB using published micro-costing data. Cost Analysis Tracer-related costs were analysed using a cost-minimisation approach, restricted to consumables directly attributable to the mapping strategy. For methylene blue (MB) single-dye SLNB, per-patient tracer cost was calculated from institutional procurement records for 2026 and included the volume of 1% MB used per case (2–3 mL) and injection materials (syringe and needle). As dual-tracer SLNB is not routinely performed at our institution, comparator tracer costs for technetium-99m sulfur colloid plus blue dye mapping were obtained from a published micro-costing study (Khadka et al.) reporting per-patient tracer costs using institutional and market radiotracer pricing (2020 USD) [ 15 ]. Costs were adjusted from 2020 to 2026 United States Dollars (USD) using the United States Consumer Price Index (CPI) inflation adjustment. Currency conversion was based on the prevailing exchange rate at the time of analysis. The analysis focused on tracer-related costs; operating theatre time, staffing, anaesthesia, and pathology costs were assumed to be comparable between mapping strategies and were therefore not included. Data Collection and Follow-Up Data were extracted retrospectively from operative records, pathology reports, and oncology clinic documentation. Variables recorded included patient age, tumour characteristics, pathological findings, nodal status, and adjuvant treatments including chemotherapy, radiotherapy, endocrine therapy, and targeted therapy. Patients were followed in surgical oncology and medical oncology outpatient clinics according to institutional protocol. Surveillance consisted of clinical examination every six months and annual mammography with breast ultrasonography. Additional imaging including computed tomography or bone scintigraphy was performed when clinically indicated. Recurrence was classified as locoregional recurrence involving the breast, chest wall, or ipsilateral axilla, or as distant metastasis. The mean follow-up duration for the analysed cohort was 36.3 months (range 12–84 months). Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA) [ 16 ]. Continuous variables were presented as means with standard deviations or medians with ranges as appropriate. Categorical variables were summarised using frequencies and percentages. Associations between sentinel lymph node positivity and clinicopathological variables were analysed using the Chi-square test or Fisher’s exact test for categorical variables and the independent t-test or Mann–Whitney U test for continuous variables, as appropriate. Correlations between clinicopathological variables were assessed using Pearson’s correlation coefficient. Recurrence-free survival was estimated using the Kaplan–Meier method and compared using the log-rank test. All statistical tests were two-sided and a p-value < 0.05 was considered statistically significant. Missing data were not imputed. Descriptive outcomes were reported with 95% confidence intervals to reflect the precision of estimates in this retrospective cohort. Results Patient and tumour characteristics A total of 115 clinically node-negative women with invasive breast carcinoma underwent sentinel lymph node biopsy (SLNB) using the methylene blue single-dye technique between December 2019 and August 2023. The mean age at diagnosis was 55.7 ± 10.2 years (median 53.5; range 35–81). Wide local excision was performed in 48 patients (43.6%), mastectomy in 44 patients (40.0%), and other breast-conserving procedures in the remainder. The mean primary tumour size on preoperative imaging was 36.8 ± 29.7 mm (median 28 mm; range 10–180 mm). Tumour size data were available for 110 patients (70 SLNB-negative and 40 SLNB-positive). There was no statistically significant difference in tumour size between SLNB-positive and SLNB-negative patients (Mann–Whitney U test, p > 0.05; Fig. 5 ). Box-and-whisker plots showing preoperative primary tumour size (mm) in SLNB-negative (n = 70) and SLNB-positive (n = 40) patients. There was no statistically significant difference in tumour size between groups (Mann–Whitney U test, p > 0.05). Sentinel lymph node biopsy performance The sentinel node identification rate was 98.3% (113/115). In two patients (1.7%), methylene blue injection did not result in visible axillary lymphatic staining, and sentinel nodes could not be identified. These patients underwent limited axillary sampling. Among patients in whom sentinel nodes were successfully identified (n = 113), the mean number of sentinel lymph nodes retrieved per patient was 3.25 ± 0.16. SLNB was positive in 40/115 patients (34.8%), while 75/115 (65.2%) had negative sentinel nodes. Among the 40 patients with positive sentinel lymph nodes, macrometastases were identified in 33 (82.5%), micrometastases in 5 (12.5%), and isolated tumour cells in 2 (5.0%). Lymphovascular invasion demonstrated a statistically significant association with sentinel lymph node positivity (χ² test, p < 0.001). In contrast, HER2 receptor status (p = 0.50) and molecular subtype classification (p = 0.51) were not significantly associated with sentinel node metastasis. Axillary Lymph Node Dissection (ALND) Outcomes All 40/40 (100%) patients with positive sentinel lymph nodes underwent completion axillary lymph node dissection (ALND). The mean number of additional axillary lymph nodes retrieved was 12.75 ± 4.9. Additional non-sentinel nodal metastases were identified in 21/40 patients (52.5%), whereas 19/40 patients (47.5%) had no further positive nodes beyond the sentinel lymph nodes. Among patients with additional axillary metastases (n = 21), the majority demonstrated limited residual nodal burden: 12/21 (57.1%) had fewer than five additional positive nodes, 6/21 (28.6%) had five to nine positive nodes, and 3/21 (14.3%) had ten or more positive nodes (Fig. 7 ). Bar chart demonstrating the number of additional metastatic axillary lymph nodes identified at ALND. Nineteen patients (47.5%) had no further nodal metastases beyond the sentinel lymph nodes, while 21 patients (52.5%) had additional positive nodes. Among those with additional metastases, the majority demonstrated limited residual nodal burden, with most patients having fewer than five additional positive nodes. Histopathological Characteristics Invasive ductal carcinoma of no special type (NST) was the predominant histological subtype, identified in 94/115 patients (81.7%). Other histological subtypes included invasive lobular carcinoma in 4/115 (3.5%), solid papillary carcinoma in 1/115 (0.9%), other rare variants in 8/115 (7.0%), and pure ductal carcinoma in situ (DCIS) without invasive disease in 8/115 (7.0%). A DCIS component coexisting with invasive carcinoma was present in 32/115 patients (27.8%). Lymphovascular invasion (LVI) was identified in 30/115 cases (26.1%), extranodal extension in 26/115 (22.6%), and Paget’s disease in 8/115 (7.0%). Clinical and Pathological Staging On initial clinical staging (n = 115), 23/115 (20.0%) patients were classified as Stage I, 51/115 (44.3%) as Stage II, and 32/115 (27.8%) as Stage III. No patients met criteria for Stage IV disease at presentation. Clinical tumour category based on imaging demonstrated a predominance of cT2 lesions. On final pathological assessment (n = 115), tumour category was pT1 in 30/115 (26.1%), pT2 in 64/115 (55.7%), pT3 in 11/115 (9.6%), and pT4 in 1/115 (0.9%). Pathological nodal staging showed pN0 in 64/115 (55.7%), pN1 (including micrometastatic disease) in 31/115 (27.0%), pN2 in 5/115 (4.3%), and pN3 in 4/115 (3.5%). According to AJCC 8th edition pathological stage grouping, the majority of cases were Stage I–II, with a smaller proportion classified as Stage III; no patients were Stage IV. Among SLNB-positive patients, micro metastases accounted for 5/40 (12.5%). Hormone Receptor and Molecular Subtypes Ooestrogen receptor (ER) expression was positive in 72/115 patients (62.6%), progesterone receptor (PR) in 51/115 (44.3%), and HER2 overexpression in 34/115 (29.6%). Based on ER, PR, and HER2 status, molecular subtypes were classified as follows: luminal (HER2-negative) in 60/115 patients (52.2%), luminal/HER2-positive in 27/115 (23.5%), HER2-enriched in 22/115 (19.1%), and triple-negative in 6/115 (5.2%). The Ki-67 proliferation index was high (> 30%) in 61/115 tumours (53.0%), intermediate (16–30%) in 19/115 (16.5%), and low (≤ 15%) in 35/115 (30.4%). There was no statistically significant association between Ki-67 category and SLNB positivity (χ² test, p > 0.05; Fig. 6 ). Adjuvant Treatment Over a mean follow-up period of 36.3 months (median 36; range 12–84 months), adjuvant therapy was administered according to tumour stage, nodal status, and receptor profile. Adjuvant chemotherapy was administered to 45 of 115 patients (39.1%), with a mean of 5.1 ± 0.9 cycles. Regimens were predominantly anthracycline-based combinations (doxorubicin plus cyclophosphamide), commonly followed by a taxane. Adjuvant radiotherapy was delivered to 49 of 115 patients (42.6%), with regional nodal irradiation including the axilla administered in five cases. Oestrogen receptor positivity was identified in 64 of 115 patients (55.7%). Among these, endocrine therapy was documented in 54 of 64 patients (84.4%). The most commonly prescribed agents were tamoxifen and aromatase inhibitors. In the remaining ER-positive cases, documentation of endocrine therapy was incomplete in the retrospective medical records. Recurrence and Survival Outcomes During a mean follow-up of 36.3 months (range 12–84 months), disease recurrence occurred in 5 of 115 patients (4.3%). Locoregional axillary recurrence was observed in 2 patients (2/115, 1.7%), both within the SLNB-positive group following completion axillary lymph node dissection (ALND). Distant metastasis occurred in 3 patients (3/115, 2.6%), including two from the SLNB-negative group and one from the SLNB-positive group. Recurrence occurred between 0.75 and 4.6 years after surgery No isolated axillary recurrences were observed among SLNB-negative patients during follow-up (0/75; 0%, 95% CI 0–4.0%). The timing and anatomical distribution of relapse are illustrated in Fig. 8 . Gantt-style plot illustrating time to recurrence for the five patients (5/115, 4.3%) who developed disease recurrence during follow-up, stratified by SLNB status and site of recurrence (axillary locoregional versus distant metastasis). Kaplan–Meier analysis demonstrated high recurrence-free survival (RFS) across the cohort. There was no statistically significant difference in RFS between SLNB-positive and SLNB-negative patients (log-rank p > 0.05; Fig. 9 ). When stratified