Combined Fluorescein and Methylene Blue Dye for Sentinel Lymph Node Biopsy in Patients of Early Carcinoma Breast: A Promising Technique

Abstract

Sentinel lymph node biopsy is currently the gold standard for clinically node-negative patients of carcinoma breast. Fluorescein is a safe, low-cost agent, and easily available. Fluorescein has shown a promising role in sentinel lymph node evaluation in carcinoma breast in combination with methylene blue dye with a detection rate of more than 90% and a false-negative rate of less than 10% in previous studies. This study aims to determine the detection rate and diagnostic accuracy of fluorescein and methylene blue dye in early breast cancer. The identification rate and false-negative rate of the combined blue and fluorescent dye method were 100% and 7.14% respectively. The accuracy of the combined blue and fluorescein dye method was 98.3%. The sensitivity, specificity, negative predictive value, and positive predictive value of the combined blue dye and fluorescein dye method were 92.8%, 100%, 97.8%, and 100% respectively. Thus, the combined blue and fluorescein dye method is an easy, safe, cost-effective, and reliable method of sentinel lymph node biopsy in early breast cancer patients.

Breast cancer is the most commonly diagnosed cancer and also the leading cause of cancer-related mortality [1]. In India, early breast cancer constitutes 30% of the breast cancer cases [2]. Early-stage breast cancer is potentially curable with more than 80% of patients having long-term survival after complete treatment [3]. Axillary lymph node involvement is an important prognostic factor for patients with carcinoma breast. Identification of axillary lymph node metastasis is important to determine stage and subsequent neoadjuvant and adjuvant treatment [4]. The incidence of axillary lymph node metastasis varies from 23.0 to 37.1% in early breast cancer [5].

Sentinel lymph node biopsy is currently the gold standard for clinically node-negative patients of carcinoma breast [6]. Numerous trials have concluded the oncological benefit and safety of SLNB for axillary staging [6]. SLNB has also led to a significant reduction in morbidity in comparison to formal axillary lymph node dissection procedure [7].

Various techniques of sentinel lymph node biopsy have been developed over time. At present, commonly used lymphatic mapping agents are Tc 99-labeled radioisotopes, blue dyes, and combination of both [6]. The American Society of Clinical Oncology (ASCO) suggests sentinel lymph node biopsy with dual tracer technique (radioisotope and blue dye) as a most widely accepted method with high detection and low false-negativity rate, i.e., > 90% and < 10% respectively [8].

Despite many advantages of the dual method, there are several limitations associated particularly with radiotracer use such as the high cost and availability of radioisotopes especially in developing countries, concerns related to radiation exposure, radioisotope handling, and safe disposal of radioactive waste, mandatory license, and facility of nuclear medicine department [9]. Owing to these several disadvantages, many new techniques have been developed for sentinel lymph node mapping such as use of fluorescent agents, superparamagnetic iron oxide, CT lymphography, and contrast-enhanced ultrasound [10].

ICG and fluorescein sodium are the two most commonly used fluorescent dyes in the field of medicine [10]. Fluorescein is widely used in the field of ophthalmology for procedures such as retinal angiography, neurological procedures, and bio-imaging. It has also been successfully used for sentinel node mapping in colorectal tumors [10–13]. Fluorescein is low-cost agent with easy availability, ease of use, and minimal adverse effects. Early studies have described its promising role in sentinel lymph node evaluation for various carcinomas particularly carcinoma breast. These studies have found the detection rate with a combination technique of fluorescein dye and methylene to be more than 90% and a false-negative rate of less than 10% [9, 14].

This study is conducted to determine the detection rate and diagnostic accuracy of fluorescein dye for sentinel lymph node biopsy with a goal to revolutionize the concept of sentinel lymph node biopsy by obviating the necessity of radiotracer. Fluorescein-guided sentinel lymph node biopsy being easy, accessible, safe, and cost-effective technique can be seamlessly integrated as a standard treatment especially in resource constrained developing nations.

