Clinical implication of metastasis in the second most radioactive sentinel lymph node with nonmetastatic most radioactive node in patients with breast cancer

Introduction

Sentinel lymph node biopsy (SNB) is the standard method for assessing axillary lymph node status in patients with clinically node-negative breast cancer. This is underpinned by robust evidence suggesting that SNB, when applied to patients with node-negative breast cancer, does not worsen survival compared with axillary lymph node dissection (ALND) (1-3). SNB is a minimally invasive procedure (4), offering significant advantages to patients with breast cancer. Specifically, it obviates unnecessary ALND, thereby reducing the risk of associated complications, such as lymphedema in the ipsilateral upper extremity.

Originally, SNB aimed to confirm that the sentinel lymph node (SN) was free of cancer cells to avoid an unnecessary ALND for patients with clinically node-negative breast cancer. Hence, initially, ALND was only omitted if the SN was negative. However, since the American College of Surgeons Oncology Group Z0011 trial demonstrated the safety of ALND omission among patients with clinically node-negative breast cancer undergoing breast-conserving surgery, systemic adjuvant therapy, and whole-breast radiotherapy in whom SNB had revealed one or two metastases, the use of completion ALND for patients who met the criteria of Z0011 trial has been decreasing (5). This strategy was developed based on the concept that both irradiation and adjuvant systemic therapy can effectively manage non-sentinel axillary lymph nodes (non-SNs), even in the presence of metastasis to these nodes (5,6). Furthermore, recently, the SENOMAC trial showed that ALND omission did not result in inferior survival, even for patients undergoing mastectomy (7). With these robust evidences supporting the safety of omitting ALND, SNB without ALND has gained acceptance as a standard treatment approach for patients with clinically node-negative breast cancer who have one or two SN metastases. However, some patients later develop metastasis to non-SNs after omission of ALND. This underscores the importance of judicious selection of ALND omissions. Therefore, preoperative or intraoperative prediction of metastasis to non-SNs can further refine the selection criteria for ALND omission. To date, few studies have investigated the predictors of metastasis to non-SNs in cases with positive SNs; hence, the risk factors for metastasis to non-SNs remain unclear.

Since Krag et al. first introduced SNB using a radioisotope (RI) method in patients with breast cancer (8), large number of studies have shown its feasibility and accuracy (9-12). As a result, RI method using ^99mTc-labeled colloid tracers (phytate, tin, or sulfur colloid) has been established as the standard technique for SNB. In this procedure, after the SN is resected, the radioactive count (RI count) is usually measured ex vivo using a gamma detection probe. Previous studies demonstrated that when multiple SNs were extracted, the SN with the most pronounced uptake of radioactive agent (the hottest SN) exhibited the highest risk of metastasis (13-15). However, in clinical practice, we sometimes encounter cases in which the hottest SN is negative, whereas the second or less radioactive SNs prove positive for metastasis. To date, the clinical significance of this phenomenon in relation to non-SN metastasis remains unclear.

This study aimed to investigate the association between the radioactivity of positive SNs and the metastatic status of non-SNs and to identify the risk factors associated with non-SN metastasis in patients with breast cancer with positive SNs. Toward this aim, we retrospectively analyzed the clinicopathological characteristics, radioactivity of SNs, and metastases to axillary lymph nodes in patients with breast cancer with positive SNs who underwent ALND and investigated the correlation between radioactivity and the prevalence of non-SN metastasis. We present this article in accordance with the STROBE reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-24-346/rc).

Methods

Patients and study design

In this study, we performed a retrospective analysis of 1,360 patients with clinical Tis-T2 node-negative breast cancer who underwent either breast-conserving surgery or mastectomy with SNB at Shinshu University Hospital between January 2015 and February 2023. All patients had pathologically confirmed breast cancer via core needle or vacuum-assisted biopsy. Patients who had received neoadjuvant chemotherapy were excluded because of concerns regarding altered lymphatic flow resulting from the treatment. Male patients were excluded from this study. Of the 1,360 patients, 132 were identified as having SNs positive for metastasis based on frozen section diagnosis. Among these 132 patients, 18 had micro metastasis in SNs. For these patients, ALND was omitted. Finally, 114 patients were included in this study (Figure S1). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The current study was approved by the Medical Ethics Committee on Clinical Investigation of Shinshu University (No. 4901). Our institution uses a form on its website to enable patients to opt out of the use of their clinical data for research purposes, and the requirement for written informed consent was waived. All patient data were anonymized.

