Contrast enhanced ultrasound of sentinel lymph nodes.

Sentinel lymph nodes are the first lymph nodes in the region that receive lymphatic drainage from a primary tumor. The detection or exclusion of sentinel lymph node micrometastases is critical in the staging of cancer, especially breast cancer and melanoma because it directly affects patient's prognosis and surgical management. Currently, intraoperative sentinel lymph node biopsies using blue dye and radioisotopes are the method of choice for the detection of sentinel lymph node with high identification rate. In contrast, conventional ultrasound is not capable of detecting sentinel lymph nodes in most cases. Contrast enhanced ultrasound with contrast specific imaging modes has been used for the evaluation and diagnostic work-up of peripherally located suspected lymphadenopathy. The method allows for real-time analysis of all vascular phases and the visualization of intranodal focal "avascular" areas that represent necrosis or deposits of neoplastic cells. In recent years, a number of animal and human studies showed that contrast enhanced ultrasound can be also used for the detection of sentinel lymph node, and may become a potential application in clinical routine. Several contrast agents have been used in those studies, including albumin solution, hydroxyethylated starch, SonoVue(®), Sonazoid(®) and Definity(®). This review summarizes the current knowledge about the use of ultrasound techniques in detection and evaluation of sentinel lymph node.

Introduction

Conventional B-mode ultrasound (US) is the first imaging method for inflammatory and neoplastic lymph node disease(. Contrast enhanced ultrasound (CEUS) techniques have been broadly established. EFSUMB guidelines( and EFSUMB recommendations( have been recently published focusing on the liver and many additional organs including lymph nodes(. In contrast to intravenous CEUS techniques dealing with lymph node examinations( the detection of sentinel lymph nodes (SLNs) using ultrasound contrast agent (UCA) is not an established technique despite some promising efforts. The article focuses on current knowledge about ultrasound techniques for the detection and evaluation of SLNs.

SLNs are the first lymph nodes in the region that receives lymphatic drainage from a primary tumor. The detection or exclusion of SLN micrometastases is critical in staging cancer, especially breast cancer and melanoma, because it directly affects patient's prognosis and surgical management(. SLNs biopsy is essential for axillary staging of the breast cancer patients who are clinically node negative, as it decreases the postoperative complications and morbidity of axillary nodal dissection(. Currently, intraoperative SLN biopsies using blue dye and radioisotopes are the method of choice for the detection of SLNs with high identification rates(. However, anaphylactic reactions to the dye, although rare, have been reported(. Radioisotope method usually needs many hours to detect SLNs after radioactive colloid is injected and it produces radiation protection problems. Moreover, potential repeat surgery is needed in up to 35% of patients who show nodal metastases on SLN biopsy(.

It is well known that conventional ultrasound is not capable of detecting SLNs in most cases, however, studies( showed that CEUS can be also used for detecting SLN, which may become a potential application in clinical routine, like lymphoscintigraphy. This method requires an US apparatus and ultrasound contrast agent (UCA).

UCA can be injected subcutaneously or intradermally or around the tumor, e.g. in patients with breast cancer, it is often injected subcutaneously in each quadrant near/ around the tumour site. After a local massage, the agent will be taken up by lymphatic channels and finally will reach the SLN, which can be imaged by low mechanical index (MI) US. So far, several contrast agents have been used in the studies, including 25% albumin solution(, hydroxyethylated starch(, SonoVue®(, Sonazoid®(, and Definity®(.

Review of literature

Experimental studies

Lymphatic CEUS with Sonazoid®

One animal study by Goldberg et al. using subcutaneous peritumoral administration of Sonazoid® showed that this agent was only confined to the SLNs and was not detected in the second echelon nodes at scanning electron microscopy(.

In 2004 and 2011, the same author also reported SLN detection using CEUS with Sonazoid® in swine models(. The accuracy of SLN detection in these studies were 90% and 81.8% for lymphatic CEUS, respectively, and were significantly higher than the 81% and 63.2% achieved with lymphoscintigraphy, respectively. Therefore, the detection of SLNs with lymphatic CEUS compared favorably with that at lymphoscintigraphy. And it is also pointed out that lymphatic CEUS can depict metastases within the SLN, which was not possible with lymphoscintigraphy. For characterization of SLN metastases, the accuracy of lymphatic CEUS in the two studies was 86% and 80%, respectively(. Kawai et al. recently evaluated the usefulness of a CEUS-guided method with Sonazoid® for imaging of the lymphatic channels and SLN of the stomach by comparing it with the conventional Evans blue dye-guided method in a porcine model(. The sensitivity for the detection of SLN was not significantly different between the CEUS- and dye-guided methods. The Evans blue dye flowed out rather quickly (≈15 min after the injection) through the SLN whereas Sonazoid® remained in the SLN until 2 h after the injection.