by molecular subtype, luminal A and luminal B tumours demonstrated numerically higher RFS compared with HER2-enriched and triple-negative subtypes; however, comparison between luminal (A/B) and non-luminal tumours did not reach statistical significance (log-rank p = 0.5819). Similarly, patients with Grade 1–2 tumours showed numerically improved RFS compared with those with Grade 3 disease, but this difference was not statistically significant (log-rank p = 0.3425). Kaplan–Meier curves comparing RFS between SLNB-negative and SLNB-positive patients during follow-up. Only five recurrence events occurred, and no statistically significant difference between groups was observed on log-rank testing. Cost comparison The tracer-related cost of MB single-dye SLNB was LKR 330 per patient, equivalent to approximately US $ 1.10 in 2026. In the published micro-costing study used for comparison, per-patient tracer costs for dual-tracer SLNB (technetium-99m sulfur colloid plus blue dye) were US $ 109.04 (institutional radiotracer pricing) and US $ 178.27 (market pricing) in 2020 [ 15 ], corresponding to approximately US $ 135 and US $ 221 after inflation adjustment to 2026 USD. All costs are reported in 2026 United States Dollars (USD). The resulting incremental tracer cost difference between MB single-dye and dual-tracer SLNB was approximately US $ 134–220 per patient, representing an approximate 99% reduction in tracer-related expenditure with MB-only mapping. Applied to the 115-patient cohort, this corresponded to an estimated cumulative tracer-related cost saving of approximately US $ 15,400–25,300 (2026 USD). Discussion This retrospective cohort study evaluated the performance and oncologic safety of methylene blue (MB) single-dye sentinel lymph node biopsy (SLNB) in clinically and radiologically node-negative early breast cancer within a resource-limited tertiary oncology centre. The principal findings were: (i) a high sentinel node identification rate of 98.3%, comparable to the upper range reported in methylene blue–only SLNB series (85–96%), with mapping failure in two patients (1.7%), (ii) a mean retrieval of 3.25 ± 0.16 SLNs per patient, (iii) nodal metastases identified in 40 of 115 patients (34.8%), and (iv) no isolated axillary recurrences among SLNB-negative patients during follow-up (0/75; 95% CI 0–4.0%). Overall recurrence occurred in 4.3% of patients over a mean follow-up of 36.3 months. In addition to favourable oncologic outcomes, methylene blue single-dye SLNB demonstrated a marked economic advantage compared with dual-tracer techniques. Tracer-specific cost analysis showed substantial per-patient savings, highlighting the potential value of MB-only SLNB in resource-limited healthcare systems [ 16 , 17 ]. Context Within Established SLNB Evidence SLNB is established as the standard axillary staging procedure for clinically node-negative breast cancer, having demonstrated equivalent survival and low axillary recurrence compared with routine ALND while significantly reducing morbidity [ 1 , 2 , 12 ]. In high-resource settings, dual-tracer mapping (radioisotope plus blue dye) is commonly used to optimise identification and minimise false-negative results [ 3 , 5 ]. However, reliance on nuclear medicine infrastructure limits the scalability of dual-tracer techniques in many low- and middle-income countries (LMICs). A systematic review and meta-analysis of MB-only SLNB reported pooled identification rates of approximately 90%, with variability across institutions [ 4 ]. Several single-centre studies from LMIC settings have reported identification rates ranging from approximately 85% to the mid-90% range [ 7 – 11 ]. The identification rate observed in the present cohort lies at the upper end of previously reported MB-only series. The small proportion of mapping failures observed (1.7%) is consistent with published methylene blue single-dye series reporting identification rates between 85% and 96% [ 4 , 7 – 11 ], and highlights the importance of surgical experience and standardized injection technique. Table 1 compares key SLNB performance metrics from the present cohort with representative dual-tracer and methylene blue single-dye series reported in global and LMIC settings. The mean nodal yield observed in this study falls within the optimal range reported in both mapping strategies, supporting adequate staging reliability. These findings suggest that, within a structured protocol and experienced surgical setting, MB single-dye SLNB can achieve technical outcomes comparable to dual-tracer approaches while maintaining feasibility in resource-constrained environments. Parameter Dual-Tracer (Radioisotope + Dye) MB Single-Dye (Global/LMIC Data) MB Single-Dye (Current Sri Lankan Cohort) Sentinel Node Identification Rate >95% 85–95% 98.3% (113/115) Mean SLNs Retrieved 2–4 1–3 3.25 ± 0.16 Axillary Recurrence After Negative SLNB <1–2% Rare 0% (0/75; 95% CI 0–4.0%) Dye-Related Complications Rare (allergic reactions reported with isosulfan/patent blue) Very low None observed Resource Requirements Nuclear medicine facility + gamma probe Low-resource setting feasible Low-resource setting feasible (routine practice) Abbreviations: SLNB, sentinel lymph node biopsy; MB, methylene blue. Table 1 . Comparative overview of sentinel lymph node biopsy (SLNB) performance metrics in dual-tracer and methylene blue (MB) single-dye series compared with the current cohort. Footnote: Comparative data summarised from representative international and LMIC series [2–4,8–12]. Retrieval of multiple sentinel nodes has been shown to improve staging accuracy and reduce the likelihood of missed nodal metastases [17]. Because completion ALND was not performed in SLNB-negative patients, a classical ALND-referenced false-negative rate could not be calculated. Axillary safety was therefore assessed clinically through follow-up, using axillary recurrence as a surrogate endpoint. Although the limited number of events precludes definitive conclusions regarding diagnostic sensitivity, the observed outcomes are consistent with the low axillary recurrence rates reported in landmark SLNB trials [1,2,12]. Axillary Disease Burden and Surgical Implications Among SLNB-positive patients who underwent completion ALND, additional non-sentinel nodal involvement was identified in a subset, but overall residual nodal burden was generally low. This pattern mirrors observations from large SLNB trials demonstrating that, in many early-stage patients, the majority of axillary disease is captured at the sentinel node level [1,2,12]. Contemporary studies evaluating axillary de-escalation strategies further highlight the evolving paradigm of risk-adapted axillary management [18]. Accurate and reproducible SLNB therefore remains central to modern axillary decision-making. Survival Outcomes and Exploratory Analyses Over a mean follow-up of approximately three years, recurrence occurred in 4.3% of patients. Kaplan–Meier analyses demonstrated high recurrence-free survival with no statistically significant subgroup differences. Subgroup comparisons by molecular subtype and tumour grade were similarly non-significant. These analyses should be interpreted cautiously because only five recurrence events occurred, limiting statistical power and increasing the risk of type II error. Longer follow-up will be required to more robustly evaluate survival outcomes. Real-World Healthcare Disruption and System Constraints The study period overlapped with the COVID-19 pandemic and national economic disruption in Sri Lanka, during which treatment delays and intermittent medication shortages occurred. The use of retrospective paper-based records may have contributed to incomplete documentation of adjuvant therapies, reflecting systemic constraints typical of resource-limited healthcare settings. Feasibility and Health System Relevance in LMIC Settings Methylene blue offers practical advantages in resource-limited environments. It is inexpensive, widely available, chemically stable, and does not require nuclear medicine facilities or radiopharmaceutical handling protocols [6,9,10]. No local tissue complications, allergic reactions, or systemic adverse events related to methylene blue injection were observed in this series, supporting the clinical safety of low-volume methylene blue use for sentinel node mapping. Tracer-specific cost analysis demonstrated a substantial reduction in expenditure compared with published dual-tracer micro-costing data [15]. Although broader perioperative costs were assumed to be comparable between mapping strategies, the principal cost differential reflects radiopharmaceutical procurement and nuclear medicine infrastructure. In settings where technetium-99m is unavailable or financially constrained, MB-only SLNB may represent a pragmatic and scalable alternative, facilitating guideline-concordant axillary staging while avoiding routine ALND-related morbidity [1,2,9,11]. While direct equivalence to dual-tracer techniques cannot be confirmed without a contemporaneous comparator group, the technical performance and early oncologic outcomes observed in this study support the feasibility of MB-only SLNB within a structured institutional protocol. Study Limitations This study has several limitations. The retrospective single-centre design introduces potential selection bias and limits generalisability. Although the calculated minimum sample size was not fully achieved due to loss to follow-up, the primary technical outcome—sentinel node identification rate—was estimated with acceptable precision. However, the study was not powered to detect small differences in recurrence or survival outcomes. The limited number of recurrence events and moderate follow-up duration restrict interpretation of long-term oncologic safety, particularly regarding late axillary relapse. Clinical data were derived from paper-based records, and incomplete documentation of adjuvant therapy cannot be excluded. The economic comparison was restricted to tracer-related costs and utilised published micro-costing data for the dual-tracer comparator. Accordingly, findings should be interpreted as tracer-specific cost minimisation rather than a comprehensive health economic evaluation. Finally, results from a high-volume tertiary centre using a standardised protocol may not be directly generalisable to lower-volume institutions. Strengths and Practical Impact This study reflects consecutive real-world practice within a defined institutional pathway using a standardised MB injection technique and systematic pathological assessment. With longitudinal follow-up exceeding three years, the findings provide context-relevant evidence supporting MB single-dye SLNB as a safe and economically rational axillary staging strategy in resource-constrained settings. Conclusion In this resource-limited tertiary oncology setting, methylene blue single-dye sentinel lymph node biopsy achieved a high identification rate with reliable nodal staging and no isolated axillary recurrence among SLNB-negative patients during follow-up. The technique demonstrated favourable short- to mid-term oncologic outcomes while offering substantial cost advantages over dual-tracer mapping. These findings support MB single-dye SLNB as a safe, accessible, and economically sustainable axillary staging strategy in healthcare systems with limited nuclear medicine infrastructure. Prospective multicentre studies with longer follow-up are warranted to confirm long-term oncologic equivalence. Declarations Ethics approval and consent to participate The study was approved by the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka and the Institutional Review Board of Teaching Hospital Anuradhapura (Ref. No. ERC/2024/22). The requirement for individual informed consent was formally waived due to the retrospective analysis of anonymised medical records. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that there are no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors and was personally funded by the principal author and corresponding author. Authors' contributions Vathsal Chinathaka Bandara : Conceptualisation, Funding, Methodology, Formal Analysis, Investigation, Supervision, Validation, Writing – Original Draft, Review & Editing. Amila Prathibha Nellihela : Conceptualisation, Funding, Methodology, Formal Analysis, Investigation, Project Administration, Statistical Analysis, Writing – Original Draft, Review & Editing. Jayanjana Asanthi : Supervision, Methodology, Validation, Writing – Review & Editing. Aruna Wijesooriya : Investigation, Data Collection, Review & Editing Ruchika Nirmalie Senevirathne Jayalathge : Formal Analysis, Investigation, Writing – Original Draft, Writing – Review & Editing. Aruna Buddhika Jayasena : Investigation, Data Collection, Review & Editing Sulakshi Karunanayake : Data Collection, Investigation, Review & Editing Pirahanthan Karunanithy : Data Collection, Investigation Abheetha Abegunasekara : Data Collection, Investigation Tharindu Jayasinghe : Data Collection, Investigation References Veronesi U, Paganelli G, Viale G, Luini A, Zurrida S, Galimberti V, et al. A randomized comparison of sentinel-node biopsy with routine axillary dissection in breast cancer. N Engl J Med. 2003;349(6):546–53. 10.1056/NEJMoa012782 . Mansel RE, Fallowfield L, Kissin M, Goyal A, Newcombe RG, Dixon JM, et al. Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC trial. J Natl Cancer Inst. 2006;98(9):599–609. 10.1093/jnci/djj158 . Niebling MG, Pleijhuis RG, Bastiaannet E, Brouwers AH, van Dam GM, Hoekstra HJ. A systematic review and meta-analysis of sentinel lymph node identification in breast cancer and melanoma: a plea for tracer mapping. Eur J Surg Oncol. 2016;42(4):466–73. Li J, Chen X, Qi M, Li Y. Sentinel lymph node biopsy mapped with methylene blue dye alone in patients with breast cancer: a systematic review and meta-analysis. PLoS ONE. 2018;13(9):e0204364. Park KU, Somerfield MR, Anne N, Brackstone M, Conlin AK, Couto HL, et al. Sentinel lymph node biopsy in early-stage breast cancer: ASCO guideline update. J Clin Oncol. 2025;43(14):1720–41. Dogan NU, Dogan S, Favero G, Köhler C, Dürsün P. The basics of sentinel lymph node biopsy: anatomical and pathophysiological considerations and clinical aspects. J Oncol. 2019;2019:3415630. Okoye CL, Ezeome ER. Use of methylene blue dye for lymphatic basin mapping and sentinel lymph node biopsy in breast cancer patients in Enugu, Nigeria. Niger J Clin Pract. 2022;25(11):1805–11. Yang S, Xiang HY, Xin L, Zhang H, Zhang S, Cheng YJ, et al. Retrospective analysis of sentinel lymph node biopsy using methylene blue dye for early breast cancer. Chin Med J (Engl). 2021;134(3):318–25. Vasudevan R, Jayalakshmi TS, Rajan S, et al. Sentinel lymph node biopsy using methylene blue dye in early breast cancer: a single-center experience from India. Indian J Surg Oncol. 2022;13(4):627–34. Subramaniam S, Wong SL, Pathmanathan S, et al. Methylene blue dye as a single agent for sentinel lymph node mapping in breast cancer: a Malaysian multicenter experience. Asian J Surg. 2018;41(3):251–6. Balasubramanian R, Mishra A, Verma GR. Evaluation of sentinel lymph node biopsy using methylene blue dye in carcinoma breast: experience at a tertiary care center in South India. Indian J Cancer. 2019;56(1):51–5. Krag DN, Anderson SJ, Julian TB, Brown AM, Harlow SP, Ashikaga T, et al. Technical outcomes of sentinel-lymph-node resection and conventional axillary-lymph-node dissection in patients with clinically node-negative breast cancer: results from the NSABP B-32 randomized phase III trial. Lancet Oncol. 2007;8(10):881–8. 10.1016/S1470-2045(07)70278-4 . Amin MB, Edge SB, Greene FL, Byrd DR, Brookland RK, Washington MK, et al. editors. AJCC Cancer Staging Manual. 8th ed. New York: Springer; 2017. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. Histopathology. 1991;19(5):403–10. 10.1111/j.1365-2559.1991.tb00229.x . Khadka S, Suresh J, Prem A, Mishra PR, Kataria K, Dhar A, et al. Sentinel node mapping in early breast cancer: a randomized comparison of fluorescein-guided technique with technetium-99m sulfur colloid plus methylene blue. Indian J Nucl Med. 2022;37(3):236–44. IBM Corp. IBM SPSS Statistics for Windows, Version 25.0. Armonk (NY): IBM Corp; 2017. Goyal A, Newcombe RG, Mansel RE, et al. Clinical relevance of multiple sentinel nodes in patients with breast cancer. Br J Surg. 2005;92(4):438–42. 10.1002/bjs.4906 . Tinterri C, Gentilini O, Veronesi U, et al. De-escalation of axillary surgery in clinically node-positive breast cancer patients treated with neoadjuvant therapy: comparative long-term outcomes of SLNB versus ALND. Cancers (Basel). 2023;16(18):3168. 10.3390/cancers16183168 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 01 May, 2026 Editor assigned by journal 12 Apr, 2026 Submission checks completed at journal 06 Apr, 2026 First submitted to journal 05 Apr, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9328110","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635521225,"identity":"3a3994eb-8bfc-4010-bec3-e3ca06a700b5","order_by":0,"name":"Vathsal Bandaranayake","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Vathsal","middleName":"","lastName":"Bandaranayake","suffix":""},{"id":635521226,"identity":"77fb22d1-5eda-401e-90e7-9a7472c51709","order_by":1,"name":"Amila Prathibha Nellihela","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYLCCBwUSCQzsDIwPgGwePqK0JBgAtTAzMBuAtLARqYUBpIVNAsQhqIV/du/BBwkGFnn8zMzPKr/m2MmwMTA/fHQDjxaJO+eSDYAOK5ZsZjO7LbstGegwNmPjHHzW3MgxkwBqSdxwmMHstuQ2ZqAWHjZpfFrkb+SY/wBp2X+Y/Vux5LZ6wloMgLYwgG1h5jFj/LjtMGEthnfOGIMdNuMwT7E047bjPGzMBPwid7vH8MOHirrE/vb2jR9/bqu252dvfvgYr/clkNjMPGASn3J0LYw/CKkeBaNgFIyCEQkA1aBCXLcRbacAAAAASUVORK5CYII=","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":true,"prefix":"","firstName":"Amila","middleName":"Prathibha","lastName":"Nellihela","suffix":""},{"id":635521227,"identity":"480c0b20-4682-4948-89fa-47f738146801","order_by":2,"name":"Jayanjana Asanthi","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Jayanjana","middleName":"","lastName":"Asanthi","suffix":""},{"id":635521232,"identity":"8c436a8b-f224-4604-a1fe-5be7fe606128","order_by":3,"name":"Aruna Wijesooriya","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Aruna","middleName":"","lastName":"Wijesooriya","suffix":""},{"id":635521235,"identity":"945a057b-227d-496b-b30d-c78036aed223","order_by":4,"name":"Ruchika Nirmalie Senevirathne","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Ruchika","middleName":"Nirmalie","lastName":"Senevirathne","suffix":""},{"id":635521237,"identity":"f0686bcf-2398-4247-b75f-0ec2a2a42c3a","order_by":5,"name":"Aruna Buddhika Jayasena","email":"","orcid":"","institution":"University of Sri Jayawardhanapura","correspondingAuthor":false,"prefix":"","firstName":"Aruna","middleName":"Buddhika","lastName":"Jayasena","suffix":""},{"id":635521251,"identity":"e03a3641-4f5d-4363-8988-faf235e5059e","order_by":6,"name":"Sulakshi Karunanayke","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Sulakshi","middleName":"","lastName":"Karunanayke","suffix":""},{"id":635521261,"identity":"28505b3b-7ea0-4915-b2cb-cde4c206b05b","order_by":7,"name":"Pirahanthan Karunanithy","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Pirahanthan","middleName":"","lastName":"Karunanithy","suffix":""},{"id":635521262,"identity":"0c48a7e5-48fe-4538-a023-6f2d7d0e82d7","order_by":8,"name":"Abheetha Abegunasekara","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Abheetha","middleName":"","lastName":"Abegunasekara","suffix":""},{"id":635521263,"identity":"9ce071e5-3bdb-4b36-82fb-b3bdd964fd36","order_by":9,"name":"Tharindu Jayasinghe","email":"","orcid":"","institution":"Teaching Hospital Anuradhapura","correspondingAuthor":false,"prefix":"","firstName":"Tharindu","middleName":"","lastName":"Jayasinghe","suffix":""}],"badges":[],"createdAt":"2026-04-05 19:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9328110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9328110/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109014397,"identity":"192059ec-b96a-4c7c-a240-30a4b61e4651","added_by":"auto","created_at":"2026-05-11 17:19:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eStudy flow diagram of patient selection and outcomes. \u003cbr\u003e\nFlow diagram illustrating patient screening (n=170), exclusions (n=35), patients undergoing methylene blue–guided SLNB (n=135), loss to follow-up (n=20), and final analysed cohort (n=115). Frozen section results and subsequent axillary management are also shown.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/9d67f59f0c0467235a81a5c5.png"},{"id":109014400,"identity":"6a2034bd-e832-4241-b01e-1737cba344ac","added_by":"auto","created_at":"2026-05-11 17:19:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMethylene blue being injected into the periareolar region of the left breast, with the tumor location marked in the upper inner quadrant.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/30dc40aecbfd7f7f0112a0a8.jpeg"},{"id":109068090,"identity":"3ee2ac6c-6d6b-4a8f-8467-cba287053389","added_by":"auto","created_at":"2026-05-12 10:03:32","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1647242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIntraoperative view of a blue-dyed sentinel lymph node during axillary dissection (blue arrow), with the left axilla indicated by the yellow arrow.