The potential of indocyanine green fluorescence detection in surgical cut margin of breast conserving surgery

Introduction

Breast cancer has the highest incidence rate among women worldwide and is the most common form of malignancy among all cancers. Each year, an estimated 2.3 million new cases (11.7%) and 700,000 deaths (6.9%) are attributed to breast cancer (1,2). Despite the growing range of treatment options available for breast cancer, surgery remains an important aspect of early-stage treatment (3). Breast-conserving surgery (BCS) is considered a minimally invasive surgical procedure that aims to remove the tumor while preserving the breast tissue. Although BCS minimizes the physical and psychological morbidity for patients, achieving clear surgical margins is crucial to reduce the risk of local recurrence (4). Positive margins triple the risk of ipsilateral recurrence and may be responsible for 75% of local recurrences and metastases (5-7).

Currently, intraoperative margin assessment predominantly relies on surgeons’ visual and tactile judgment, followed by frozen section analysis for confirmation (8-10). However, frozen section analysis also has several disadvantages—it is time-consuming and labor-intensive, and requires skilled professionals for processing (11). Therefore, researchers are searching for other sensitive and convenient methods to reduce the positive surgical margin rate.

Fluorescence-guided surgery (FGS) is an innovative technique that uses fluorescent agents to improve surgical outcomes by enhancing intraoperative visualization of desired tissues and structures. By labelling certain tissues or structures with a fluorescent dye, the surgeon is able to visualize and distinguish them more easily during a surgical procedure (12). It shows great potential in cardiac surgery, oncological surgery, or neurosurgery (13,14). Indocyanine green (ICG) is a water-soluble tricarbocyanine dye that can be detected by near-infrared fluorescence (NIF) imaging equipment. Its use in the assessment of cardiac and hepatic function, retinal imaging, and choroidal vasculopathy is well documented (15). In recent years, ICG has also been used to locate lymph nodes, such as in stomach (16-18), as well as for surgical localization of sites and metastases involving the tumor (19-21). The purpose of this study was to investigate whether ICG fluorescence can be used as a supplementary tool for frozen section analysis. In this study, we evaluated the value of different doses of ICG in determining surgical margins by injecting 0.5 mg/kg of ICG the morning before surgery and 1 mg/kg of ICG on the night before surgery. We present this article in accordance with the STROBE reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-24-195/rc).

Methods

Study methods

A non-randomized prospective cohort study was conducted. According to previous literature, the fluorescence visualization rate of ICG on tumor tissue is 0.893 and its 95% confidence interval is 0.808–0.978. Using the target value of 80.9%, a unilateral test level of 0.025, and a test efficacy of 80%, the sample size of the current study was calculated to be at least 51 cases (12,15). Taking into account the possibility of test shedding, 60 samples were expected to be included in this study. Eventually, based on the inclusion and exclusion criteria, 69 patients with early-stage breast cancer who underwent BCS in the Division of Breast Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Medical School, Nanjing University from August 2021 to October 2022 were selected.

The inclusion criteria were patients aged 18–75 years with breast cancer who were eligible for BCS. Exclusion criteria were inflammatory breast cancer, previous allergy to ICG and iodine, cardiovascular and cerebrovascular disease, significant organ damage, and inability to tolerate surgery. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital (No. 2020-024) and informed consent was taken from all the patients.

Surgery

Participants who met the inclusion criteria received ICG dissolved in sterile water at a dose of 0.5 or 1 mg/kg according to body weight mixed in 100 mL dextrose solution before surgery, and the infusion was completed by peripheral intravenous drip for 30 minutes. Patients received an injection of 0.5 mg/kg ICG at 6:00 on the day prior to surgery or 1 mg/kg ICG at 20:00 on the first day before surgery. The patient’s vital signs were closely monitored.