Lymphatic CEUS with SonoVue®

Lymphatic CEUS with SonoVue® also has a high sensitivity for the detection of SLN, it is reported that the sensitivity of CEUS was 89.5% in a rabbit model( and 91.3% in a dog model(. On CEUS, the lymphatic channels were demonstrated as hyperechoic linear structures deriving from the injection site which could be readily followed to their SLNs. The enhancement pattern of SLN could be divided into complete enhancement and partial enhancement. Compared with the histopathologic results, proliferation of lymphatic follicles or lymphatic sinus were found in partial enhanced SLNs while normal lymphatic tissue was demonstrated in completely contrast enhanced SLNs(.

In a swine model subcutaneous injections of 1 ml SoneVue® were given below the mammilla and the contrast could be detected in the draining lymph nodes after five minutes(. In 22 of 26 injections the drainage was confirmed with blue dye to go to an inguinal lymph node, in the remaining ones, the drainage was towards the neck. Also, the lymphatic tracts were visualized (fig. 1).

Fig. 1

Contrast enhanced images of the injection site and lymphatic channels. After injection of SonoVue®, contrast enhancement is shown as a hyperechoic area in the subcutaneous tissues (A). Contrast enhanced images of two superficially located contrast-filled lymphatic channels (echogenic) in a swine model (B)(. The figures are from J Ultrasound Med 2008; 27: 1203–1209, reproduced with permission from the American Institute of Ultrasound in Medicine

Animal study also showed that CEUS with SonoVue® could guide the percutaneous removal of the entire SLN. Sever et al.( injected SonoVue® that was mixed with blue dye around the mammary papillae in two young pigs. The enhancing superficial inguinal lymph nodes were easily identified and successfully removed percutaneously by the breast lesion excision system (BLES). This method was safe and timesaving, and thus may have some potential for the clinical use.

The sensitivity of lymphatic CEUS for the detection of SLN in animal studies is summarized in tab. 1.

Tab. 1

The sensitivity of lymphatic CEUS for the detection of SLN in animal studies

Author	Year	Subjects	Tumor	Number of subjects	Contrast agent	Sensitivity	Gold standard
Sever et al.(31)	2012	Swine	Normal	2	SonoVue® mixed with blue dye	100% (3/3)	-
Nielsen et al.(30)	2008	Swine	Normal	13	SonoVue®	84% (22/26)	Blue dye
Goldberg et al.(18)	2011	Swine	Melanoma	63	Sonazoid®	81,8% (293/351)	Scintigraphy + blue dye
Wang et al.(25)	2009	Rabbit	Breast cancer	12	SonoVue®	89,5% (17/19)	Blue dye
Wang et al.(26)	2009	Dog	Normal	5	SonoVue®	91,3% (21/23)	Blue dye
Lurie et al.(27)	2006	Dog	Head or neck tumor	10	Definity®	80% (8/10)	Scintigraphy
Goldberg et al.(28)	2005	Swine, rabbit, dog, monkey	Normal	8 pigs, 4 rabbits, 7 dogs, 1 monkey	Sonazoid®	100% (20/20)	Scintigraphy + blue dye
Goldberg et al.(17)	2004	Swine	Melanoma	6	Sonazoid®	90% (28/31)	Scintigraphy + blue dye

Patient studies

Breast cancer

Lymphatic CEUS with Sonazoid®

UCAs have been proven to be safe in human applications which has been widely shown in liver and non-liver organs(. Two more recent studies show that UCA could be reliably taken in the SLN and suggest the SLN detection with CEUS is feasible for breast cancer patients(. In 2009, Omoto et al. published their results of preliminary clinical study on SLN detection using lymphatic CEUS with subareolar Sonazoid® injection in 20 breast cancer patients(, the sensitivity of lymphatic CEUS, blue dye and γ-probe-guided methods were 70%, 70%, and 100%, respectively. Lymphatic CEUS has the same sensitivity as blue dye, but significantly lower than that of γ-probe detection method.

