Sentinel lymph node biopsy in early breast carcinoma using fluorescein sodium and methylene blue dye: A validation study

Abstract

The availability of radioisotopes for sentinel lymph node biopsy (SLNB) in breast carcinoma is limited in low- and middle-income countries and thus the need for other reliable tracers exists. We aimed to validate the effectiveness of fluorescein sodium (FS) together with methylene blue dye (MBD) for patients with node-negative early breast carcinoma in a prospective cross-sectional study. Patients underwent SLNB using FS and MBD followed by axillary dissection to validate results. Sentinel lymph node (SLN) identification rate and false negative rate were assessed for all three tracers/combinations (MBD, FS, and MBD + FS). We concluded that SLNB using a combination of FS and MBD has an acceptable rate of SLN identification but the addition of FS provided no additional benefit.

Keywords

Sentinel lymph node biopsy fluorescein sodium methylene blue early breast cancer node-negative breast cancer

Introduction

The presence or absence of axillary lymph node metastases is the most important prognostic factor in patients with potentially curable breast carcinoma. Axillary lymph node dissection (ALND) has been the gold standard for axillary staging but is associated with potential morbidity, including lymphoedema, shoulder dysfunction, pain, and paraesthesia.

In clinically node-negative disease, the rate of nodal metastases is only 20% to 35% and ALND is considered unnecessary for most patients, adding only to the morbidity without any therapeutic benefit. Therefore, sentinel lymph node biopsy (SLNB), a more selective approach, was developed. The status of the sentinel lymph node (SLN) reflects the status of the entire nodal basin and permits accurate staging of the axilla. Seven trials examining outcomes following SLNB policy versus standard ALND have consistently shown that morbidity is lower and quality of life better in patients who undergo SLNB compared to standard ALND, with no adverse effects on survival from lesser axillary surgery.^1–7 A level I/level II axillary dissection is generally recommended.⁸

Radioactive tracers are expensive, involve radiation exposure, have limited availability, and require statutory permission and licensing.⁹ The standard blue dyes, isosulfan blue and patent blue, are often not available in our practice, and the only blue dye available is methylene blue.

Fluorescein sodium (FS), an organic fluorophore, which provides intense yellow–green fluorescence on excitation by the blue light of a wavelength of 480 nm, has been investigated as a tracer.^10–14 It is cheap, widely available, lymphophilic, trapped selectively by lymphatics, and rapidly transported to and concentrated in the lymph nodes. After subcutaneous or intra-parenchymal injection, FS rapidly reaches the SLN.¹⁵ It provides visual input of high contrast to surrounding tissues by its fluorescence and also by its colour.

Patients and methods

Our prospective cross-sectional study was conducted between July 2021 and July 2023 after obtaining approval from our Institute Ethical Committee. All patients with biopsy-proven, clinically node-negative (negative on palpation and axillary ultrasound examination) early breast carcinoma presenting to the Department of General Surgery were included, after obtaining informed consent. Exclusion criteria for enrolment were: prior surgery or radiotherapy to the breast or axilla, neoadjuvant chemotherapy, inflammatory breast cancer, allergy to FS or methylene blue, pregnancy, and refusal to undergo an axillary clearance for validation.

The sample size for our study was calculated to be 77 using the online software Statulator (assuming a 95% SLN-identification rate (SLN-IR) and a 5% false negative rate (FNR)). Enrolment of 77 node-negative early breast cancer patients would have required >3 years (based on our departmental statistics, hence an adjusted sample size was calculated with a confidence level of 95% and a margin of error of 5% to be 30.