All surgeries in this trial were performed by senior consultants. The surgeries were performed according to the BCS procedure for breast cancer. After the specimen was removed from the body, the NIF imaging device called FLI-10B (Nanjing Nuoyuan Medical Devices, Co., Ltd., Nanjing, China) was used to image 30–40 cm above the patient’s primary tumor sites and the surrounding surgical field in accordance with the requirements of the Fluorescence Image Navigation System clinical trial programme. If an incisal margin is considered positive after NIF detection, supplementary excision is performed at the patient’s surgical site corresponding to the “positive” margin. The supplementary margin will be detected again by NIF detection. The images were captured under optimal lighting conditions in the operating room, using automatic exposure settings. Each side of the specimen was positioned and photographed according to preset parameters. The NIF detection was performed after completion of the examination, with specimens sent to frozen section analysis for detection after examination. Highlighted areas were individually labelled and sent for frozen section analysis. The specimen was selected from the lesion site. OCT embedding medium was applied, and the specimen was rapidly frozen at −20 ℃ for 3 minutes. The section thickness was maintained at 5–6 µm, and the sections were fixed in a 10% formalin solution, subjected to hematoxylin and eosin (HE) staining, mounted with rhamsan gum and evaluated by pathologists. All surgical specimens were followed by routine pathological examination, and the final status of the margins was determined according to the postoperative pathological results. All pathologies were analyzed by two experienced senior pathologists.

Quantitative analysis of fluorescence imaging

During the procedure, fluorescence measurements were taken using the FLI-10B instrument (Nanjing Nuoyuan Medical Devices, Co., Ltd.). The device has a color scale analysis system that can be quantitatively analyzed. During the procedure, the FLI-10B instrument’s built-in software was used to measure the fluorescence values on the specimen surface and the adjacent breast tissue, which was considered the ‘background’. Finally, the surface-to-background fluorescence ratio (SBR) was calculated for each specimen. We defined SBR >1.5 is the cut off value.

Medicines

ICG for injection, specification: 25 mg, manufacturer: Dandong Medical Chuang Pharmaceutical Co., Ltd. (Donggang, China), storage conditions: shielded from light, airtight, stored in a cold place (2–10 ℃). Glucose injection, specification: 100 mL, manufacturer: Otsuka (Zhangjiagang, China).

Statistical analysis

Quantitative variables were described by mean ± standard deviation (normal distribution) or median ± interquartile spacing (non-normal distribution). Categorical variables were described as numbers and proportions. For between-group comparisons, one-way analysis of variance (ANOVA) was used for analyses of continuous variables conforming to a normal distribution, the Kruskal-Wallis test for analyses of continuous non-normally distributed or hierarchical data, and Pearson’s chi-square test or Fisher’s exact test for analyses of categorical variables, with post hoc tests if significant P values were available (Bonferroni test) to determine where the differences were located. Kappa coefficient was used to assess the internal consistency and reliability of the measurement tools used in this study. The results of Kappa coefficient were classified as slight (0.0 to 0.20), fair (0.21 to 0.40), moderate (0.41 to 0.60), and substantial (0.61 to 0.80) and almost perfect (0.81 to 1). All analyses were performed under good clinical practice standards using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA).

Results

From August 2021 to October 2022, a total of 73 patients were recruited to the study. After excluding 3 patients due to loss of imaging data, a total of 69 patients were finally enrolled in the study. Patients received an injection of 0.5 mg/kg ICG at 6:00 on the day prior to surgery or 1 mg/kg ICG at 20:00 on the first day before surgery. Routine pathological examination of surgical margins was positive in 11 out of 69 patients. The NIF detection and frozen sections analysis detected 9 positive surgical margins respectively. At a dose of 0.5 mg/kg, NIF detected a positive incisal margin in 2/3 of cases, and at a dose of 1 mg/kg, NIF detected a positive incisal margin in 7/8 cases. Patient and tumor characteristics are shown in Table 1. Some of the indicators that may be relevant to the outcome are analyzed in Table 2. According to the effects of different doses, we conducted a separate analysis in Table 3. At the dose of 0.5 mg/kg, tumor grade, histologic type, and tumor size may affect the results of NIF detection. We cannot rule out this is due to the small sample size. No statistically significant factors were found at the dose of 1 mg/kg.

After removal of the mammary segment, we bisected the isolated specimens and placed it under the FLI-10B. It could be clearly seen that the fluorescence intensity gradually decreased from the center of the tumor to the surrounding, showing that the concentration of ICG in the tumor was much higher than that in the normal tissue (Figure 1).