Data collection

We collected clinical information from the patients’ medical records, including age, sex, primary tumor laterality, location of primary tumor, clinical stage, surgical procedure, histological type, pathological tumor size, presence of lymphovascular invasion, and the status of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor type 2 (HER2). Breast cancer subtypes were defined as follows: luminal (ER- or PgR-positive and HER2-negative), luminal-HER2 (ER- or PgR-positive and HER2-positive), HER2-enriched (ER- and PgR-negative, HER2-positive), and triple-negative (TN) (ER-, PgR-, and HER2-negative).

Sentinel node biopsy

SNB was performed using the radio isotope (RI) method in combination with a vital dye and a fluorescent method using indigo carmine blue dye and indocyanine green (ICG) for patients with clinical N0 breast cancer. Preoperative imaging work-up included ultrasonography, contrast-enhanced magnetic resonance imaging, and contrast-enhanced computed tomography. Any LNs suspicious for metastasis on at least one imaging modality were further evaluated using fine-needle aspiration cytology to confirm the presence of metastasis. One day before surgery, phytate sodium hydrate and ^99mTc (1.32 mCi at a volume of 0.2 mL) were subdermally injected above the primary tumor and within 1 cm of its periphery. Lymphoscintigraphy was performed 5 min and 3 h after injections of ^99mTc (Figure S2). Planar static images were also captured. A hotspot in the axillary region was considered an SN, and the number of SNs detected by lymphoscintigraphy was recorded. At the onset of the surgical procedure, a 1.5 mL mixture containing indigo carmine blue dye (4 mg) and ICG (2.5 mg) was subdermally administered around the primary tumor site. Before the resection of the primary tumor, SNB was performed. SNB began after a 2-minute massage at the injection site. SNs were identified using vital dyes, fluorescence, and RI method. In the vital dye and fluorescent method, SNs were either identified by their blue color or fluorescence detected using a handheld camera imaging device (PDE-NEO; Hamamatsu Photonics, Hamamatsu, Japan) or when a blue or fluorescent lymphatic vessel was directly afferent to it. In the RI method, SNs were detected by radioactivity captured using a gamma detection probe (Neoprobe; Devicor Medical Japan, Tokyo, Japan). Any nodes exhibiting blue staining, fluorescence, and/or radioactivity were excised and designated as SNs. Upon node extraction, we verified the ex vivo fluorescence of SNs. In addition, the axilla was inspected for any residual radioactivity from ^99mTc and fluorescence intensity from ICG to ensure that there were no SN remnants. The ex vivo RI counts of the resected SNs were measured using a gamma detection probe. All fat-trimmed SNs were numbered in order of RI count and intraoperatively evaluated by frozen section diagnosis.

Pathological examination

For frozen section diagnosis, SNs were bisected at their center, serially sectioned into 2 mm slices, and stained with hematoxylin and eosin. In this study, the presence of metastatic lesion within the SNs ≥0.2 mm was defined as SN metastasis. Isolated tumor cells, which are metastatic lesions <0.2 mm, were not considered to have lymph node metastasis. Metastatic lesions measuring between 0.2 and 1.9 mm were defined as micro metastases, while those larger than 2 mm were classified as macro metastases. At our institution, ALND was omitted only for patients who have one or two SN metastases with micro metastasis. If macro metastasis was detected through the frozen section diagnosis, we performed ALND regardless of the surgical procedure (mastectomy of breast-conserving surgery) during the study period.