All included patients planned for mastectomy or breast-conserving surgery (BCS) underwent SLNB using a combination of FS and MBD. After induction of anaesthesia, 2 ml of 1% methylene blue dye (MBD) and 2 ml of 20% FS dye were injected into the sub-areolar location, one after another. The injection site was massaged for 5 min. In the case of BCS, an axillary skin crease incision was used for SLNB, while in patients planned for mastectomy, the axilla was entered after creating the upper flap. All blue nodes and those at the end of a blue lymph vessel were sampled and then the room was darkened and a blue light-emitting diode (LED) light source (wavelength: 480 nm) or an ultraviolet LED light source (wavelength: 390 nm) was illuminated. All likewise fluorescent nodes were excised. These were also assessed for fluorescence under blue light and findings were recorded. SLNB was followed by an ALND in all cases to validate the SLNB result. The blue (MBD), fluorescent (FS), and blue-fluorescent (MBD + FS) SLNs and the rest of the axillary dissection specimens were sent separately for histopathology. Evaluation of SLNs was done only on haematoxylin and eosin-stained sections under light microscopy.

The identification rate was defined as the number of successful SLNB procedures divided by the total number of SLNB attempted. The results from each successfully identified SLNB procedure were categorised as true positives, true negatives, or false negatives, taking the outcome of the complete ALND as a reference. A true negative SLN was defined as a negative SLN and a negative axilla after ALND. A false negative SLN was defined as a negative SLN with metastasis detected in one or more lymph nodes in the ALND specimen or nodes identified by another tracer. A true positive SLN was defined as a positive SLN with or without a positive axilla. Based on these definitions, there were no false positives in this study, hence specificity of SLNB could not be calculated. Accuracy was computed as the sum of all true positives and true negatives, divided by the total number of patients with a successfully identified SLN.

Continuous variables were expressed as mean with standard deviation. Categorical variables were expressed as frequencies or percentages. Statistical analysis was performed using SPSS software (Version 24.0, IBM Corp., Armonk, NY). The values of the diagnostic parameters related to techniques of SLNB were described in terms of sensitivity, negative predictive value, FNR, and accuracy by applying basic descriptive statistical methods: Sensitivity of SLNB = true positives/(true positives + false negatives); FNR = false negatives/(false negatives + true positives); negative predictive value = true negative/(true negative + false negative); accuracy = (true positive + true negative)/no. of patients with successfully identified SLN.

Results

A total of 31 women with node-negative breast carcinoma were included. Their demographic and clinicopathological profile is described in Table 1. We detected a total of 82 nodes in 29 patients; 67 blue-fluorescent (MBD + FS) nodes in 27 patients, three fluorescent nodes (FS) nodes in three patients and 12 blue nodes (MBD) in five patients. The mean number of SLN identified in 29 patients using the MBD, FS, and MBD + FS was 2.4 ± 1.6 (range: 1–7), 2.4 ± 1.6 (range: 1–7), and 2.8 ± 1.9 (range: 1–7), respectively.

Table 1.

Demographic and clinicopathological profile.

Parameters (n = 31)	Values (percentage)
Age in years (mean ± SD), range: 21–68 years	46.2 ± 12.3
Menstrual status
Pre-menopausal	12 (38.7)
Post-menopausal	19 (61.3)
Clinical tumour size (mean + SD)	3.2 ± 1.3
Tumour stage (clinical)
cT1	08 (25.8)
cT2	20 (64.5)
cT3	03 (09.7)
Surgery performed
BCS	24 (77.4)
Mastectomy	07 (22.6)
Pathological tumour size (mean + SD)	3.7 ± 2.4
Tumour stage (pathological)
pT1	10 (32.3)
pT2	16 (51.6)
pT3	05 (16.1)
Nodal stage (pathological)
pN0	24 (77.4)
pN1	07 (22.6)
Histology
IDC	30 (96.7)
Metaplastic carcinoma	01 (03.2)
IHC subtype
ER- and/or PR-positive, HER-2 negative	18 (58.0)
ER- and/or PR-positive, HER-2 positive	07 (22.5)
Triple-negative	06 (19.5)
Histological grade
I	07 (22.6)
II	13 (41.9)
III	11 (35.5)
Lymphovascular invasion
Yes	08 (25.8)
No	23 (74.2)

BCS: breast-conserving surgery; IDC: invasive ductal carcinoma; IHC: immunohistochemistry; ER: estrogen receptor; PR: progesterone receptor; HER-2: human epidermal growth factor receptor 2.

