The value of percutaneous contrast-enhanced ultrasound in the detection and diagnosis of sentinel lymph nodes in breast cancer: comparison with pathological features

Introduction

Breast cancer is the most common malignant tumor in women all over the world (1). The prognosis of breast cancer is closely linked to the status of axillary lymph nodes (ALN) (2,3). Initially, breast cancer cells spread to sentinel lymph nodes (SLNs) via lymphatic channels (LCs) before metastasizing to other ALNs (4). If two or fewer metastatic SLNs are found in sentinel lymph node biopsy (SLNB), patients with breast cancer are spared from axillary lymph node dissection (ALND) (5-9) to reduce side effects such as lymphedema and arm paresthesia (7,10). Moreover, the risk of locoregional recurrence and decrease in disease-free survival does not increase with additional radiotherapy and systemic treatment (11).

Percutaneous contrast-enhanced ultrasound (P-CEUS) is a preoperative method for detecting SLN and assessing their status (12-16), offering advantages over other detection techniques such as blue dye-guided, radioisotope, and indocyanine green fluorescence imaging methods (17-19). The classification of SLNs using P-CEUS remains a topic of debate, with varying diagnostic performances among different classification methods (12-15,20). A novel P-CEUS classification approach based on lymph node structural characteristics has demonstrated superior diagnostic accuracy compared to the widely used classification method that does not consider lymph node structure (21). Despite this, there is limited research on the relationship between P-CEUS patterns and the pathological characteristics of SLNs. Therefore, this study aims to evaluate the diagnostic efficacy of P-CEUS in SLNs and investigate the correlation between P-CEUS patterns and the pathological features of SLNs in breast cancer. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2024-2631/rc).

Methods

Patients

A retrospective study was conducted on consecutive breast cancer patients admitted to our hospital between June 2019 and March 2021. Patients were included if they met the following criteria: (I) a confirmed breast cancer diagnosis through pathological examination; (II) underwent pre-surgery or pre-biopsy P-CEUS of SLNs; and (III) underwent biopsy or surgery to obtain pathological results of SLNs. Patients who had received radiotherapy, chemotherapy, axillary surgery, or had a history of allergy to ultrasound contrast agents were excluded. Ultimately, 238 patients were included in the study (Figure 1). This study was approved by the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University [No. (2020)316], and all patients provided informed consent. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study has been registered on Chinese Clinical Trial Registry (No. ChiCTR2200057903).

Figure 1 Flowchart of study selection. P-CEUS, percutaneous contrast enhanced ultrasound; SLN, sentinel lymph node.

Instruments and methods

The study utilized ultrasound instruments from Siemens ACUSON Sequoia Redwood equipped with a 10L4 linear array probe, Mindray Resona 7 equipped with an L9-3 linear array probe, and Philips iU22 equipped with an L9-3 linear array probe. A microbubble continuous real-time contrast-enhanced ultrasound (CEUS) imaging mode was employed with a mechanical index lower than 0.15. The ultrasound examinations were conducted by two experienced physicians (Y.Z. and J. Luo), each with over a decade of expertise in CEUS. Each ultrasound examination was conducted by only one of these two physicians. Both physicians were blinded to the clinical and pathological details of the patients. The ultrasound contrast agent used was SonoVue (Bracco, Milan, Italy), a lyophilized powder containing 59 mg of sulfur hexafluoride microbubbles, which was reconstituted with 5 mL of normal saline and shaken vigorously for 30 seconds. Patients underwent conventional ultrasound in the supine position with their upper extremities abducted. Following skin disinfection, 0.6–0.8 mL of ultrasound contrast agent suspension was injected at the edge of the outer upper quadrant of the areola on the affected side. When the lesion was located at the nipple-areola complex, injection was ensured to be away from the breast lesion. The SLN was confirmed by tracing the enhanced lymphatic drainage route immediately post-injection. When no enhanced lymphatic vessels or SLN were initially detected, multi-point injections (4–6 injection points, 3.2–4.4 mL of ultrasound contrast agent) around the areola and skin surface massage were performed. The lymphatic drainage route and SLNs identified via P-CEUS were marked on the skin surface, and details such as number, location, size, cortex thickness, and enhancement pattern of the SLNs were documented.