All SNs and lymph nodes resected during subsequent ALND were evaluated by histopathological examination. Any lymph nodes resected during ALND following SNB were considered non-SNs. Then, we divided the patients into two groups; the hottest SN group, consisting of patients who had metastasis to the hottest SN (n=88) and the second SN group, consisting of patients who had metastasis to the second or less radioactive SNs but no metastasis to the hottest SN (n=26).

Statistical analyses

Categorical variables were analyzed using the chi-squared test, whereas continuous variables were analyzed using two-sided t-tests or one-way analysis of variance with Tukey’s multiple comparison test. Univariate and multivariate analyses using a logistic regression model were performed to determine the significant factors associated with non-SN metastasis. Multivariate analysis was performed for parameters with P<0.30 in the univariate analysis. All statistical analyses were performed using GraphPad Prism version 9.3.1 (GraphPad Software, CA, USA), and P<0.05 was considered statistically significant.

Results

Patient characteristics

The clinicopathological characteristics of the studied patients are shown in Table 1. The patients had a mean age of 55.9±12.1 years. Lymphoscintigraphy detected an average of 1.75±0.85 (range, 1–5) lymph nodes, whereas an average of 2.45±1.30 (range, 1–8) lymph nodes was resected during SNB. Regarding surgical procedures, 74 (64.9%) patients underwent mastectomy, and 40 (35.1%) patients opted for breast-conserving surgery. Histologically, 101 (88.6%) patients presented with invasive ductal carcinoma, 7 (6.1%) with invasive lobular carcinoma, and 6 (5.3%) with other special types. Seventeen (14.9%) patients had tumors <1 cm; 51 patients (44.7%) had tumors between 1 and <2 cm; and 46 (40.4%) patients had tumors ≥2 cm in size. Non SNs were pathologically involved in 38 (33.3%) patients. Lymphatic invasion was observed in 70 (61.4%) patients, and vascular invasion was observed in 33 (28.9%) patients.

Metastasis to the SNs according to radioisotope count

To investigate the association between RI count and metastasis to SNs, we numbered the excised SNs in the order of RI count (hottest SN, second SN, or third or subsequent SNs) and investigated the metastatic status in each SN. A total of 244 SNs were excised, comprising 114 of the hottest SN, 82 of second SN, and 48 of third or subsequent SNs (up to the fifth SN). Of the 114 hottest SNs, metastases were observed in 88 (77.2%) nodes (macro and micro metastases had 71 and 17 nodes, respectively). The frequency of SN metastasis gradually decreased toward the second (61.0%, 50 nodes; macro and micro metastases had 42 and 8 nodes, respectively) and third or subsequent SNs (35.4%, 17 nodes; macro and micro metastases had 14 and 3 nodes, respectively). This decline in metastatic frequency was statistically significant (P<0.01) (Figure 1).

Figure 1 Frequency of positive SNs according to the RI accumulation. The 3^rd SN group included 3^rd SN and subsequent SNs. The number of excised SNs and metastatic SNs are indicated in each group. LN, lymph node; SN, sentinel lymph node; RI, radioisotope.

Comparison of clinicopathological characteristics between patients with positive hottest SN and those having positive second or less radioactive SNs with nonmetastatic hottest SN

Next, to explore the differences in clinicopathological features between patients who presented with a positive hottest SN and those who did not exhibit metastasis in the hottest SN but did so in the second or less radioactive SNs, we compared the clinicopathological characteristics of the hottest SN group (n=88) and the second SN group (n=26), the data for which are shown in Table 2. Across parameters, including age, sex, primary tumor laterality, location of primary tumor, clinical stage, number of lymph nodes detected by lymphoscintigraphy, surgical procedure, histological type, pathological tumor size, lymphovascular invasion, subtype, and pathological stage, no significant differences were observed between the hottest SN and second SN groups. However, the number of resected lymph nodes by SNB was significantly higher in the second SN group (2.96±1.51; range, 1–8) than in the hottest SN group (2.30±1.20; range, 1–6) (P=0.01). The incidence rate of metastasis to non-SNs was significantly higher in the hottest SN group (38.6%) than in the second SN group (15.4%) (P=0.03). Additionally, among the patients with positive non-SNs, the percentage of those who had more than two non-SN metastases was significantly higher in the hottest SN group (67.6%) than in the second SN group (0%) (P=0.01).