Aims and Objectives

Aim of Study

The study aims to evaluate the detection rate and false-negative rate of combined fluorescein and methylene blue technique in patients of early carcinoma breast.

Objective of Study

The objective of this study is to find out the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of combined fluorescein and methylene blue technique for sentinel lymph node biopsy in patients of early carcinoma breast.

Material and Methods

This study was a descriptive type of observational study with a cross-sectional study design. This study protocol was approved by the Institutional Scientific Committee & Institutional Ethics Committee. The study was conducted among 60 patients with early breast cancer who fulfilled the inclusion criterion. Inclusion criteria included patients with ages between 18 and 70 years, diagnosed as (T1, T2) clinico-radiological node negative, histopathological confirmed cases of carcinoma breast with Karnofsky Performance Status (KPS) > 70. Exclusion criteria included patients with locally advanced breast cancer, multicentric/multifocal tumor, axillary nodes confirmed for malignancy on cytology (FNAC), previous history of lumpectomy/NACT/irradiation for this specific cancer, pregnant patients/lactating mothers, second malignancy/recurrence in same breast, patients with known allergies or positive for allergy to any of the dye on sensitivity testing, and non-cooperative patients.

Histopathologically confirmed cases of early breast cancer with essential workup including ultrasonography/mammography of bilateral breast, USG bilateral axilla, and metastatic workup (if indicated) for accurate staging of the disease were enrolled. Patients were evaluated for fitness for surgery. Suspicious axillary nodes on clinical examination or sonography were confirmed by FNAC. Patients with FNAC reports of positive for malignancy were excluded from the study. Surgical procedure for carcinoma breast either BCS or mastectomy was decided by patients and senior consultants as per findings and counseling.

After prior allergy testing and induction of general anesthesia. Two milliliters of sterilized 1% methylene blue was injected intraparenchymal peri tumoral region at 12, 3, 6, and 9 o’clock position in 0.5-ml divided doses. The injection site was massaged for 5 min toward the axilla. This was followed by an injection of 10% fluorescein sodium (1 ml of 20% fluorescein sodium in 1 ml of distilled water). Two milliliters of 10% fluorescein sodium was injected sub-dermally in the peri-areolar region at 12, 3, 6, and 9 o’clock position in 0.5-ml divided doses. Injection site was massaged for another 5 min.

The axilla was entered through a skin crease incision. A lymphatic tract of blue dye was followed to identify the blue sentinel nodes. After turning off the OT lights, with the use of ultraviolet (wavelength 395 nm) LED torch, the direction of lymph vessels seen as green fluorescent and the location of their disappearance was traced to remove the fluorescent sentinel lymph nodes. The SLNs were excised and labeled as blue or fluorescent or their combination depending on which dye identified such SLNs. Besides this, any palpable nodes were also removed and classified as other palpable nodes. Meanwhile, mastectomy/breast conservation surgery (BCS) along with complete axillary lymph node dissection was done in all the cases (level III dissection done for any palpable node at level II or III). This specimen was sent to the department of pathology labeled as “Rest of ALND Specimen.”

Histopathological evaluation of all the lymph nodes, i.e., the SLNs and the axillary nodes were done. They were dissected off the axillary tissue/fat and fixed in 10% neutral-buffered formalin, embedded (whole lymph node if small, bisected along the long axis if large) in paraffin, sectioned serially to make three sections from each block. Thus, the total number of sections examined from each node is either 3 (for small nodes which are embedded non-bisected) or 6 (for large nodes which are bisected and then embedded). Sections were stained with hematoxylin and eosin and evaluated for evidence of metastasis by light microscopy. The detection rate, false-negative rate, sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were calculated using standard formulas Figs. 1, 2, 3, 4, 5, 6.

Fig. 1.

Peri-areolar injection of fluorescein dye

Fig. 2.

Peritumoral injection of methylene blue dye

Fig. 3.