The SLN-IR was the same (93.5%) with all three tracers and combinations. The FNR with MBD was 12.5%, FS was 25%, and MBD + FS was 14.3% (Table 2). The diagnostic parameters of the SLNB using MBD + FS are summarised in Table 3.

Table 2.

SLNB identification rate and false negative rate with different tracers/combinations.

Tracer	Identification rate	False negative rate
MBD alone	29/31 (93.5)	1/8 (12.5)
FS alone	29/31 (93.5)	2/8 (25.0)
Combination of MBD and FS	29/31 (93.5)	1/7 (14.3)

SLNB: sentinel lymph node biopsy; MBD: methylene blue dye; FS: fluorescein sodium.

Table 3.

Diagnostic parameters of SLNB using FS and MBD combination.

Parameters	Value in percentage
Sensitivity	85.7
Negative predictive value	95.7
Accuracy	96.6

SLNB: sentinel lymph node biopsy; FS: fluorescein sodium; MBD: methylene blue dye.

Discussion

An SLN-IR of 90% and a FNR of 10% is considered optimal. Our study found an acceptable rate of SLN identification (93.5%) using a combination of FS and MBD. Although the FNR of 14.3% found in our study with the combination (MBD + FS) is slightly higher than the acceptable value of 10%, it must be added that there was only one false negative in our study with the combination and the higher FNR may well be due to a small sample size.

Enrolment of a large enough number of patients in low- and middle-income countries becomes an issue, since the majority of our patients present with node-positive breast cancer. Also, patients who have node-negative disease may not consent to undergo an axillary dissection to validate SLNB results, since SLNB is already an established procedure for axillary staging in EBC.

FS has been proposed as a tracer agent with promising results. A recently published meta-analysis, which also included data from our study, found a pooled SLN-IR of 93%, and an FNR of 5.6% and concluded that FS-guided SLNB is a viable option in node-negative early breast cancer patients.¹⁶ However, analysing such results is problematic as varied techniques are used; thus it is imperative to provide the details of the technique used in the study. We used 2 ml of 20% FS, but various researchers have utilised variable concentrations of FS. Khadka et al.¹³ reported that reducing the concentration of FS improved the fluorescence. They obtained the best results by diluting 0.125 ml of 10% solution in 4 ml of normal saline. It seems a wide range of concentrations of FS can be used to detect SLNs, but the optimum concentration needs to be investigated further. We waited 15 min after the injection of dyes for our skin incision. This was done as per our protocol for SLNB using methylene blue, and it also worked for FS. Most authors have used blue light to induce fluorescence, but some have used ultraviolet (UV) light successfully.¹¹ We could detect fluorescence with UV as well as blue light (Figs. 1 to 5). Some authors have claimed that transcutaneous fluorescence of the lymphatic and lymph nodes can be visualised under blue light, but we could not visualise the dye movement transcutaneously.¹¹

Figure 1.

A fluorescent and blue SLN (dotted circle) under UV light. Black arrow points to the fluorescent lymphatic leading to the SLN.

Figure 2.

A fluorescent and blue SLN (dotted circle) under blue light.

Figure 3.

A black arrow pointing to a fluorescent and blue SLN under white light.

Figure 4.

A fluorescent lymphatic (dotted arrow) leading to a fluorescent SLN (solid arrow) seen under blue light.

Figure 5.

A FS stained SLN (dotted circle) under white light.

Our conclusion is that SLNB using a combination of FS and MBD has an acceptable identification rate, but the addition of FS provided no additional benefit.

Footnotes

Authors’ contributions

CKJ, US, MK, and PKS contributed to the study concept and design; CKJ and PKS contributed to the acquisition of data; KP, CKJ, and US contributed to the analysis and interpretation of data; CKJ, KP, and CKJ contributed to the drafting of the manuscript; PKS, MK, and US contributed to the critical revision of the manuscript. All authors reviewed and approved the final version of the manuscript.