Impact of metastasis to the hottest SN as a risk factor for metastasis to non-sentinel axillary lymph node

Univariate and multivariate analyses were performed to determine the factors independently associated with the risk of metastasis to non-SNs. Univariate analysis revealed that more than two SNs detected by lymphoscintigraphy [hazard ratio (HR), 2.43; 95% confidence interval (CI), 1.09–5.66; P=0.03] and the hottest SN group (HR, 3.63; 95% CI, 1.25–13.2; P=0.03) were significantly associated with metastasis to non-SNs (Table 3). On multivariate analysis using logistic regression analysis, more than two SNs detected by lymphoscintigraphy (HR, 3.36; 95% CI, 1.41–8.56; P=0.01) and the hottest SN group (HR, 4.93; 95% CI, 1.57–19.3; P=0.01) were independent predictive factors for metastasis to non-SNs.

Prediction of non-SN metastasis using the radioactive count along with the number of lymphoscintigraphy-visualized SNs

We found that the detection of more than two SNs by lymphoscintigraphy and metastasis to the hottest SN were independent predictive factors for non-SN metastasis and stratified the patients into four groups according to these two factors (n=17, one SN detected by lymphoscintigraphy in the hottest SN group; n=45, more than two SNs detected by lymphoscintigraphy in the hottest SN group; n=8, one SN detected by lymphoscintigraphy in the second SN group; n=18, more than two SNs detected by lymphoscintigraphy in the second SN group). The incidence rate of non-SN metastasis was highest in patients who had more than two SNs detected by lymphoscintigraphy in the hottest SN group (51.1%) and gradually decreased in those who had one SN detected by lymphoscintigraphy in the hottest SN group (23.5%), those who had more than two SNs detected by lymphoscintigraphy in the second SN group (22.2%), and those who had one SN detected by lymphoscintigraphy in the second SN group (0%), with statistical significance (P<0.001) (Table 4).

Comparison of SN identification rate by vital dye and fluorescent method using indigo carmine blue dye and ICG between the hottest SN and second SN groups

SNB using indigo carmine blue dye and ICG are widely used in the treatment of breast cancer (16-18), and previous studies have shown that they have the highest accuracy for detecting SNs when used in combination with the RI method (17,19,20). However, it is generally accepted that there can be discordance in the accumulation of indigo carmine blue dye, ICG, and RI in SNs (21), and it remains unknown whether the frequency of such discordance may be associated with the status of radioactivity in positive SNs. To address this, we investigated the identification rate of the hottest and second SN using indigo carmine blue dye and ICG and compared it between the hottest and second SN groups (Table S1). Regarding the indigo carmine blue dye, there was no significant difference in the frequency of visualization of the hottest SN between the hottest SN (90.9%) and second SN groups (76.9%) (P=0.08). The second SN visualization rate did not differ significantly between the two groups (hottest SN group, 71.2%; second SN group, 69.2%; P=0.99). Similar results were obtained in the ICG method (hottest SN group, 98.9%; second SN group, 92.3%; P=0.12 for hottest SN visualization, hottest SN group, 83.1%; second SN group, 88.5%; P=0.74 for second SN visualization).

Discussion

This study demonstrates that metastasis to the second or less radioactive SNs without metastasis to the hottest SN is significantly associated with a reduced risk of non-SN metastasis. Our findings suggest that data regarding the order of radioactivity of SNs and their metastatic status may provide helpful information for predicting non-SN metastasis in patients with breast cancer undergoing SNB.