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/728a58805e96a3b9925359b4.jpeg"},{"id":109068212,"identity":"0b7cd8bc-202c-4cea-93b5-86fdbfec918d","added_by":"auto","created_at":"2026-05-12 10:04:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":118654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHarvested sentinel lymph node with adjacent lymph nodes; the yellow arrow indicates the blue-stained sentinel node following methylene blue injection\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/a89d255e0c42ee41dd6e14c7.jpeg"},{"id":109067780,"identity":"6b4f1a75-1527-4825-88d9-015ea28595e7","added_by":"auto","created_at":"2026-05-12 10:00:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":106124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDistribution of primary tumour size according to sentinel lymph node biopsy (SLNB) status.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBox-and-whisker plots showing preoperative primary tumour size (mm) in SLNB-negative (n = 70) and SLNB-positive (n = 40) patients. There was no statistically significant difference in tumour size between groups (Mann–Whitney U test, p \u0026gt; 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/fedd3ea488fc23ad0c0d0e68.png"},{"id":109068085,"identity":"1ea08701-911b-4614-b5ca-0dd5d63d5a99","added_by":"auto","created_at":"2026-05-12 10:03:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":117209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDistribution of SLNB-positive and SLNB-negative patients according to Ki-67 proliferation index categories (low ≤15%, intermediate 16–30%, high \u0026gt;30%). No statistically significant association was observed between Ki-67 category and sentinel lymph node positivity (χ² test, p \u0026gt; 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/9d6e40a57577361162c83395.png"},{"id":109068184,"identity":"19f11a6f-2934-4d5a-9a00-702034f05b4a","added_by":"auto","created_at":"2026-05-12 10:04:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDistribution of additional metastatic axillary lymph nodes identified at completion axillary lymph node dissection (ALND) in patients with positive sentinel lymph nodes (n = 40).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBar chart demonstrating the number of additional metastatic axillary lymph nodes identified at ALND. Nineteen patients (47.5%) had no further nodal metastases beyond the sentinel lymph nodes, while 21 patients (52.5%) had additional positive nodes. Among those with additional metastases, the majority demonstrated limited residual nodal burden, with most patients having fewer than five additional positive nodes.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/fd1201c625a8abffed357477.png"},{"id":109068171,"identity":"1e84fdaa-0041-491b-94ee-64fd8089a106","added_by":"auto","created_at":"2026-05-12 10:04:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":173306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTiming and pattern of recurrence following methylene blue single-dye sentinel lymph node biopsy (SLNB).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGantt-style plot illustrating time to recurrence for the five patients (5/115, 4.3%) who developed disease recurrence during follow-up, stratified by SLNB status and site of recurrence (axillary locoregional versus distant metastasis).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/73f33a04ef2ea7889963f8ac.png"},{"id":109067567,"identity":"30b4bc75-33a1-4a36-b214-ff82a92db9ac","added_by":"auto","created_at":"2026-05-12 09:56:21","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":104317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRecurrence-free survival (RFS) by sentinel lymph node biopsy (SLNB) status.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKaplan–Meier curves comparing RFS between SLNB-negative and SLNB-positive patients during follow-up. Only five recurrence events occurred, and no statistically significant difference between groups was observed on log-rank testing.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/d60b1a37faec2f5de39e4863.png"},{"id":109081875,"identity":"e87a3295-386b-44b2-8cc3-ea77b1229193","added_by":"auto","created_at":"2026-05-12 12:29:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2774973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9328110/v1/dff70f4b-ac61-4675-80ab-04e23e826c4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Outcomes of Methylene Blue Single-Dye Sentinel Lymph Node Biopsy in Early Breast Cancer: A Retrospective Cohort Study from a Resource-Limited Tertiary Centre","fulltext":[{"header":"Background","content":"\u003cp\u003eSentinel lymph node biopsy (SLNB) has become the standard approach for axillary staging in clinically node-negative early breast cancer, replacing routine axillary lymph node dissection (ALND) in most patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. By selectively identifying and excising the first draining lymph node(s), SLNB provides accurate nodal staging while significantly reducing surgical morbidity, including lymphedema and shoulder dysfunction. Landmark randomized trials, including those by Veronesi et al. and the ALMANAC study, demonstrated equivalent oncologic outcomes between SLNB and ALND in node-negative disease, with superior postoperative quality of life following SLNB [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn high-resource settings, SLNB is typically performed using a dual-tracer technique combining a radiolabeled colloid and a blue dye. Dual mapping improves sentinel node identification rates and reduces false-negative rates (FNRs) compared with either modality alone [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Meta-analyses report identification rates exceeding 95% and FNRs generally below 5\u0026ndash;8% when dual tracers are used in experienced centres [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The most recent American Society of Clinical Oncology (ASCO) guideline update continues to recommend dual-tracer mapping where feasible and emphasizes adherence to established quality benchmarks to ensure oncologic reliability [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, dual-tracer SLNB requires nuclear medicine infrastructure, radiopharmaceutical supply chains, gamma probes, and trained personnel. These requirements increase cost and logistical complexity and may not be available in many healthcare systems, particularly in low- and middle-income countries (LMICs) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Regulatory constraints related to radioactive handling further limit widespread adoption. While blue dyes such as isosulfan blue are commonly used adjunctively, they carry a small but clinically relevant risk of anaphylaxis. In contrast, methylene blue (MB) is inexpensive, widely accessible, and does not require specialized equipment, making it an attractive single mapping agent in resource-constrained environments [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsequently, many centres in LMICs perform SLNB using 1% methylene blue alone as a pragmatic adaptation to limited infrastructure. This approach enables broader access to axillary staging while avoiding the morbidity associated with routine ALND. Nonetheless, concerns persist regarding the diagnostic accuracy of single-dye techniques. Earlier pooled analyses suggested that blue dye\u0026ndash;only mapping achieved identification rates of approximately 85\u0026ndash;91% and FNRs approaching 10\u0026ndash;13%, exceeding commonly accepted surgical quality thresholds [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A systematic review of MB-only SLNB reported a pooled identification rate of 91% but highlighted variability in FNR across studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these concerns, accumulating contemporary data suggest that outcomes with MB-only SLNB may improve with structured technique and surgical experience. A large retrospective series from China reported identification rates approaching 96% using methylene blue alone [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies from Nigeria, India, Malaysia, and other LMIC settings have demonstrated identification rates between 85% and 92%, with acceptable sensitivity for nodal metastasis detection [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several of these reports describe improvements in performance metrics with increased operator experience and retrieval of multiple sentinel nodes, suggesting that technique standardization may narrow the performance gap between single- and dual-tracer methods [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOncologic safety remains the critical benchmark. Long-term data from major SLNB trials indicate that axillary recurrence after a negative SLNB is uncommon, typically below 2%, and comparable to outcomes following ALND [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although most of these data derive from dual-tracer cohorts, contemporary MB-only series have not demonstrated excess axillary relapse when appropriate patient selection and surgical protocols are applied [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings suggest that, in clinically node-negative early breast cancer, carefully executed single-dye SLNB may provide acceptable regional control.\u003c/p\u003e \u003cp\u003eThe relevance of this issue extends beyond technical considerations. Breast cancer incidence continues to rise across LMICs, where healthcare systems often face limited access to nuclear medicine services. In such contexts, omission of SLNB due to lack of radiotracers may lead either to unnecessary ALND with associated morbidity or to inadequate nodal staging. A validated, low-cost single-dye protocol could therefore facilitate guideline-concordant care while minimizing treatment-related harm.\u003c/p\u003e \u003cp\u003eNevertheless, high-quality real-world data from LMIC tertiary centres remain limited, and variability in technique, follow-up duration, and reporting standards complicates interpretation of existing literature. Few studies have evaluated identification rate, false-negative rate, nodal burden, clinicopathologic correlates, and recurrence outcomes within a standardized institutional protocol.