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Sentinel lymph node biopsy (SLNB) is currently used in the UK for axillary staging in breast cancer. Node localisation is achieved with the combination of injected dye and radioactive tracer.1 The commonly used dyes are the isomers isosulphan blue and Patent blue V (PBV).1,2,3 Methylene blue (MB) is also used in some centres as it is cheaper and easily available.4 In the Far East, other dyes such as Indigocarmine (IDC) are also being used.5
In the 1980s, when dyes were used to localise occult breast cancers before surgery, studies were carried out on the effect of the dyes on oestrogen receptor (ERα) assessment by ligand‐binding assay. These showed a decrease in the binding capacity after treatment with MB but not with isosulphan blue.6,7 Given the importance of the determination of ERα expression in the treatment of breast cancer and the wider use of SLNB to stage the axilla, we investigated whether the dyes used change the immunocytochemical profile of these receptors.
The ERα‐positive and progesterone receptor (PR)‐positive malignant breast cell line MCF‐7 was cultured under standard conditions and exposed to the dyes diluted 1:10, 1:100 and 1:1000 in triplicate for 4 and 24 h. The dyes used were 2.5% PBV (Guerbert, France), 1% MB (Martindale Pharmaceuticals, UK) and 4% IDC (Amino), which are the concentrations used clinically for the SLNB procedure. Appropriate controls were established in parallel. After incubation, cells were trypsinised and embedded in paraffin wax blocks.8
Sections were cut and stained as per the procedures routinely used for ERα, PR and cytokeratin MNF116. The primary antibodies used ER‐6F11 (Novocastra, Newcastle upon Tyne, UK) diluted 1:30, PgR 636 (DakoCytomation, Glostrup, Denmark) diluted 1:50 and MNF116 (DakoCytomation) diluted 1:100. Secondary detection was achieved using the ABC technique (DakoCytomation) for ERα and Envision (DakoCytomation) for PR and MNF116. Slides were incubated with DAB and counterstained with haematoxylin. Negative and positive controls were included with each batch. MNF116 was used to determine whether there was evidence to the fact that general immunoreactivity of the samples was affected by the overall effect of the dyes.
Slides were scored blindly using the Allred (quick) score for ERα and PR by two specialist breast histopathologists (AMH and AMS), and for any evidence of diminution in staining intensity for MNF116 by AMH.8 Statistical analysis was performed using InStat software V.3.06 with Dunn's multiple comparison test comparing the different dyes and dilutions with the controls. p Values of <0.05 were considered significant.
With higher concentrations of MB dye (1:10 and 1:100 dilutions of the 1% MB preparation) there was a significant decrease in both ERα and PR scores after 4 and 24 h exposure except for the PR 1:100 dilution, 4 h exposure (fig 11).). No significant change was seen with the MB 1:1000 dilution (table 11).). On 24 h exposure, cells incubated with PBV showed a non‐significant decrease in ERα score on the 1:10 dilution, whereas IDC showed a non‐significant decrease in PR score with the 1:10 dilution.
All the slides stained strongly positive for the MNF116 on 24 h exposures, and, as no effect was seen, they were not stained for the shorter exposure.
The presence of ERα in breast cancer is an established prognostic marker determining the likelihood of response to anti‐hormonal therapy. PR is a marker of ERα functionality. ERα‐positive and PR‐positive tumours are more likely to respond to anti‐hormonal treatment than ERα‐positive and PR‐negative tumours; there is some evidence that ERα‐positive and PR‐negative tumours are more likely to respond to aromatase inhibitors than tamoxifen.9,10,11 In this study, we have explored the hypothesis that the dyes used in SLNB could interfere with hormonal receptor determination. A significant diminution in scoring was seen with cells treated with MB at 1:10 and 1:100 dilutions, with some samples rendered negative. This was not seen with PBV and IDC (table 11).). These results suggest that an inherently weak ERα expresser could be rendered negative after exposure to MB with consequent withholding of potentially beneficial treatment. However, it is difficult to accurately relate in vitro findings to an in vivo situation. No similar effect was seen on the results of MNF116 immunolabelling, and the effects of these dyes on other markers would need case‐by‐case evaluation.
Hirsch et al6 measured MB tissue concentrations in breast tissue samples from a patient who had 0.1–0.2 ml of 1% MB injected, using spectrophotometry. This showed tissue concentration of 0.08, which is roughly equal to 1:10 dilution of the dye. The volume used for SLNB is larger, with 2 ml routinely injected for PBV and up to 5 ml for MB.1,4,12,13 Although the recommended site of injection for PBV in the UK is subdermal, around the areola, this is not the case with MB because of potential injection site complications, especially skin necrosis.14 The site of injection for MB is deeper in the breast tissue.13,15 Deep subareolar injections of MB have been previously described.12 Probably, with deeper peritumoral injections, the concentration of MB in the tumour could be in the region of 1:10 or even higher because of the larger volume used, whereas it should be lower with injections at sites more distant from the tumour. These concentrations are comparable to those that reduced ERα and PR status in our experimental system. The time durations of exposure to the chosen dyes were pragmatic: the shorter 4 h exposure to represent the duration after a straightforward excision transferred promptly to pathology, whereas the longer time was chosen to represent delayed fixation.
The concentration dynamics and the cell time exposures are likely to be highly complex. In cell line studies, direct and rapid exposure of the cells to the dye occurs. In the context of a complex tissue, even when the dye can be directly visualised pervading the tumour on gross analysis, the uniformity and certainty of cell exposure can only be hypothesised.
In summary, MB; which is one of the dyes used for SLNB worldwide, can reduce ERα and PR staining levels but not MNF116, in vitro. We suggest that if MB is used for SLNB, ERα should be checked on the core biopsy, if it was negative in the excised tumour.
Funding: We thank the Breast Cancer Research Action Group for their continuous support.
Competing interests: None.