Ultraviolet (blue light) LED torch

Fig. 4.

Fluorescent lymphatic channel leading to its fluorescent sentinel lymph node visualized using UV (BLUE) LED Torch

Fig. 5.

Dissected fluorescent sentinel lymph node

Fig. 6.

Dissected blue sentinel lymph node

Results

A total of 60 patients were included in the study. The mean age of patients in the study was 49.4 years. Blue nodes were identified in 51 patients, fluorescent nodes were identified in 59 patients, and patients identified by combined blue and fluorescent dye method were 60 patients out of a total of 60 patients. The identification rate of blue dye alone was 85%, fluorescent dye alone was 95%, and the combined blue and fluorescent dye method was 100%.

Sentinel blue nodes were found to be negative on histopathology in 3 out of 14 pathological positive patients. The false-negative rate for the blue dye method was 21.4%. Sentinel fluorescent nodes were negative on histopathology in 1 out of 14 pathological node-positive patients. The false-negative rate for the fluorescein dye method was 7.14%%. The false-negative rate for the combined blue and fluorescein dye method was 7.14%

The accuracy of the blue dye alone and fluorescein dye alone method was 94.1% and 98.3% respectively. The accuracy of the combined blue and fluorescein dye method was 98.3%. The sensitivity, specificity, negative predictive value, and positive predictive value of blue dye alone were 78.6%, 100%, 92.5%, and 100% respectively. The sensitivity, specificity, negative predictive value, and positive predictive value of fluorescein dye alone were 92.8%, 100%, 97.8%, and 100% respectively. The sensitivity, specificity, negative predictive value, and positive predictive value of the combined blue dye and fluorescein dye method were 92.8%, 100%, 97.8%, and 100% respectively Tables 1, 2, 3, 4, 5, 6, 7.

Table 1.

Identification rate

Method	Identified (patients) N = 60	Not identified (patients) N = 60	Identification rate (sentinel node visualized in patients/total no. of patients)
Blue dye	51	9	85%
Fluorescent dye	59	1	95%
Combined blue and fluorescent dye	60	0	100%

Table 2.

False-negative rate

Method	False-negative SLN (number of patients)	True-positive patients	False-negative rate (total no. of false-negative patients by respective method/total no. of TP + FN patients × 100%)
Blue dye alone	3	11	21.4%
Fluorescent dye alone	1	13	7.14%
Combined blue and fluorescent dye	1	13	7.14%

Table 3.

Accuracy of method

Method	Accuracy
Blue dye	94.1%
Fluorescent dye	98.3%
Combined blue and fluorescein dye	98.3%

Table 4.

Sensitivity, specificity, negative predictive value, and positive predictive value

Method	Sensitivity	Specificity	NPV	PPV
Blue dye	78.6%	100%	92.5%	100%
Fluorescein dye	92.8%	100%	97.8%	100%
Combined blue and fluorescein dye	92.8%	100%	97.8%	100%

Table 5.

Characteristics of patients with non-visualized and false-negative nodes by the combined fluorescent and blue dye method

Characteristics	Not identified	False negative
Age  < 55  ≥ 55	1	1
Tumor location (Breast Quadrant) a) UO b) UI c) LO d) LI e) RA Total	1 1	1
Histology a) IDC b) ILC c) Mucinous d) Medullary Total	1 1	1
T Stage a) T1 b) T2 Total	1 1	1

Table 6.

Distribution of patients according to pathological nodal stage

Pathological nodal stage	Total patients
	No	%
pN0	46	76.67
pN1	14	23.33
Total	60	100.00

Table 7.