P-CEUS pattern of SLNs was divided into eight types (Figure 2):

• Type I: only part of the cortex was enhanced, others were non-enhanced, and the lymph node cortex was unevenly thickened;
• Type II: partial cortical filling defect;
• Type III: non-enhancement;
• Type IV: homogeneous high enhancement;
• Type V: diffuse inhomogeneous high enhancement;
• Type VIa: non/low enhancement of lymphatic hilus, homogeneous high enhancement of cortex;
• Type VIb: one half showed Type IV, V or VIa, and the other showed non-enhancement;
• Type VIc: only part of the cortex was enhanced, others were not enhanced, and lymph node cortex was evenly thickened.

Figure 2 Percutaneous contrast-enhanced ultrasound classification based on the structural characteristics of sentinel lymph nodes. (A) Type I: only part of the cortex was enhanced, others were non-enhanced, and the lymph node cortex was unevenly thickened. (B) Type II: partial cortical filling defect. (C) Type III: non-enhancement. (D) Type IV: homogeneous high enhancement. (E) Type V: diffuse inhomogeneous high enhancement. (F) Type VIa: non/low enhancement of lymphatic hilus, homogeneous high enhancement of cortex. (G) Type VIb: one half showed Type IV, V or VIa, and the other showed non-enhancement. (H) Type VIc: only part of the cortex was enhanced, others were not enhanced, and lymph node cortex was evenly thickened.

Type I–III were diagnosed as metastatic lymph nodes, and type IV–VI as non-metastatic lymph nodes (21). When conducting P-CEUS classification of SLNs, the size and shape of the lymph nodes were not referred to.

Patients scheduled for neoadjuvant therapy underwent ultrasound-guided SLNB, while the others received surgical treatment. Blue staining was utilized during surgery to identify SLNs, and the consistency with SLNs labeled by P-CEUS was examined. Hematoxylin-eosin staining was applied to the pathological sections of SLNs. A pathologist with 15 years of experience observed the structural characteristics of SLNs under a microscope and compared them with the P-CEUS characteristics of SLNs.

Statistical methods

Statistical analysis was performed using SPSS 27.0. Consistency between body surface labeling and blue staining indicated the correct location of P-CEUS in SLNs and LCs. The status of SLN was determined based on pathology. For the lymph nodes with questionable types of P-CEUS, the classification was determined through discussion by two physicians. Inter-observer agreement on P-CEUS patterns of SLNs was assessed using Cohen’s kappa. A Kappa value between 0.6 and 0.8 was deemed good, while a value between 0.4 and 0.6 was considered moderate. The diagnostic efficacy of P-CEUS in SLNs was evaluated against pathology as the gold standard. A two-sided P value of less than 0.05 was considered statistically significant.

Results

Participant characteristics

A total of 348 breast cancer patients were collected, and finally 238 cases were included, of which 237 were females and 1 was male (Figure 1). Among them, five patients with bilateral breast cancer underwent P-CEUS of SLNs on both sides. The average age of participants was 51.0±11.3 years, ranging from 25 to 79 years. The clinical characteristics of the participants are detailed in Table 1. No adverse reactions, such as allergic responses to ultrasound contrast agents, were reported during the study.

Location and diagnostic efficacy of P-CEUS in SLNs

The SLN detection rate of P-CEUS was 96.29% (234/243). Table 1 shows the number of enhanced lymph nodes and enhanced lymphatic vessels tracked by P-CEUS. Among the nine cases for which SLN were not detected, five did not show enhanced LCs. Of the remaining four cases, one underwent a suspicious lymph node biopsy without blue staining, while the other three cases underwent blue staining. P-CEUS revealed an average of 1.24 enhanced LCs per side (301/243), and an average of 1.28 enhanced SLNs per side (311/243) (Table 1). In cases where patients were scheduled for neoadjuvant therapy, only one SLN underwent biopsy, resulting in the exclusion of 18 SLNs without specific pathological results. A total of 293 SLNs were included in the analysis (Figure 1).