In patients with positive SNs, if the absence of non-SN metastasis can be accurately predicted preoperatively or intraoperatively, ALND can be safely omitted. In this regard, the results of this study showed that only 15.4% of patients with metastasis to the second or less radioactive SNs with the hottest nonmetastatic SN had positive non-SNs. Therefore, 84.6% of these patients may have undergone unnecessary ALND. In contrast, 39.8% of the patients with metastasis to the hottest SN had non-SN metastasis. These findings imply a lower risk of non-SN metastasis in patients with metastasis solely to the second or less radioactive SN without metastasis in the hottest SN. Consequently, these patients may be more suitable candidates for ALND omission than those with metastases to the hottest SN.

The underlying mechanisms for the low risk of non-SN metastasis in patients with metastasis to the second or less radioactive SN, but not to the hottest SN, are not fully understood. The SN is defined as the first lymph node receiving lymphatic drainage from the primary tumor site (22). However, multiple SNs are often identified. In the RI method, the size of colloid particle combined with ^99mTc is a critical factor for the distribution of radioactivity in the SN and the ability to visualize SN in the scintigraphy. The ideal size of colloid particle is between 20 and 500 nm (23). In this regard, phytate and tin colloid fall within this range and have been most widely utilized for SNB (24) . However, several reports demonstrated that ^99mTc-phytate colloid is superior to a ^99mTc-tin colloid in terms of identification rate for SN in patients with breast cancer (24,25). Hence, we use ^99mTc-phytate colloid sodium hydrate in the RI method. ^99mTc-labeled phytate sodium hydrate that reaches the SN is phagocytosed by macrophages and remains there (26,27). The highest accumulation level of RI is found in the lymph node first encountered by lymphatic vessels draining the breast cancer (the first SN), with second or subsequent SNs showing diminishing radioactivity (26). On the other hand, when the SN is involved with cancer cells, few normal macrophages remain in the metastatic SN (26,28). Furthermore, LN metastasis drives LN-residing macrophages towards an immune suppressive phenotype, leading to a decrease in their phagocytic ability (29,30). Consequently, the capacity to retain the RI in the involved SN is impaired, resulting in diminished radioactivity (Figure 2) (26,28).

Figure 2 Schematic presentation of nonmetastatic SN and metastatic SN. In metastatic SN, LN-residing macrophages decrease and the phagocytic ability of macrophages is reduced as the metastasis forms, leading to a loss of capacity to retain the RI, which results in diminished radioactivity. SN, sentinel lymph node; LN, lymph node; RI, radioisotope.

Previous studies have demonstrated four types of anatomical classifications on the numbers of lymphatic routes and SNs: (I) single route/single SN; (II) multiple routes/single SN; (III) single route/multiple SNs; and (IV) multiple routes/multiple SNs (Figure 3A-3D) (31,32). The single route/single SN type is the most common (65%) among these patterns, followed by multiple routes/single SN (11%), single route/multiple SNs (11%), and multiple routes/multiple SNs (13%) (32). In cases of single route/multiple SNs or multiple routes/multiple SNs, metastasis to the second or less radioactive SNs, but not to the hottest SN, may occur because the hottest and second radioactive SNs receive lymphatic flow from distinct lymphatic vessels (Figure 3E). In contrast, in cases of single route/single SN or multiple routes/single SN, because metastasis may develop progressively along the specific lymphatic pathway, metastasis is unlikely to form in the second or less radioactive SNs if there is no metastasis in the hottest SN.

Figure 3 Schematic presentation of anatomical pattern of drainage lymphatic patterns connecting to SN, (A) single route/single SN, (B) multiple routes/single SN, (C) single route/multiple SNs, and (D) multiple routes/multiple SNs. (E) Hypothesis of metastatic pattern to SN according to the radioactivity in each drainage system. SN, sentinel lymph node.

However, in patients with breast cancer having positive SN, the radioactivity of SNs may not truly reflect the order of SNs because of metastasis-induced loss of radioactivity. Considering this, we speculate one possible mechanism when metastasis is found in the second radioactive SN but not in the hottest SN, even in cases of single route/single SN or multiple routes/single SN. When metastasis is detected solely in the second radioactive SN, this SN may actually be the first SN because of diminished radioactivity attributed to metastasis. In this case, the hottest SN may appear to be the first SN but is actually the second SN. In this scenario, the true second SN (apparent hottest SN) is metastasis-free (Figure 3E). Hence, metastasis to further subsequent lymph nodes (non-SNs) is unlikely to be formed. Given the high frequency of these anatomical patterns, this hypothesis can explain why metastasis to the second or less radioactive SNs, but not to the hottest SN, is significantly associated with a low risk of non-SN metastasis.