\u003c/p\u003e \u003cp\u003e In this context, we conducted a retrospective cohort study evaluating methylene blue\u0026ndash;guided SLNB performed as the sole mapping technique in clinically and radiologically node-negative early breast cancer at a tertiary referral centre in Sri Lanka. We assessed sentinel node identification rate, nodal burden, clinicopathologic predictors of sentinel node positivity, and short- to mid-term oncologic outcomes, including axillary recurrence. By situating these findings within the broader global literature, we aim to clarify the feasibility and oncologic safety of MB-only SLNB in a resource-limited setting.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study was conducted at the Surgical Oncology Unit, Teaching Hospital Anuradhapura (THA), Sri Lanka, a tertiary referral centre serving a predominantly rural population. The study period extended from December 2019 to August 2023.\u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, and the Institutional Review Board of Teaching Hospital Anuradhapura (Ref. No. ERC/2024/22, dated July 15, 2024). As this study involved retrospective analysis of anonymized medical records without direct patient contact, the requirement for individual informed consent was formally waived. All eligible consecutive patients during the study period were included in order to minimise selection bias and to reflect real-world clinical practice. A summary of patient screening, exclusions, sentinel lymph node biopsy (SLNB) procedures, follow-up, and the final analysed cohort is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFlow diagram illustrating patient screening (n\u0026thinsp;=\u0026thinsp;170), exclusions (n\u0026thinsp;=\u0026thinsp;35), patients undergoing methylene blue\u0026ndash;guided SLNB (n\u0026thinsp;=\u0026thinsp;135), loss to follow-up (n\u0026thinsp;=\u0026thinsp;20), and final analysed cohort (n\u0026thinsp;=\u0026thinsp;115). Frozen section results and subsequent axillary management are also shown.\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eA total of 170 patients with invasive breast carcinoma were initially assessed for eligibility during the study period.\u003c/p\u003e \u003cp\u003eClinically node-negative (cN0) status was defined as:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAbsence of palpable axillary lymphadenopathy on clinical examination, and\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNo suspicious axillary lymph nodes on preoperative ultrasonography.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAll patients underwent standard breast imaging with mammography and ultrasound. Breast MRI was performed selectively when clinically indicated due to resource limitations.\u003c/p\u003e \u003cp\u003eThirty-five patients were excluded for the following reasons:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBiopsy-proven axillary nodal metastasis (cN1 or higher)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMultifocal or multicentric invasive lobular carcinoma requiring separate mapping injections\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eLocally advanced disease not suitable for SLNB\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePrior ipsilateral breast or axillary surgery\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eReceipt of neoadjuvant chemotherapy or radiotherapy\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAfter exclusions, 135 consecutive eligible patients underwent primary breast surgery with sentinel lymph node biopsy (SLNB) using 1% methylene blue as the sole mapping agent.\u003c/p\u003e \u003cp\u003eOf the 135 patients who underwent SLNB, 20 (14.8%) were excluded from final outcome analysis due to incomplete follow-up data, relocation, or failure to attend postoperative oncology surveillance clinics. No direct patient contact was attempted in accordance with ethics approval. The final study cohort consisted of 115 patients included in the outcome analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSample Size\u003c/h3\u003e\n\u003cp\u003eThis study was designed as a retrospective consecutive cohort; therefore, no a priori sample size was specified. All eligible consecutive patients undergoing sentinel lymph node biopsy (SLNB) using methylene blue single-dye technique between December 2019 and August 2023 at Teaching Hospital Anuradhapura were included.\u003c/p\u003e \u003cp\u003eThe final analysed cohort comprised patients with complete clinical records and adequate follow-up data. The precision of key outcome estimates was assessed using 95% confidence intervals.\u003c/p\u003e\n\u003ch3\u003eSLNB Surgical Technique\u003c/h3\u003e\n\u003cp\u003eAll procedures were performed under general anesthesia by consultant surgical oncologists specializing in breast surgery, using a standardized institutional protocol. After induction, 2\u0026ndash;3 mL of 1% methylene blue was injected subcutaneously in the periareolar or peritumoral region, according to tumor location. The injection site was gently massaged for 2\u0026ndash;5 minutes to facilitate lymphatic uptake as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn axillary incision was then made, and the blue-stained lymphatic channels were traced toward the axilla. All blue-stained lymph nodes and any palpably suspicious nodes were excised. An intraoperative view of a blue-dyed sentinel lymph node during axillary dissection is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and the harvested sentinel lymph node with adjacent lymph nodes following methylene blue injection is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn cases where no blue-stained lymphatic channels or nodes were identified intraoperatively, sentinel node mapping was considered unsuccessful. In such instances, limited axillary sampling was performed at the discretion of the operating surgeon.\u003c/p\u003e\n\u003ch3\u003eSentinel Lymph Node Pathology and Axillary Management\u003c/h3\u003e\n\u003cp\u003eExcised sentinel lymph nodes were immediately sent for intraoperative frozen section analysis. The histopathology laboratory and on-call pathologist were pre-notified before surgery to ensure availability. Sentinel lymph node biopsy was performed first, and while awaiting frozen section results definitive breast tumour resection was undertaken.\u003c/p\u003e \u003cp\u003eIf metastatic disease was identified on frozen section analysis, immediate completion axillary lymph node dissection (ALND) involving Levels I and II was performed during the same operation. Patients had been counselled preoperatively and consented for possible completion ALND if sentinel lymph node metastasis was detected. When frozen section analysis was negative, no additional axillary surgery was performed. Final paraffin-embedded histology confirmed sentinel lymph node status.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePathological Assessment\u003c/h2\u003e \u003cp\u003eHistopathologic evaluation was performed using hematoxylin\u0026ndash;eosin staining, with immunohistochemistry of all tumour and axillary dissections including sentinel samples.\u003c/p\u003e \u003cp\u003ePathological staging followed the Tumour\u0026ndash;Node\u0026ndash;Metastasis (TNM) classification of the American Joint Committee on Cancer (AJCC), 8th edition [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Tumour grading was performed using the Nottingham histological grading system (Elston\u0026ndash;Ellis grading system) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecorded clinicopathological variables included tumour size, histological subtype, tumour grade, lymphovascular invasion (LVI), extranodal extension, ooestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2) status, Ki-67 proliferation index, number and status of sentinel lymph nodes, axillary lymph node dissection findings when performed, and adjuvant treatment details.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOutcomes and Definitions\u003c/h3\u003e\n\u003cp\u003ePrimary outcomes were sentinel node identification rate and axillary recurrence among SLNB-negative patients. Sentinel lymph node identification rate was defined as the proportion of patients in whom at least one sentinel lymph node was successfully identified. Axillary safety was assessed using the axillary recurrence rate among sentinel lymph node\u0026ndash;negative patients during follow-up.\u003c/p\u003e \u003cp\u003eBecause completion axillary lymph node dissection was performed only in patients with positive intraoperative sentinel lymph node findings, a classical false-negative rate defined against axillary dissection as a reference standard could not be calculated. Axillary safety was therefore assessed clinically using the axillary recurrence rate among sentinel lymph node\u0026ndash;negative patients.\u003c/p\u003e \u003cp\u003eSecondary outcomes included nodal yield, additional nodal burden, recurrence-free survival, and tracer-related cost comparison with dual-tracer SLNB using published micro-costing data.\u003c/p\u003e\n\u003ch3\u003eCost Analysis\u003c/h3\u003e\n\u003cp\u003eTracer-related costs were analysed using a cost-minimisation approach, restricted to consumables directly attributable to the mapping strategy. For methylene blue (MB) single-dye SLNB, per-patient tracer cost was calculated from institutional procurement records for 2026 and included the volume of 1% MB used per case (2\u0026ndash;3 mL) and injection materials (syringe and needle).\u003c/p\u003e \u003cp\u003eAs dual-tracer SLNB is not routinely performed at our institution, comparator tracer costs for technetium-99m sulfur colloid plus blue dye mapping were obtained from a published micro-costing study (Khadka et al.) reporting per-patient tracer costs using institutional and market radiotracer pricing (2020 USD) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Costs were adjusted from 2020 to 2026 United States Dollars (USD) using the United States Consumer Price Index (CPI) inflation adjustment. Currency conversion was based on the prevailing exchange rate at the time of analysis. The analysis focused on tracer-related costs; operating theatre time, staffing, anaesthesia, and pathology costs were assumed to be comparable between mapping strategies and were therefore not included.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Collection and Follow-Up\u003c/h2\u003e \u003cp\u003eData were extracted retrospectively from operative records, pathology reports, and oncology clinic documentation. Variables recorded included patient age, tumour characteristics, pathological findings, nodal status, and adjuvant treatments including chemotherapy, radiotherapy, endocrine therapy, and targeted therapy.