The inter-observer agreement in P-CEUS of SLNs was good (Kappa value =0.886). Pathology results indicated that 64 SLNs (21.8%) were metastatic and 229 SLNs (78.2%) were non-metastatic. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of P-CEUS in SLNs were 76.56% (49/64), 97.38% (223/229), 89.09% (49/55), 93.70% (223/238), and 92.83% (272/293), respectively (Table 2). Additionally, six non-SLNs in four patients were metastatic, while the SLNs (identified by P-CEUS) were non-metastatic.

Comparison between P-CEUS and pathology of SLNs

Pathology sections of non-metastatic lymph nodes revealed varying degrees of adipose tissue and fibrous tissue hyperplasia. Based on the amount and distribution of adipose tissue and fibrous connective tissue in lymph nodes, distinct P-CEUS patterns were identified. SLNs with minimal and scattered fibrous adipose tissue exhibited homogeneous high enhancement (Type IV, Figure 3A-3C) and diffuse inhomogeneous enhancement (Type V, Figure 3D-3F); lymph nodes with patchy fat tissue distribution near the lymph hilus displayed homogeneous high enhancement in the cortex and no/low enhancement near the lymph hilus (Type VIa, Figure 3G-3I).

Figure 3 The non-metastatic sentinel lymph node images of three breast cancer patients. The sentinel lymph node images of a 49-year-old female with right breast cancer show non-metastatic results (A-C). (A) The sentinel lymph node shows homogeneous high enhancement (Type IV) in P-CEUS imaging. (B) It shows uniform cortical thickness of the lymph node in conventional ultrasound. (C) Pathological section reveals only a small amount of adipose fiber tissue can be seen at the lymphatic hilum. The sentinel lymph node images of a 63-year-old female with right breast cancer show non-metastatic results (D-F). (D) The P-CEUS imaging shows sentinel lymph node is diffuse inhomogeneous high enhancement. (E) The lymph node shows uniform cortical thickness in conventional ultrasound. (F) Pathological examination reveals that scattered fibrous adipose tissue exhibited in the sentinel lymph node. The sentinel lymph node images of a 43-year-old female with right breast cancer show non-metastatic results (G-I). (G) The P-CEUS imaging shows low enhancement of the lymphatic hilus and homogeneous high enhancement of the cortex (Type VIa). (H) It shows evenly thickened lymph node cortex in conventional ultrasound. (I) Pathological analysis indicates flaky adipose tissue near the lymph hilus and the absence of cancer cells in the lymph node. (C,F,I) Hematoxylin-eosin-stained histopathological sections. P-CEUS, percutaneous contrast enhanced ultrasound.

In addition, there were some non-metastatic lymph nodes with unique characteristics. Firstly, P-CEUS revealed that half of the lymph nodes exhibited non-enhancement, while the other half displayed Type IV, V, or VIa (Type VIb) enhancement patterns. Some lymph nodes showed high enhancement only in certain areas of the cortex, with the remaining areas non-enhanced and uniformly thickened cortex (Type VIc). No metastatic lesions were identified in the pathological sections of these lymph nodes. Furthermore, some lymph nodes exhibited narrow or occluded LCs near the capsule. Secondly, in three SLNs examined, adipose tissue was found not only near the lymph hilus but also beneath the capsule. P-CEUS showed minimal to no enhancement in the fat tissue beneath the capsule. Lastly, certain lymph nodes displayed cortical thickening due to reactive hyperplasia, with some showing uneven cortical thickening and presenting Type IV, V, or VIa enhancement patterns on P-CEUS.