Mean number of sentinel lymph nodes identified according to dye

	Total no. of lymph nodes harvested	Mean nodes	Standard deviation	Minimum nodes	Maximum nodes
Blue nodes	111	1.85	1.12	0	4
Fluorescent nodes	189	3.15	0.92	0	6
Blue and fluorescent nodes	81	1.35	0.92	0	4
Combined blue and fluorescent nodes	219	3.65	0.90	2	6

Discussion

Detection Rate

In our study, the detection rate of the blue dye alone, fluorescent dye alone, and combined blue and fluorescent dye method was 85%, 95%, and 100% respectively. The detection rate using methylene blue dye only has been reported in the range of 65 to 96.1% in published literature [15]. Golshan et al. [15], Nikhils et al. [15], Kasula et al. [15], and Mathelin et al. [15] reported identification rate using methylene blue dye as 96.1%, 86.7%, and 65% respectively. The identification rate using methylene blue dye and radio colloid has been reported in the range of 94 to 100% by Cox et al. [16] and Somashekar et al. [17] respectively.

Lidong Ren et al. [18] reported a 100% identification rate in an animal experimental study to investigate the feasibility of using 10% fluorescein sodium as a new dye for sentinel lymph node biopsy in 30 rabbits. Young Woo Chang et al. [9] reported identification rate as 93.4%. Valiveru Ramya C. et al. [14] reported identification rate methylene blue as 82% and using fluorescein dye as 92% while by combined methylene blue dye and radio colloid as 96.9% and combined methylene and fluorescein dye was 95.4%. Liang Li et al. [12] reported an identification rate using methylene blue dye as 83.9% and using fluorescein dye as 87.5% while using both methylene blue and fluorescein dye was 92.9%.

False-Negative Rate

In our study, false-negative rate for blue dye, fluorescein dye, and combined blue and fluorescent dye method was 21.4%, 7.14%, and 7.14% respectively. The false-negative rate using methylene blue dye only technique ranges from 3 to 30% in the literature. Bakhtiyar et al. [15], Ozdemir et al. [15], and Chen et al. [15] have reported false-negative rate using methylene blue dye only as 3%, 15%, and 30% respectively. The false-negative rate using combined methylene blue dye and radiotracer is reported in the range of 8 to 29% with an average false-negative rate of 7.5%. Valiveru Ramya C. et al. [14] reported a false-negative rate with methylene blue dye, fluorescein dye and combined methylene blue and radiotracer and methylene blue, and fluorescein dye combined fluorescein as 25%, 12.5%, 6.3%, and 6.3% respectively. Liang Li et al. [12] reported a false-negative rate of combined methylene blue dye and fluorescein dye method as 7.5%. The results of our study are comparable with the previous literature on methylene blue and radio colloid method and similar studies of fluorescein dye method.

Accuracy

We reviewed the literature, and due to paucity of data on sentinel lymph node biopsy using fluorescein dye, we compared the accuracy, sensitivity, specificity, negative predictive value, and positive predictive value of fluorescein dye alone and combined fluorescein and methylene blue dye with the current standard sentinel lymph node biopsy technique, i.e., radio colloid and methylene blue dye technique. In our study, the accuracy of blue dye alone and fluorescein dye alone method was 94.1% and 98.3% respectively. The accuracy of combined blue and fluorescein dye method was 98.3%. Zaazou et al. [15], Sohail et al. [15], and Ozdemir et al. [15] have reported the accuracy of methylene blue technique as 95.3%, 93.4%, and 92.7% respectively. The accuracy of the combined radiotracer and blue dye technique ranges from 91.5 to 99% in published literature [17]. Deo et al. [17] and Somashekhar et al. [17] reported the diagnostic accuracy of radio colloid and methylene blue as 91.3% and 99% respectively.

Sensitivity, Specificity, NPV, and PPV

In our study, the sensitivity, specificity, negative predictive value, and positive predictive value of blue dye alone, fluorescein dye alone, and combined blue dye and fluorescein dye method were 78.6%, 100%, 92.5%, and 100% respectively; 92.8%, 100%, 97.8%, and 100% respectively; and 92.8%, 100%, 97.8%, and 100% respectively.