\u003c/p\u003e \u003cp\u003ePatients were followed in surgical oncology and medical oncology outpatient clinics according to institutional protocol. Surveillance consisted of clinical examination every six months and annual mammography with breast ultrasonography. Additional imaging including computed tomography or bone scintigraphy was performed when clinically indicated. Recurrence was classified as locoregional recurrence involving the breast, chest wall, or ipsilateral axilla, or as distant metastasis. The mean follow-up duration for the analysed cohort was 36.3 months (range 12\u0026ndash;84 months).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContinuous variables were presented as means with standard deviations or medians with ranges as appropriate. Categorical variables were summarised using frequencies and percentages.\u003c/p\u003e \u003cp\u003eAssociations between sentinel lymph node positivity and clinicopathological variables were analysed using the Chi-square test or Fisher\u0026rsquo;s exact test for categorical variables and the independent t-test or Mann\u0026ndash;Whitney U test for continuous variables, as appropriate. Correlations between clinicopathological variables were assessed using Pearson\u0026rsquo;s correlation coefficient. Recurrence-free survival was estimated using the Kaplan\u0026ndash;Meier method and compared using the log-rank test.\u003c/p\u003e \u003cp\u003eAll statistical tests were two-sided and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Missing data were not imputed. Descriptive outcomes were reported with 95% confidence intervals to reflect the precision of estimates in this retrospective cohort.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePatient and tumour characteristics\u003c/h2\u003e \u003cp\u003eA total of 115 clinically node-negative women with invasive breast carcinoma underwent sentinel lymph node biopsy (SLNB) using the methylene blue single-dye technique between December 2019 and August 2023.\u003c/p\u003e \u003cp\u003eThe mean age at diagnosis was 55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 years (median 53.5; range 35\u0026ndash;81). Wide local excision was performed in 48 patients (43.6%), mastectomy in 44 patients (40.0%), and other breast-conserving procedures in the remainder.\u003c/p\u003e \u003cp\u003eThe mean primary tumour size on preoperative imaging was 36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;29.7 mm (median 28 mm; range 10\u0026ndash;180 mm). Tumour size data were available for 110 patients (70 SLNB-negative and 40 SLNB-positive). There was no statistically significant difference in tumour size between SLNB-positive and SLNB-negative patients (Mann\u0026ndash;Whitney U test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eBox-and-whisker plots showing preoperative primary tumour size (mm) in SLNB-negative (n\u0026thinsp;=\u0026thinsp;70) and SLNB-positive (n\u0026thinsp;=\u0026thinsp;40) patients. There was no statistically significant difference in tumour size between groups (Mann\u0026ndash;Whitney U test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSentinel lymph node biopsy performance\u003c/h2\u003e \u003cp\u003eThe sentinel node identification rate was 98.3% (113/115). In two patients (1.7%), methylene blue injection did not result in visible axillary lymphatic staining, and sentinel nodes could not be identified. These patients underwent limited axillary sampling. Among patients in whom sentinel nodes were successfully identified (n\u0026thinsp;=\u0026thinsp;113), the mean number of sentinel lymph nodes retrieved per patient was 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16. SLNB was positive in 40/115 patients (34.8%), while 75/115 (65.2%) had negative sentinel nodes. Among the 40 patients with positive sentinel lymph nodes, macrometastases were identified in 33 (82.5%), micrometastases in 5 (12.5%), and isolated tumour cells in 2 (5.0%).\u003c/p\u003e \u003cp\u003eLymphovascular invasion demonstrated a statistically significant association with sentinel lymph node positivity (χ\u0026sup2; test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, HER2 receptor status (p\u0026thinsp;=\u0026thinsp;0.50) and molecular subtype classification (p\u0026thinsp;=\u0026thinsp;0.51) were not significantly associated with sentinel node metastasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAxillary Lymph Node Dissection (ALND) Outcomes\u003c/h2\u003e \u003cp\u003eAll 40/40 (100%) patients with positive sentinel lymph nodes underwent completion axillary lymph node dissection (ALND). The mean number of additional axillary lymph nodes retrieved was 12.75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9. Additional non-sentinel nodal metastases were identified in 21/40 patients (52.5%), whereas 19/40 patients (47.5%) had no further positive nodes beyond the sentinel lymph nodes.\u003c/p\u003e \u003cp\u003eAmong patients with additional axillary metastases (n\u0026thinsp;=\u0026thinsp;21), the majority demonstrated limited residual nodal burden: 12/21 (57.1%) had fewer than five additional positive nodes, 6/21 (28.6%) had five to nine positive nodes, and 3/21 (14.3%) had ten or more positive nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eBar chart demonstrating the number of additional metastatic axillary lymph nodes identified at ALND. Nineteen patients (47.5%) had no further nodal metastases beyond the sentinel lymph nodes, while 21 patients (52.5%) had additional positive nodes. Among those with additional metastases, the majority demonstrated limited residual nodal burden, with most patients having fewer than five additional positive nodes.\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Characteristics\u003c/h2\u003e \u003cp\u003eInvasive ductal carcinoma of no special type (NST) was the predominant histological subtype, identified in 94/115 patients (81.7%). Other histological subtypes included invasive lobular carcinoma in 4/115 (3.5%), solid papillary carcinoma in 1/115 (0.9%), other rare variants in 8/115 (7.0%), and pure ductal carcinoma in situ (DCIS) without invasive disease in 8/115 (7.0%).\u003c/p\u003e \u003cp\u003eA DCIS component coexisting with invasive carcinoma was present in 32/115 patients (27.8%). Lymphovascular invasion (LVI) was identified in 30/115 cases (26.1%), extranodal extension in 26/115 (22.6%), and Paget\u0026rsquo;s disease in 8/115 (7.0%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eClinical and Pathological Staging\u003c/h2\u003e \u003cp\u003eOn initial clinical staging (n\u0026thinsp;=\u0026thinsp;115), 23/115 (20.0%) patients were classified as Stage I, 51/115 (44.3%) as Stage II, and 32/115 (27.8%) as Stage III. No patients met criteria for Stage IV disease at presentation. Clinical tumour category based on imaging demonstrated a predominance of cT2 lesions.\u003c/p\u003e \u003cp\u003eOn final pathological assessment (n\u0026thinsp;=\u0026thinsp;115), tumour category was pT1 in 30/115 (26.1%), pT2 in 64/115 (55.7%), pT3 in 11/115 (9.6%), and pT4 in 1/115 (0.9%). Pathological nodal staging showed pN0 in 64/115 (55.7%), pN1 (including micrometastatic disease) in 31/115 (27.0%), pN2 in 5/115 (4.3%), and pN3 in 4/115 (3.5%). According to AJCC 8th edition pathological stage grouping, the majority of cases were Stage I\u0026ndash;II, with a smaller proportion classified as Stage III; no patients were Stage IV. Among SLNB-positive patients, micro metastases accounted for 5/40 (12.5%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHormone Receptor and Molecular Subtypes\u003c/h2\u003e \u003cp\u003eOoestrogen receptor (ER) expression was positive in 72/115 patients (62.6%), progesterone receptor (PR) in 51/115 (44.3%), and HER2 overexpression in 34/115 (29.6%).\u003c/p\u003e \u003cp\u003eBased on ER, PR, and HER2 status, molecular subtypes were classified as follows: luminal (HER2-negative) in 60/115 patients (52.2%), luminal/HER2-positive in 27/115 (23.5%), HER2-enriched in 22/115 (19.1%), and triple-negative in 6/115 (5.2%).\u003c/p\u003e \u003cp\u003eThe Ki-67 proliferation index was high (\u0026gt;\u0026thinsp;30%) in 61/115 tumours (53.0%), intermediate (16\u0026ndash;30%) in 19/115 (16.5%), and low (\u0026le;\u0026thinsp;15%) in 35/115 (30.4%). There was no statistically significant association between Ki-67 category and SLNB positivity (χ\u0026sup2; test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAdjuvant Treatment\u003c/h2\u003e \u003cp\u003eOver a mean follow-up period of 36.3 months (median 36; range 12\u0026ndash;84 months), adjuvant therapy was administered according to tumour stage, nodal status, and receptor profile.\u003c/p\u003e \u003cp\u003eAdjuvant chemotherapy was administered to 45 of 115 patients (39.1%), with a mean of 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 cycles. Regimens were predominantly anthracycline-based combinations (doxorubicin plus cyclophosphamide), commonly followed by a taxane.\u003c/p\u003e \u003cp\u003eAdjuvant radiotherapy was delivered to 49 of 115 patients (42.6%), with regional nodal irradiation including the axilla administered in five cases.\u003c/p\u003e \u003cp\u003eOestrogen receptor positivity was identified in 64 of 115 patients (55.7%). Among these, endocrine therapy was documented in 54 of 64 patients (84.4%). The most commonly prescribed agents were tamoxifen and aromatase inhibitors. In the remaining ER-positive cases, documentation of endocrine therapy was incomplete in the retrospective medical records.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRecurrence and Survival Outcomes\u003c/h2\u003e \u003cp\u003eDuring a mean follow-up of 36.3 months (range 12\u0026ndash;84 months), disease recurrence occurred in 5 of 115 patients (4.3%). Locoregional axillary recurrence was observed in 2 patients (2/115, 1.7%), both within the SLNB-positive group following completion axillary lymph node dissection (ALND). Distant metastasis occurred in 3 patients (3/115, 2.6%), including two from the SLNB-negative group and one from the SLNB-positive group. Recurrence occurred between 0.75 and 4.6 years after surgery\u003c/p\u003e \u003cp\u003eNo isolated axillary recurrences were observed among SLNB-negative patients during follow-up (0/75; 0%, 95% CI 0\u0026ndash;4.0%). The timing and anatomical distribution of relapse are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eGantt-style plot illustrating time to recurrence for the five patients (5/115, 4.3%) who developed disease recurrence during follow-up, stratified by SLNB status and site of recurrence (axillary locoregional versus distant metastasis).