Metastatic lymph nodes generally exhibited a consistent background compared to non-metastatic lymph nodes in the pathological sections of metastatic SLNs, characterized by varying degrees of adipose tissue and fibrous tissue hyperplasia. Different P-CEUS patterns were observed in metastatic lymph nodes based on the size and distribution of metastatic lesions within the lymph nodes. Micrometastatic lesions located in the lymphatic vessels or beneath the capsule of SLNs posed challenges in distinguishing them from non-metastatic lymph nodes using P-CEUS. In cases where nested metastatic lesions were identified in the cortical area of SLNs, P-CEUS revealed filling cortical defects (indicative of metastatic lesions), while the remaining normal lymph node tissue exhibited characteristics consistent with non-metastatic lymph nodes (Type II, Figure 4A-4C). When metastatic lesions occupied most of the lymph node and only part of the cortex exhibited normal lymph node tissue, P-CEUS demonstrated high enhancement in that specific area of the cortex, with the rest showing non-enhancement, resulting in uneven cortical thickening (Type I, Figure 4D-4F). In situations where lymph nodes were nearly entirely occupied by metastatic lesions, P-CEUS revealed non-enhancement (Type III, Figure 4G-4I). However, when metastatic lesions were diffusely distributed throughout the lymph nodes without a clear boundary with normal lymph tissues or were distributed in multiple small focal areas, the P-CEUS of SLNs could resemble that of non-metastatic lymph nodes.

Figure 4 The sentinel lymph node images of a 46-year-old female with right breast cancer, and three SLNs are metastatic. The first SLN: (A) a partial cortical filling defect is observed, with the other nodes showing Type IV (Type II) in P-CEUS imaging; (B) the lymph node cortex appears unevenly thickened in conventional ultrasound; (C) pathological section reveals nested metastases in the cortex (arrow). The second SLN: (D) in P-CEUS, only part of the cortex is enhanced while others were not, yet the lymph node cortex appears unevenly thickened (Type I); (E) conventional ultrasound also shows uneven thickening of the lymph node cortex; (F) pathological examination confirms that cancer tissue had infiltrated most of the lymph node. The third SLN: (G) the P-CEUS imaging does not show any enhancement (Type III); (H) the lymph node cortex appears unevenly thickened in conventional ultrasound; (I) pathological examination reveals that the cancer tissue had almost completely invaded the lymph node. (C,F,I) Hematoxylin-eosin-stained histopathological sections. P-CEUS, percutaneous contrast enhanced ultrasound; SLN, sentinel lymph node.

Discussion

P-CEUS can assist in diagnosing the status of SLNs while simultaneously locating them (12-15). The SLN detection rate achieved in this study was 96.29%, closely aligning with findings from previous research (12-14). Among the total cases, nine did not exhibit enhanced LC/SLN, potentially due to lymphatic vessel blockage caused by cancer emboli or obstruction from lymphatic reflux due to factors such as inflammation or injury (13,22). During P-CEUS procedures, blockages in lymphatic vessels can impede the passage of both lymph and contrast agents, resulting in interrupted enhancement or the inability to visualize LC/SLN, thus hindering tracing. The commonly used P-CEUS classification system for SLN categorizes enhancement patterns into three types: homogeneous enhancement, heterogeneous enhancement, and no enhancement. However, variations in diagnostic efficacy exist across different studies (12-14). One study has demonstrated that combining the P-CEUS classification method with SLN structure yields superior diagnostic performance compared to the three classification methods (21). Another study identified seven types of enhancement patterns for SLNs (15), offering a more detailed classification system that is somewhat similar to the combined lymph node structure method, except that some subtypes do not specify the specific location of filling defects in lymph nodes or whether there is uneven cortical thickening. In this current study, the P-CEUS classification method, integrated with SLN structure, was utilized and categorized into six types with a diagnostic accuracy of 92.83%. In this study, different instruments were used for ultrasound examination, reflecting the actual clinical working situation. Similar CEUS manifestations of SLNs could be observed across examinations using different instruments.

