Methylene blue, chemically known as methylthioninium chloride, is a green powder that becomes a blue solution when dissolved in water. It is a derivative of phenothiazine that is used as a dye. Its medical use has been approved by the FDA for the treatment of methemoglobinemia [1] resulting from exposure to dapsone, benzocaine, nitroglycerin, or amyl nitrite.
The remaining indications, including vasoplegic syndrome, ifosfamide-induced encephalopathy, or Plasmodium falciparum infections are not approved by the FDA [2-4]. Methylene blue is also used off-label in oncology, particularly for breast cancer.
Methylene blue applications in breast cancer
Breast cancer has become the most diagnosed cancer worldwide in 2020. Its incidence is continuously increasing and the number of patients with breast cancer is estimated to reach 364,000 in 2040 in the United States [5]. Breast cancer mortality decreases with early-stage diagnosis. Therefore, mammography screening and women’s awareness of breast cancer lead to better outcomes. In addition, improvement of breast cancer treatments increased survival and contributed to the reduction or stabilization of breast cancer mortality in developed countries [6].
However, breast cancer has recently become the fifth leading cause of mortality from cancer among women worldwide [7]. It causes one in six cancer-related deaths making breast cancer the leading cause of cancer mortality in many countries [8].
Methylene blue has various applications as a dye in breast cancer. It is used in sentinel lymph node biopsies. In perioperative settings, methylene blue is used to identify non-palpable breast lesions. In intraoperative imaging, it is used to assess surgical margins. Finally, methylene blue may be used as a photosensitizer in experimental photodynamic therapy for breast cancer.
Methylene blue in sentinel lymph node biopsy (SLNB)
Methylene blue is a dye used in sentinel lymph node biopsy (SLNB) in the context of breast cancer. SLNB is a procedure that identifies the first lymph node ("sentinel" lymph node) that cancer cells are likely to spread to from a primary tumor. This technique helps to determine whether cancer has spread to the lymphatic system, which is critical for staging the disease and planning treatment. For this purpose, methylene blue is injected near the tumor site. It then follows the lymphatic channels to the sentinel lymph node, the first node that drains the specific tumor location. Once dyed, this sentinel lymph node can be removed surgically for pathological examination [9,10].
While other dyes have been proposed for SLNB, including patent blue or isosulfan blue, methylene blue remains a good option given its properties and cost. A single-blinded two-arm parallel design randomized control trial was conducted in India. It involved 237 patients with breast cancer who underwent SLNB performed with either patent blue violet or methylene blue in combination with a radioactive tracer. The results showed that methylene blue and patent blue violet were very similar in identifying cancer lymph nodes. However, methylene blue costs much less, making it the dye technique of choice in low-resource settings [11]. Another clinical study involving 415 patients with early breast cancer evaluated the combined use of indocyanine green with methylene blue for SLNB. All patients underwent SLNB, half of them were injected indocyanine green with methylene blue, while the other half received methylene blue only. All sentinel lymph nodes were harvested for pathological examination and detection and the false negative rates were compared between groups. Results showed that the detection rate was significantly higher in the indocyanine green with methylene blue group compared to the methylene blue group while the false negative rate showed no differences between groups. Therefore, the combination of indocyanine green with methylene blue is much more effective than methylene blue alone in detecting sentinel lymph nodes [12]. Another trial compared three tracer methods for SLNB: indocyanine green combined with methylene blue, methylene blue alone, and carbon nanoparticles. Patients with early breast cancer were divided into three groups of 60 patients each before receiving one of the three tracer methods. The results showed that indocyanine green combined with methylene blue treatment was superior to methylene blue only and carbon nanoparticles in predicting axillary lymph node metastasis [13]. Finally, a prospective, randomized trial involving 142 patients with invasive breast carcinoma compared methylene blue with patent blue to detect the sentinel lymph node. Results showed that methylene blue performed as well as patent blue in identifying the SLNB in breast cancer patients while being more widely available, less expensive, and with a lower risk of anaphylaxis than patent blue [14].
Many studies have shown that methylene blue is effective in identifying the sentinel lymph node, with identification rates comparable to other dyes. It is also often used in conjunction with a radioactive tracer to increase the accuracy of the procedure. Beyond its availability and cost-effectiveness compared to other dyes, methylene blue is also easier to handle and store, making it a practical option in various clinical settings.
Methylene blue in the identification of non-palpable lesions
Methylene blue is also used in pre-operative settings in combination with imaging techniques like ultrasound or mammography to identify non-palpable lesions. Indeed, non-palpable lesions, highly suggestive of malignancy by imaging examination, require excision for pathological examination. The dye is injected into the area of interest for the surgeon to locate and accurately remove the possible cancer lesion. Detection of non-palpable breast masses has increased with the widespread use of mammography. Therefore, perioperative localization has become critical to reduce the number of false negatives and decrease the volume of tissue resection.