\u003c/em\u003e \u003c/p\u003e \u003cp\u003eKaplan\u0026ndash;Meier analysis demonstrated high recurrence-free survival (RFS) across the cohort. There was no statistically significant difference in RFS between SLNB-positive and SLNB-negative patients (log-rank p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). When stratified by molecular subtype, luminal A and luminal B tumours demonstrated numerically higher RFS compared with HER2-enriched and triple-negative subtypes; however, comparison between luminal (A/B) and non-luminal tumours did not reach statistical significance (log-rank p\u0026thinsp;=\u0026thinsp;0.5819). Similarly, patients with Grade 1\u0026ndash;2 tumours showed numerically improved RFS compared with those with Grade 3 disease, but this difference was not statistically significant (log-rank p\u0026thinsp;=\u0026thinsp;0.3425).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eKaplan\u0026ndash;Meier curves comparing RFS between SLNB-negative and SLNB-positive patients during follow-up. Only five recurrence events occurred, and no statistically significant difference between groups was observed on log-rank testing.\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCost comparison\u003c/h2\u003e \u003cp\u003eThe tracer-related cost of MB single-dye SLNB was LKR 330 per patient, equivalent to approximately US\u003cspan\u003e$\u003c/span\u003e1.10 in 2026. In the published micro-costing study used for comparison, per-patient tracer costs for dual-tracer SLNB (technetium-99m sulfur colloid plus blue dye) were US\u003cspan\u003e$\u003c/span\u003e109.04 (institutional radiotracer pricing) and US\u003cspan\u003e$\u003c/span\u003e178.27 (market pricing) in 2020 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], corresponding to approximately US\u003cspan\u003e$\u003c/span\u003e135 and US\u003cspan\u003e$\u003c/span\u003e221 after inflation adjustment to 2026 USD. All costs are reported in 2026 United States Dollars (USD).\u003c/p\u003e \u003cp\u003eThe resulting incremental tracer cost difference between MB single-dye and dual-tracer SLNB was approximately US\u003cspan\u003e$\u003c/span\u003e134\u0026ndash;220 per patient, representing an approximate 99% reduction in tracer-related expenditure with MB-only mapping. Applied to the 115-patient cohort, this corresponded to an estimated cumulative tracer-related cost saving of approximately US\u003cspan\u003e$\u003c/span\u003e15,400\u0026ndash;25,300 (2026 USD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis retrospective cohort study evaluated the performance and oncologic safety of methylene blue (MB) single-dye sentinel lymph node biopsy (SLNB) in clinically and radiologically node-negative early breast cancer within a resource-limited tertiary oncology centre. The principal findings were: (i) a high sentinel node identification rate of 98.3%, comparable to the upper range reported in methylene blue\u0026ndash;only SLNB series (85\u0026ndash;96%), with mapping failure in two patients (1.7%), (ii) a mean retrieval of 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 SLNs per patient, (iii) nodal metastases identified in 40 of 115 patients (34.8%), and (iv) no isolated axillary recurrences among SLNB-negative patients during follow-up (0/75; 95% CI 0\u0026ndash;4.0%). Overall recurrence occurred in 4.3% of patients over a mean follow-up of 36.3 months.\u003c/p\u003e \u003cp\u003eIn addition to favourable oncologic outcomes, methylene blue single-dye SLNB demonstrated a marked economic advantage compared with dual-tracer techniques. Tracer-specific cost analysis showed substantial per-patient savings, highlighting the potential value of MB-only SLNB in resource-limited healthcare systems [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eContext Within Established SLNB Evidence\u003c/h2\u003e \u003cp\u003eSLNB is established as the standard axillary staging procedure for clinically node-negative breast cancer, having demonstrated equivalent survival and low axillary recurrence compared with routine ALND while significantly reducing morbidity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In high-resource settings, dual-tracer mapping (radioisotope plus blue dye) is commonly used to optimise identification and minimise false-negative results [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, reliance on nuclear medicine infrastructure limits the scalability of dual-tracer techniques in many low- and middle-income countries (LMICs).\u003c/p\u003e \u003cp\u003eA systematic review and meta-analysis of MB-only SLNB reported pooled identification rates of approximately 90%, with variability across institutions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Several single-centre studies from LMIC settings have reported identification rates ranging from approximately 85% to the mid-90% range [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The identification rate observed in the present cohort lies at the upper end of previously reported MB-only series. The small proportion of mapping failures observed (1.7%) is consistent with published methylene blue single-dye series reporting identification rates between 85% and 96% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and highlights the importance of surgical experience and standardized injection technique.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e compares key SLNB performance metrics from the present cohort with representative dual-tracer and methylene blue single-dye series reported in global and LMIC settings. The mean nodal yield observed in this study falls within the optimal range reported in both mapping strategies, supporting adequate staging reliability. These findings suggest that, within a structured protocol and experienced surgical setting, MB single-dye SLNB can achieve technical outcomes comparable to dual-tracer approaches while maintaining feasibility in resource-constrained environments.\u003c/p\u003e \u003c/div\u003e\n\u003ctable border=\"1\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDual-Tracer (Radioisotope + Dye)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMB Single-Dye (Global/LMIC Data)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMB Single-Dye (Current Sri Lankan Cohort)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSentinel Node Identification Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85\u0026ndash;95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.3% (113/115)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMean SLNs Retrieved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u0026ndash;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u0026ndash;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.25 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAxillary Recurrence After Negative SLNB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;1\u0026ndash;2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRare\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0% (0/75; 95% CI 0\u0026ndash;4.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDye-Related Complications\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRare (allergic reactions reported with isosulfan/patent blue)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVery low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNone observed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eResource Requirements\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNuclear medicine facility + gamma probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLow-resource setting feasible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLow-resource setting feasible (routine practice)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: SLNB, sentinel lymph node biopsy; MB, methylene blue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. \u003cem\u003eComparative overview of sentinel lymph node biopsy (SLNB) performance metrics in dual-tracer and methylene blue (MB) single-dye series compared with the current cohort.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFootnote: Comparative data summarised from representative international and LMIC series [2\u0026ndash;4,8\u0026ndash;12].\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRetrieval of multiple sentinel nodes has been shown to improve staging accuracy and reduce the likelihood of missed nodal metastases [17].\u003c/p\u003e\n\u003cp\u003eBecause completion ALND was not performed in SLNB-negative patients, a classical ALND-referenced false-negative rate could not be calculated. Axillary safety was therefore assessed clinically through follow-up, using axillary recurrence as a surrogate endpoint. Although the limited number of events precludes definitive conclusions regarding diagnostic sensitivity, the observed outcomes are consistent with the low axillary recurrence rates reported in landmark SLNB trials [1,2,12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAxillary Disease Burden and Surgical Implications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong SLNB-positive patients who underwent completion ALND, additional non-sentinel nodal involvement was identified in a subset, but overall residual nodal burden was generally low. This pattern mirrors observations from large SLNB trials demonstrating that, in many early-stage patients, the majority of axillary disease is captured at the sentinel node level [1,2,12]. Contemporary studies evaluating axillary de-escalation strategies further highlight the evolving paradigm of risk-adapted axillary management [18]. Accurate and reproducible SLNB therefore remains central to modern axillary decision-making.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival Outcomes and Exploratory Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOver a mean follow-up of approximately three years, recurrence occurred in 4.3% of patients. Kaplan\u0026ndash;Meier analyses demonstrated high recurrence-free survival with no statistically significant subgroup differences. Subgroup comparisons by molecular subtype and tumour grade were similarly non-significant. These analyses should be interpreted cautiously because only five recurrence events occurred, limiting statistical power and increasing the risk of type II error. Longer follow-up will be required to more robustly evaluate survival outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-World Healthcare Disruption and System Constraints\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study period overlapped with the COVID-19 pandemic and national economic disruption in Sri Lanka, during which treatment delays and intermittent medication shortages occurred. The use of retrospective paper-based records may have contributed to incomplete documentation of adjuvant therapies, reflecting systemic constraints typical of resource-limited healthcare settings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeasibility and Health System Relevance in LMIC Settings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethylene blue offers practical advantages in resource-limited environments. It is inexpensive, widely available, chemically stable, and does not require nuclear medicine facilities or radiopharmaceutical handling protocols [6,9,10]. No local tissue complications, allergic reactions, or systemic adverse events related to methylene blue injection were observed in this series, supporting the clinical safety of low-volume methylene blue use for sentinel node mapping.