Data availability

The datasets generated and analysed during the current study are available from the corresponding author upon reasonable request.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval and informed consent

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Approval was granted by the Ethics Committee of All India Institute of Medical Sciences, Patna, India (Date: 16.06.2021/No. AIIMS/Pat/IEC/2021/710). Informed consent was obtained from all individual participants included in the study.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Chandan Kumar Jha

References

Purushotham

Upponi

Klevesath

, et al. Morbidity after sentinel lymph node biopsy in primary breast cancer: Results from a randomized controlled trial. J Clin Oncol 2005; 23: 4312–4321.

Mansel

Fallowfield

Kissin

, 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: 599–609.

Zavagno

De Salvo

Scalco

, et al. A randomized clinical trial on sentinel lymph node biopsy versus axillary lymph node dissection in breast cancer: Results of the Sentinella/GIVOM trial. Ann Surg 2008; 247: 207–213.

Canavese

Catturich

Vecchio

, et al. Sentinel node biopsy compared with complete axillary dissection for staging early breast cancer with clinically negative lymph nodes: Results of randomized trial. Ann Oncol 2009; 20: 1001–1007.

Gill

, SNAC Trial Group of the Royal Australasian College of Surgeons (RACS) and NHMRC Clinical Trials Centre. Sentinel-lymph-node-based management or routine axillary clearance? One-year outcomes of sentinel node biopsy versus axillary clearance (SNAC): A randomized controlled surgical trial. Ann Surg Oncol 2009; 16: 266–275.

Krag

Anderson

Julian

, et al. Sentinel-lymph-node resection compared with conventional axillary-lymph-node dissection in clinically node-negative patients with breast cancer: Overall survival findings from the NSABP B-32 randomised phase 3 trial. Lancet Oncol 2010; 11: 927–933.

Veronesi

Viale

Paganelli

, et al. Sentinel lymph node biopsy in breast cancer: Ten-year results of a randomized controlled study. Ann Surg 2010; 251: 595–600.

Schwartz

D’Ugo

Rosenberg

. Extent of axillary dissection preceding irradiation for carcinoma of the breast. Arch Surg 1960 1986; 121: 1395–1398.

Tandon

. Regulatory requirements for designing PET-CT facility in India. Indian J Nucl Med 2010; 25: 39–43.

10.

Chang

Lee

, et al. Sentinel lymph node detection using fluorescein and blue light-emitting diodes in patients with breast carcinoma: A single-center prospective study. Asian J Surg 2020; 43: 220–226.

11.

Gao

Yang

, et al. Application of fluorescein combined with methylene blue in sentinel lymph node biopsy of breast cancer. Sci Rep 2021; 11: 12119.

12.

Valiveru

Agarwal

Agrawal

, et al. Low-cost fluorescein as an alternative to radio-colloid for sentinel lymph node biopsy – a prospective validation study in early breast cancer. World J Surg 2020; 44: 3417–3422.

13.

Khadka

Suresh

Prem

, et al. Sentinel node mapping in early breast cancer: a randomized comparison of fluorescein guided All India Institute of Medical Sciences, Anurag’s technique with Technetium-99 m Sulfur colloid plus methylene blue. Indian J Nucl Med 2022; 37: 236–244.

14.

Chavda

Mishra

Silodia

, et al. Validation sentinel lymph node biopsy study in cN0 axilla using low-cost dual dye technique: Potential solution for resource poor settings. Breast Cancer Res Treat 2022; 193: 105–110.

15.

Kersey

Van Eyk

Lannin

, et al. Comparison of intradermal and subcutaneous injections in lymphatic mapping. J Surg Res 2001; 96: 255–259.

16.

Yadav

Bharath

Sharma

, et al. A systematic review and meta-analysis of diagnostic performance of fluorescein-guided sentinel lymph node biopsy in early breast cancer. Breast Cancer Res Treat 2024; 206: 19–30.