A clinical trial involving 51 patients with non-palpable masses in the breasts evaluated methylene blue in combination with ultrasound to localize the lesions for resection. All resections were successful. Only one mass was not found during surgery because of a rapid dye washout; however, another methylene blue injection during the operation resulted in its successful removal. No allergic reactions were observed. Therefore, methylene blue injection is a safe and low-cost method to use in combination with ultrasound to localize non-palpable lesions before surgery [15]. Another study evaluated methylene blue injection into non-palpable lesions under mammographic guidance. A small dose of methylene blue was injected into each of the 138 patients before lesion resection. All suspicious lesions were accurately excised and the results demonstrated that methylene blue is a safe and simple procedure with high diagnostic accuracy [16].
Methylene blue in the assessment of surgical margins
Methylene blue is similarly used after excising a breast tumor to stain surgical margins and assess whether these are cancer-free and determine whether all cancerous tissue has been removed. Therefore, in combination with other imaging agents, methylene blue is used to improve the accuracy of tumor resection.
A preliminary study evaluated methylene blue-based near-infrared fluorescence imaging for breast cancer resection and involved 30 patients. Methylene blue injection was performed three hours before tumor resection. After surgery, the resected breast tissues were used to identify the tumor by the fluorescence contrast. No severe adverse events were observed and this technique showed a sensitivity of 63% and a positive predictive value of 0.79. Therefore, methylene blue fluorescence imaging could offer great possibilities in breast-conserving surgery for tumor-positive margin detection [17]. Another clinical trial involving 24 patients with breast cancer with a planned surgical resection evaluated methylene blue's ability to detect breast cancer intraoperatively using near-infrared fluorescence imaging. The results demonstrated that methylene blue reached an overall cancer identification rate of 83%. Additionally, the real-time guidance with methylene blue may lead to more accurate or effective management decisions during surgery [18].
Methylene blue in photodynamic therapy
Photodynamic therapy relies on the uptake of a photosensitizer that will, upon excitation by light in a specific wavelength, react with oxygen to generate oxidant species in targeted tissues and lead to photo-oxidative stress. Methylene blue is currently being explored as a photosensitizer in experimental photodynamic therapy for breast cancer to induce cancer cell death.
Different studies in breast cancer cells have been performed in vitro and several methylene blue mechanisms were identified. Photo-oxidation using methylene blue leads to lysosomal damage and triggers ferroptosis (an iron-dependent form of regulated cell death [19]) as well as necroptosis in tumor cells, particularly in highly aggressive triple-negative breast cancer cells. On the other hand, non-tumor cells can develop an efficient antioxidant response against methylene blue [20]. Therefore, methylene blue differentially induces massive cell death of breast tumor cells and appears as an efficient strategy that could be used as an adjunct therapy to surgery to minimize the risks of cancer recurrence [21]. An in vitro study shows that the efficiency of photodynamic therapy significantly improves when methylene blue is chemically combined with salicylic acid, an anticancer agent [22]. Cellular uptake of the dye through the cell membrane is difficult and limits cancer cell apoptosis. Several drug carriers have been successfully tested to improve methylene blue uptake including liposome [23], poly(N-isopropylacrylamide) microgel [24], and graphene oxide nano-sheets [25]. By increasing methylene blue uptake, its cytotoxicity is enhanced in vitro suggesting great potential for breast cancer therapy.
Lack of treatments for aggressive breast cancer is a major health problem and photodynamic therapy with methylene blue appears as a valuable therapeutic strategy for these highly aggressive tumors that should be investigated further.
Methylene blue safety profile
Although methylene blue has a long history of use and is thought to be safe, there is a lack of preclinical toxicology data depending on its applications. Its toxic effects are dose-dependent and include severe symptoms such as hemolysis, methemoglobinemia, nausea, chest pain, dyspnea, dizziness, confusion, or headaches. Methylene blue can also trigger skin irritation and urine discoloration. Additionally, it may increase the risk of serotonin syndrome, especially when combined with other serotonergic drugs, that manifests through neuromuscular hyperactivity, including diaphoresis, clonus, and tremors. Methylene blue is generally well-tolerated when used as a dye in oncology settings. It can cause localized skin reactions or, in rare cases, allergic reactions. If the dye extravasates from the injection site, methylene blue can trigger skin necrosis [26,27].
Conclusion
Methylene blue is a valuable tool in breast cancer management given its large range of applications from aiding in the precise localization and removal of tumors to experimental uses in photodynamic therapy. Its cost-effectiveness, availability, and ease of use make it a popular choice in many surgical settings, contributing to the accurate staging of breast cancer and informed treatment decisions.
References
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