Multivariate analysis revealed that the number of lymph nodes detected by lymphoscintigraphy was independently correlated with non-SN metastasis. Regarding the association between the number of lymph nodes visualized by lymphoscintigraphy and the status of axillary lymph nodes, Ogasawara et al. demonstrated that the high number of visualized axillary nodes on lymphoscintigraphy was positively associated with the risk of metastasis to the SNs in patients with breast cancer undergoing breast surgery with SNB (28). Considering that phytate sodium hydrate, which is combined with ^99mTc, is phagocytosed by macrophages residing in SNs and retained once accumulated in the SNs (26,27), they proposed that compromised phagocytosis because of the development of metastasis might facilitate the migration of RI agents to other SNs and, hence, is associated with an increased number of lymph nodes visualized by lymphoscintigraphy. This scenario can also explain our finding that the number of lymph nodes detected by lymphoscintigraphy was independently associated with non-SN metastasis. Consequently, together with our findings, patients in whom lymphoscintigraphy revealed more than two SNs and who had metastasis to the hottest SN might be at the highest risk of metastasis to non-SNs. Utilizing information on the number of lymphoscintigraphy-visualized SNs along with the radioactivity of the histologically involved SNs would enable a more accurate prediction of the status of non-SNs.

The results of this study demonstrate that indigo carmine blue dye and ICG can visualize SNs at a relatively high rate, as reported in previous studies (16,33). Importantly, this high identification rate was observed irrespective of the radioactivity status of the positive SNs, as indicated by the results showing no significant differences in the visualization rates of these two agents (indigo carmine blue dye and ICG) between the hottest and second SN groups. In particular, even for patients in the second SN group, ICG detected the second SN, which turned out to be pathologically involved with tumor cells, at a high rate of 88.5%. These findings suggest that information from indigo carmine blue dye and ICG, in combination with RI, would be helpful in locating the second SN.

This study has some limitations. First, the relatively small number of patients included in the analysis may have limited the generalizability of the findings. Second, the data were obtained from a single institution, which could have introduced potential institutional bias. Multicenter studies would provide a broader perspective and enhance the robustness of findings. Third, this study used a retrospective design, which may have been susceptible to selection bias. Further studies are warranted to validate and extend these findings to larger and more diverse patient populations. Additionally, patients who received neoadjuvant chemotherapy were excluded from this study. Although this exclusion was due to concerns about altered lymphatic flow caused by neoadjuvant chemotherapy, growing evidence suggests that SNB after neoadjuvant chemotherapy is feasible (34). Future studies involving patients who received neoadjuvant chemotherapy are necessary to broaden the applicability of our findings.

Conclusions

Patients with metastasis in the second or less radioactive SNs, without metastasis in the hottest SN, are at a significantly lower risk of non-SN metastasis. Despite the small sample size and the need for further validation, our findings suggest that information regarding the order of radioactivity of SNs and their metastatic status may provide useful insights for predicting non-SN status in patients with breast cancer undergoing SNB.

Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://gs.amegroups.com/article/view/10.21037/gs-24-346/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-346/dss

Peer Review File: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-346/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-24-346/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 Medical Ethics Committee on Clinical Investigation of Shinshu University (No. 4901) and individual consent for this retrospective analysis was waived. The institution listed in the author list, the facility where the patient data were collected, and the institution that granted Institutional Review Board (IRB) approval are all part of Shinshu University. Specifically, while patient data were collected at Shinshu University Hospital, IRB approval was obtained from Shinshu University, as Shinshu University Hospital is a subsidiary unit of Shinshu University. Therefore, these three units are effectively identical for the purposes of this study.

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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