Figure 1 Fluorescence of dissected tumor. NIF detection of isolated tumors in white light, fluorescence, color scale and merger modes. ICG can be found accumulating in the tumor tissue after sectioning the specimen, which is different from the paracancerous tissue. (A) Patient was injected 4 hours prior to surgery at a dose of 0.5 mg/kg. The tumor size was 1.5 cm. (B) Patient was injected 3 hours prior to surgery at a dose of 0.5 mg/kg with a tumor size of 1.8 cm. (C) Patient was injected 8 hours prior to surgery at a dose of 1 mg/kg with a tumor size of 2.3 cm. (D) Patient was injected 2 hours prior to surgery at a dose of 0.5 mg/kg with a tumor size of 1.9 cm. NIF, near-infrared fluorescence; ICG, indocyanine green.

A special case showed abnormal fluorescence images at the surgical cut margin (Figure 2A). The frozen section analysis of this sample did not reveal tumor at the surgical cut margin, while NIF detected high light fluorescence. After a supplementary resection, an intraoperative frozen section analysis was performed. The frozen section analysis showed the supplementary margin was negative, but routine pathology revealed a carcinoma in situ component 0.7 mm from the margin (Figure 2B).

Figure 2 Tumor specimens with positive margin. (A) Example of an ex vivo (after resection) whole specimen, abnormal fluorescence images were observed at the surgical cut margin and identified as residual tumor tissue. (B) Hematoxylin and eosin stains are used to determine tumor cells from normal cells. Postoperative pathology showed a carcinoma in situ component 0.7 mm from the margins whereas frozen biopsy margins showed no tumor tissue.

SBR >1.5 is the cut off value of this study. NIF detection groups refer to the detection of the primary tumor site and surrounding area by FLI-10B above 30–40 cm after the specimen has been isolated from the patient. Frozen section analysis groups refer to the analysis of sections of tissue by pathologists through freezing techniques. The true positive (TP) rate for NIF detection was 81.82%, whereas the true negative (TN) rate was 79.31%. The positive predictive value (PPV) was 42.85%, and the negative predictive value (NPV) was 95.83%. Frozen section analysis provided a TP rate of 81.82% and a TN rate of 98.28%, with a PPV of 90% and an NPV of 96.61% (Table 4). At a dose of 0.5 mg/kg, the TP rate was 66.67% whereas the TN rate was 93.33%. At the dose of 1 mg/kg, the TP rate was 87.5%, and the TN was 74.42%. Specifically, the NPV of NIF detection at 0.5 mg/kg dose was 93.33% and that of NIF detection at 1 mg/kg dose was 96.97% (Table 5). When calculating the sensitivity and specificity of different concentrations of ICG, it was discovered that the dose of 1 mg/kg increased the sensitivity of NIF detection compared to the dose of 0.5 mg/kg, but at the same time reduced its specificity. The Kappa coefficient for ICG and routine pathological tests was 0.447, while that for 0.5 mg/kg was 0.600, and for 1 mg/kg was 0.410, all indicating moderate consistency.

Discussion

Thanks to the advances in screening and examination techniques, breast cancer detection rates have steadily increased and the majority of breast cancers can now be detected at an early stage (22). BCS is a surgical procedure aimed at removing tumor lesions while preserving breast tissue. BCS aids in maintaining the patient’s physical and mental wellbeing, as well as promoting the appearance of their breast, when compared to radical mastectomy (23-25). However, BCS needs to ensure that the margin is negative to prevent recurrence (26). In addition to pathological methods, some new methods are also being studied, such as genetic testing, imaging evaluation, and optically- or artificial intelligence-assisted prediction models (27,28).

The main purpose of this study was to evaluate the accuracy of intraoperative NIF imaging in removing tumors of BCS. Conventional pathology showed that the final NIF NPV was 95.83%, which was slightly lower than the NPV of intraoperative frozen section analysis (98.28%). This result shows that intraoperative NIF imaging has certain potential in rapid identification of negative margins. Moreover, we have also increased our understanding of the effects of ICG at different time points and doses on tumor detection. This is crucial for the implementation of the technology and the interpretation of the results in future research.