The sensitivity and specificity of the methylene blue dye technique have been reported in the range of 81.8 to 100% and 45.5 to 100% in the previous literature [15, 19]. The NPV and PPV of methylene blue technique are reported in the range of 75 to 100% and 75 to 100% in the previous literature [13, 20]. Varghese et al. [19] in their study reported sensitivity of blue dye as 75%. Radovanovic et al. [21] in their study reported sensitivity of methylene blue technique as 82%, specificity as 60%, and NPV as 86.9%. Ozdemir et al. [15] and Somashekhar et al. [17] reported positive predictive value of methylene blue dye technique as 90 and 100% respectively. Radovanovic et al. [21] reported sensitivity, specificity, and NPV of combined radiotracer and blue dye as 95%, 96.2%, and 95.3%respectively. Somashekhar et al. [17] reported sensitivity, specificity, NPV, and PPV of combined radiotracer and blue dye as 93%, 100%, 98.6%, and 100% respectively.

Characteristics of Patients with Low Detection Rate and False-Negative Sentinel Lymph Nodes

In our study, fluorescein dye failed to identify sentinel lymph nodes in 1 patient, with age more than 55 years, mucinous carcinoma in upper inner quadrant with stage pT2. Older age has been reported as an important factor in non-visualization of the sentinel lymph node in several studies. The reason behind this is the with advancing age breast tissue is replaced with fatty tissue which leads to sluggish movement of dye in the lymphatics [22]. Straalman et al. [23] reported sentinel node identification failure rate using radio colloid and blue dye as 15.2% for patients older than 65 years, 8.3% for patients aged 55–65, and 6.2% for patients younger than 55 years. The SN identification failure rate was 13.7% when tumor was located in medial quadrants and 7.7% when tumor was located centrally or in lateral quadrants. Yu ling Liu et al. [24] reported that lymphatic drainage in tumor size larger than 2.5 cm may be directed to a node other than true sentinel nodes.

In our study, fluorescein dye alone and combined methylene blue and fluorescein dye technique was false negative in single patient with lump in upper inner quadrant, invasive lobular carcinoma histopathology, T2 pathological T stage, and 3 sentinel lymph nodes harvested. Goyal et al. [25] demonstrated that tumor location, tumor size, tumor histology, and presence of multifocality did not alter the false-negative rate. However, high-grade tumors have a higher incidence of nodal metastases, thereby have an increased risk of lymphatic obstruction and re-routing of tracer leading to a false-negative result. Wei et al. [26] also found that high-grade and lobular carcinoma is associated with a high false-negative rate. Yi M. et al. [27] found that mixed ductal and lobular histology, inner quadrant tumor location, and T1 tumor stage significantly increased the number of SLNs that needed to be removed to achieve 99% recovery of all positive SLNs.

Adverse Effects and Complications

Common adverse effects with use of fluorescein and methylene blue are skin inflammation or necrosis at the injection site, anaphylaxis, and allergic manifestations such as rashes and urticaria [13, 28]. We did not find any of the adverse effects related to either methylene blue or fluorescein dye in our patients. However, we did notice skin discoloration with fluorescein and blue dye which lasted for a few days and urine discoloration with fluorescein dye which lasted for 24 h.

Conclusion

This study concludes that the combined blue and fluorescein dye method has an identification rate of 100% and false-negative rate of 7.14% only. Thus, this technique is an easy, safe, cost-effective method of sentinel lymph node biopsy in early breast cancer patients with an accuracy of 98.3%. This study also demonstrates that the combined blue and fluorescein dye method in early breast cancer is comparable to the current standard combined radio colloid and blue dye method in terms of identification rate, false-negative rate, accuracy, sensitivity, specificity, and negative and positive predictive value. This study also highlights the potential of fluorescein dye as an easy, safe, and cost-effective single-agent sentinel lymph node biopsy method for early breast cancer. This study also indicates that addition of methylene blue dye to fluorescein dye method only improves the yield of sentinel nodes and does not affect the identification rate and false-negative rates.

Declarations

Conflict of Interest

The authors declare no competing interests.