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTracer-specific cost analysis demonstrated a substantial reduction in expenditure compared with published dual-tracer micro-costing data [15]. Although broader perioperative costs were assumed to be comparable between mapping strategies, the principal cost differential reflects radiopharmaceutical procurement and nuclear medicine infrastructure. In settings where technetium-99m is unavailable or financially constrained, MB-only SLNB may represent a pragmatic and scalable alternative, facilitating guideline-concordant axillary staging while avoiding routine ALND-related morbidity [1,2,9,11].\u003c/p\u003e\n\u003cp\u003eWhile direct equivalence to dual-tracer techniques cannot be confirmed without a contemporaneous comparator group, the technical performance and early oncologic outcomes observed in this study support the feasibility of MB-only SLNB within a structured institutional protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Limitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. The retrospective single-centre design introduces potential selection bias and limits generalisability. Although the calculated minimum sample size was not fully achieved due to loss to follow-up, the primary technical outcome\u0026mdash;sentinel node identification rate\u0026mdash;was estimated with acceptable precision. However, the study was not powered to detect small differences in recurrence or survival outcomes.\u003c/p\u003e\n\u003cp\u003eThe limited number of recurrence events and moderate follow-up duration restrict interpretation of long-term oncologic safety, particularly regarding late axillary relapse. Clinical data were derived from paper-based records, and incomplete documentation of adjuvant therapy cannot be excluded.\u003c/p\u003e\n\u003cp\u003eThe economic comparison was restricted to tracer-related costs and utilised published micro-costing data for the dual-tracer comparator. Accordingly, findings should be interpreted as tracer-specific cost minimisation rather than a comprehensive health economic evaluation.\u003c/p\u003e\n\u003cp\u003eFinally, results from a high-volume tertiary centre using a standardised protocol may not be directly generalisable to lower-volume institutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrengths and Practical Impact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study reflects consecutive real-world practice within a defined institutional pathway using a standardised MB injection technique and systematic pathological assessment. With longitudinal follow-up exceeding three years, the findings provide context-relevant evidence supporting MB single-dye SLNB as a safe and economically rational axillary staging strategy in resource-constrained settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this resource-limited tertiary oncology setting, methylene blue single-dye sentinel lymph node biopsy achieved a high identification rate with reliable nodal staging and no isolated axillary recurrence among SLNB-negative patients during follow-up. The technique demonstrated favourable short- to mid-term oncologic outcomes while offering substantial cost advantages over dual-tracer mapping. These findings support MB single-dye SLNB as a safe, accessible, and economically sustainable axillary staging strategy in healthcare systems with limited nuclear medicine infrastructure. Prospective multicentre studies with longer follow-up are warranted to confirm long-term oncologic equivalence.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka and the Institutional Review Board of Teaching Hospital Anuradhapura (Ref. No. ERC/2024/22). The requirement for individual informed consent was formally waived due to the retrospective analysis of anonymised medical records.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors and was personally funded by the principal author and corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVathsal Chinathaka Bandara\u003c/strong\u003e: Conceptualisation, Funding, Methodology, Formal Analysis, Investigation, Supervision, Validation, Writing \u0026ndash; Original Draft, Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmila Prathibha Nellihela\u003c/strong\u003e: Conceptualisation, Funding, Methodology, Formal Analysis, Investigation, Project Administration, Statistical Analysis, Writing \u0026ndash; Original Draft, Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJayanjana Asanthi\u003c/strong\u003e: Supervision, Methodology, Validation, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAruna Wijesooriya\u003c/strong\u003e: Investigation, Data Collection, Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRuchika Nirmalie Senevirathne Jayalathge\u003c/strong\u003e: Formal Analysis, Investigation, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAruna Buddhika Jayasena\u003c/strong\u003e: Investigation, Data Collection, Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSulakshi Karunanayake\u003c/strong\u003e: Data Collection, Investigation, Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePirahanthan Karunanithy\u003c/strong\u003e: Data Collection, Investigation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbheetha Abegunasekara\u003c/strong\u003e: Data Collection, Investigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTharindu Jayasinghe\u003c/strong\u003e: Data Collection, Investigation\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVeronesi U, Paganelli G, Viale G, Luini A, Zurrida S, Galimberti V, et al. 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Cancers (Basel). 2023;16(18):3168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers16183168\u003c/span\u003e\u003cspan address=\"10.3390/cancers16183168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sentinel lymph node biopsy, methylene blue, single-dye technique, breast neoplasms, axillary staging, low- and middle-income countries","lastPublishedDoi":"10.21203/rs.3.rs-9328110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9328110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSentinel lymph node biopsy (SLNB) is the standard method of axillary staging in clinically node-negative early breast cancer and is commonly performed using dual-tracer mapping with radioisotope and blue dye. However, access to nuclear medicine facilities is limited in many low- and middle-income countries (LMICs), restricting widespread implementation. This study evaluated the technical performance, oncologic safety, and economic implications of methylene blue (MB) single-dye SLNB in a resource-limited tertiary oncology centre.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA retrospective cohort study included 115 consecutive patients with clinically and radiologically node-negative early breast cancer who underwent MB single-dye SLNB between 2019 and 2023. Sentinel nodes were identified using subcutaneous injection of 2\u0026ndash;3 mL of 1% methylene blue. Primary outcomes were sentinel node identification rate and axillary recurrence among SLNB-negative patients. The sentinel lymph node identification rate was defined as the proportion of procedures in which at least one sentinel lymph node was successfully identified. Secondary outcomes included nodal yield, additional nodal burden, recurrence-free survival, and tracer-related cost comparison with dual-tracer SLNB using published micro-costing data. Statistical analyses included Chi-square testing, independent t-tests or Mann\u0026ndash;Whitney U tests, and Kaplan\u0026ndash;Meier survival analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSentinel nodes were successfully identified in 113 of 115 patients (98.3%). The mean number of sentinel lymph nodes retrieved was 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 per patient. Nodal metastases were detected in 40 patients (34.8%). No isolated axillary recurrences were observed among SLNB-negative patients during follow-up (0/75; 95% CI 0\u0026ndash;4.0%). Overall recurrence occurred in 5 patients (4.3%) over a mean follow-up of 36.3 months. No methylene blue-related adverse reactions were recorded. The estimated tracer cost per patient was approximately US\u003cspan\u003e$\u003c/span\u003e0.50 for MB single-dye mapping compared with US\u003cspan\u003e$\u003c/span\u003e135\u0026ndash;221 for dual-tracer techniques, representing a reduction exceeding 99% in tracer-related costs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMB single-dye SLNB achieved high identification rates with favourable short- to mid-term oncologic outcomes while providing substantial cost savings. These findings support MB single-dye SLNB as a safe, feasible, and economically sustainable approach to axillary staging in resource-limited settings where dual-tracer techniques are not readily available.\u003c/p\u003e","manuscriptTitle":"Outcomes of Methylene Blue Single-Dye Sentinel Lymph Node Biopsy in Early Breast Cancer: A Retrospective Cohort Study from a Resource-Limited Tertiary Centre","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 17:19:45","doi":"10.21203/rs.3.rs-9328110/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-01T11:13:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-12T14:02:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-06T09:02:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Surgical Oncology","date":"2026-04-05T18:55:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc12598b-5ebb-40b9-8e75-b73ab2568f37","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"5","date":"2026-05-01T11:13:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T17:19:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 17:19:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9328110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9328110","identity":"rs-9328110","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}