Intraoperative frozen section analysis is a more accurate detection method and a key tool for BCS (9). However, it has disadvantages such as being time-consuming and labor-intensive (11,29). Previous studies have shown that the use of ICG can be used to identify tumors and normal tissues in colon cancers (30). ICG has been used to identify the surgical margins of gastrointestinal tumors, lung cancer, and so on (31-33). In breast cancer surgery, ICG has also been tried for tumor localization or margin determination with satisfactory results (34-36). At the same time, due to the existence of some special types of tumors in breast cancer, such as invasive micropapillary carcinoma, ductal carcinoma in situ, etc., it is difficult to identify the surgical margin (37,38). Therefore, this study explored the identification of surgical cut margin of BCS to find a more effective means of detection. During the operation, we removed the tumor completely and cut it. It could be clearly seen that the fluorescence intensity gradually decreases from the center of the tumor to the surrounding, which shows that ICG can specifically accumulate in the tumor tissue. Due to the characteristics of frozen sections, it is more difficult to identify the components of carcinoma in situ than those of invasive carcinoma for most breast cancers (39). Residual tumor is an important cause of postoperative recurrence (22,40). Among the 69 patients, 2 cases of frozen section analysis failed to identify positive surgical cut margin. One case was in situ carcinoma 0.7 mm away from the surgical cut margin, and the other case was 1 mm away from the surgical cut margin, and ICG was identified as positive surgical cut margin. A special case showed the first frozen section analysis failed to identify the positive surgical cut margin during the operation while NIF found the positive fluorescence. We performed an extended excision at the location of the margin during surgery. The second frozen section analysis showed that the margin was positive, and the total resection was finally selected. Similarly, frozen section analysis failed to detect positive incisal margins in two patients’ surgical cut margin, including 1 case of carcinoma in situ from the nearest margin of 0.2 mm, and the other case of carcinoma in situ from the nearest margin of <0.5 mm, and intraoperative frozen section analysis showed positive outcome. Therefore, ICG shows potential in the choice of surgical methods and prognosis of patients during surgery, which can be combined with frozen section analysis to reduce the possibility of secondary trauma and provide help for treatment.

In terms of ICG dose selection, we initially tried to use a dose of 0.5 mg/kg to conduct experiments around 6 hours before surgery, but this time point tends to be in the early hours of the morning, which has a certain impact on the conduction of the experiment. Therefore, the subsequent change to a dose of 1 mg/kg was carried out around 14 hours before surgery. The results showed that the sensitivity increased and the specificity decreased at 1 mg/kg compared to the 0.5 mg/kg dose. All patients had no adverse reactions after injection. Although the number of patients is small, we can still see the relationship between ICG dose and recognition rate, which will also provide help for subsequent research.

In this study, one patient’s data were excluded due to environmental factors, and two patients’ data were incomplete. This issue was caused by operator error, which can be rectified with simple training. In comparison to the extensive professional training required for frozen section analysis, the diagnostic aspect of NIF surgery is significantly less demanding, which is a substantial advantage.

There are some limitations in this study. Firstly, the sample size was insufficient to fully validate the reliability of BCS margin recognition using ICG Fluorescence detection. Secondly, although fluorescence can be quantified using machines, determining whether the margin is positive still requires subjective judgment, which might lead to confounding variables.

Although there are several limitations, the findings of this study demonstrate that ICG fluorescence detection can function as a supplementary tool to efficiently detect positive surgical cut margin. It can be applied in BCS for breast cancer, offering a novel approach to detect surgical margins whilst presenting a favorable clinical outlook.

Conclusions

The findings of the validation presented here suggest that ICG could be useful for surgical cut margin in BCS, particularly in terms of sensitivity similar to frozen section analysis. ICG can detect cancerous tissue not identified by frozen section analysis. The sensitivity increased and the specificity decreased at 1 mg/kg compared to the 0.5 mg/kg dose. ICG fluorescence can be used as a supplementary tool for frozen section analysis.

Although the results are encouraging for ICG, more trials are needed to investigate whether the current experimental protocols can positively impact intra-operative decision making and patient prognosis.

Acknowledgments

The authors are grateful to the Nanjing Nuoyuan Medical Devices, Co., Ltd. for providing products.

Funding: This study was supported by the Medical Science and Technology Development Foundation, Nanjing Department of Health (No. ZKX20025).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://gs.amegroups.com/article/view/10.21037/gs-24-195/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-195/dss

Peer Review File: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-195/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-24-195/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital (No. 2020-024) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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