The P-CEUS patterns of SLN were closely related to their pathological characteristics. The different pathological features of non-metastatic lymph nodes may be associated with age-related degenerative changes in lymph nodes. Human lymph node development progresses rapidly during childhood and then regresses after puberty (23). Early lymph nodes consist of cellular networks with free cells and blood vessels, featuring more extracellular meshwork, lymphocytes, and blood vessels (24). Aging alters lymph node architecture and function (24-27), leading to signs of adipocyte accumulation and fibrosis since puberty (24,28). The degenerative process affects the medulla first, gradually spreading to all tissue layers. Conventional ultrasound shows slightly hyperechoic adipose and fibrous connective tissue near the lymph node hilum, and hypoechoic lymphoid tissue near the cortex in degenerate lymph nodes. Therefore, SLNs exhibit Type IV in P-CEUS when lymph node degeneration is mild, and Type VIa when degeneration is significant. This study revealed that adipose tissue in the three SLNs could also be observed under the medulla and capsule, making it challenging to differentiate its filling defect from Type II SLNs in P-CEUS, especially the smaller ones. This difficulty contributed to the false negative diagnosis of SLNs by P-CEUS. SLNs classified as Type V appeared to be associated with the scattered distribution of fibrous adipose tissue within lymph nodes, possibly due to the limited entry of contrast agents into the SLNs. The stenosis or occlusion of subcapsular sinus without tumor invasion was identified in Type VIb and Type VIc SLNs, resulting in the obstruction of lymph and contrast medium drainage. It was crucial in this study to differentiate between Type VIc and Type I SLNs by considering whether the lymph node cortex exhibited uneven thickening in conventional ultrasound. Only two cases were misdiagnosed, one involving SLN biopsy without a complete lymph node pathological section during SLN biopsy, and another related to SLN reactive hyperplasia leading to cortical thickening. Furthermore, some patients underwent invasive procedures such as breast tumor biopsy or local resection before undergoing P-CEUS examination, potentially causing reactive proliferation of ALNs and thickening of the lymph node cortex. Performing P-CEUS was helpful in distinguishing cortical thickening caused by inflammatory hyperplasia from that caused by tumor growth in metastatic lymph nodes.

The different enhancement patterns of metastatic lymph nodes were closely associated with the distribution of metastatic lesions within the lymph nodes. Tumor cells can enter lymphatic vessels by passing through intercellular openings between endothelial cell junctions or by inducing larger discontinuities in the endothelial cell layer (29). Lymph from the afferent duct travels through the subcapsular sinus, follicles, deep cortical unit, medullary sinus, hilum, and exits the lymph node through the efferent duct (30). Upon entering the lymph node via the afferent lymphatic vessel, cancer cells may give rise to micrometastases, often found in the cortex of lymph nodes (31). Distinguishing micrometastases was challenging for P-CEUS, leading to false negatives in eight cases. As metastatic lesions grew, they gradually formed nest-like structures in the cortex, eventually occupying most or the entire SLN, resulting in P-CEUS patterns of Type II, Type I, and Type III. Diffuse distribution of metastatic lesions without clear boundaries with normal lymphatic tissue or multiple small focal distributions made it difficult to differentiate between metastatic and non-metastatic lymph nodes, also contributing to false negatives. Further research is necessary to determine if the occurrence of these SLNs, which were relatively rare in this study, is associated with the specific pathological type of cancer.

The study utilized P-CEUS pattern classification to diagnose the status of SLNs based on lymph node structural characteristics, and conducted a comprehensive comparison between the P-CEUS pattern and SLNs pathology. A limitation of the study is the inability to ensure complete consistency between the P-CEUS pattern of SLN and the pathological section, potentially excluding very small metastatic lesions not captured on the pathological sections.

Conclusions

P-CEUS proved to be an effective method for accurately locating and diagnosing SLNs prior to surgery. The P-CEUS pattern demonstrated a close correlation with pathological characteristics, aiding in accurate diagnosis.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2024-2631/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2024-2631/dss

Funding: This work was supported by the Major Research Plan of the National Natural Science Foundation of China (No. 92059201).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2024-2631/coif). All authors report that this work was supported by the Major Research Plan of the National Natural Science Foundation of China (No. 92059201). The authors have no other 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 and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University [No. (2020)316] 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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