Lymphatic CEUS with SonoVue®

Sever et al. ( published three papers about the detection of SLN using CEUS with SonoVue®, the sensitivity in the three studies were 93% (126/136), 89% (71/80) and 89% (48/54), respectively. After SLNs were identified, guidewires could be inserted. Operative findings using conventional blue dye and radioisotope techniques confirmed that the wired nodes in these patients were SLNs. These results suggest that the lymphatic CEUS may become a useful new modality for sentinel node identification (fig. 2).

Fig. 2

Lymph node before (A) and after (B) subcutaneous injection of SonoVue®. The contrast enhancement was limited to a smaller part of the lymph node

Comparison of SonoVue® and Sonazoid®

In the studies on humans(, between one or two SLNs could be detected in each patient. The transit time from injection of UCA to arrival in the SLN was 15–45 seconds with SonoVue® but it needed an average time of 5 minutes with Sonazoid®. The time of UCA remaining in the SLN was 1–3 minutes with SonoVue®, whereas Sonazoid® could stay for much longer time in the nodes, which may allow for sufficient time for intraoperative ultrasound localization of the SLNs during resection. Sever reported that the whole procedure including detection of SLN with SonoVue® and the insertion of guidewire could be completed in 15 minutes(. In addition, both UCAs allow the real-time visualization of contrast agent flowing from the tumor region to the lymphatic vessels and entering SLN(.

Melanoma

Although CEUS has been proven useful in the detection of SLN in breast cancer patients, one study( using SonoVue® revealed disappointing results in 10 patients with melanoma. The author used a similar method to that reported by Goldberg with Sonazoid® in a swine model(, but SLNs were visualized only in one patient (1/10). This suggested that CEUS might not be used for the SLN detection in patients with melanoma, despite the high sensitivity in a swine model with melanoma. The possible explanations, made by the authors, included: 1) the structure of the lymph nodes and the afferent lymphatic vessels are different than those of humans, 2) the echogeneity of the hilum in humans is more hyperechoic than in swine which makes it more difficult to decide whether the hyperechogeneity is normal or due to the contrast agent, 3) Sonazoid® seems to fill the lymph nodes better than SonoVue® giving higher contrast to the surroundings. However, the exact reasons for the different results are still not clear. The sensitivity of lymphatic CEUS for the detection of SLN in patient studies is summarized in the tab. 2.

Tab. 2

The sensitivity of lymphatic CEUS for the detection of SLN in patient studies

Author	Year	Tumor	Number of subjects	Contrast agent	Sensitivity	Gold standard
Sever et al.(32)	2012	Breast cancer	136	SonoVue®	93% (126/136)	-
Sever et al.(24)	2011	Breast cancer	80	SonoVue®	89% (71/80)	-
Rue Nielsen et al.(33)	2009	Melanoma	10	SonoVue®	10% (1/10)	Scintigraphy + γ-probe + blue dye
Sever et al.(19)	2009	Breast cancer	54	SonoVue®	89% (48/54)	Radioisotope + blue dye
Omoto et al.(20)	2009	Breast cancer	20	Sonazoid®	70% (14/20)	Scintigraphy + γ-probe + blue dye
Omoto et al.(21)	2006	Breast cancer	23	25% albumin solution	52% (12/23)	Scintigraphy + γ-probe + blue dye

Challenges

Even though there are promising results from the above studies and the advantages of lymphatic CEUS (e.g. realtime, minimal invasive, capability of guidance puncture), some challenges and questions concerning its clinical application are still open.

First of all, preliminary studies suggest that intravenous CEUS can improve the differential diagnosis of malignant lymph nodes from reactive nodes and provide a more accurate selection of nodes to be submitted to fine-needle aspiration biopsy (fig. 3)(. However, there is a controversy on whether lymphatic CEUS for detection of SLN is capable to differentiate benign from malignant nodes, and delineate the perfusion defect which is useful for guidance of needle biopsy to preoperatively stage more accurately. In the two studies by Goldberg in a swine model, CEUS could delineate the intranodal perfusion defects, which may indicate SLN metastases, and the accuracy of lymphatic CEUS in two studies was 86% and 80%, respectively. However, Wang reported that CEUS might not be helpful for detecting metastases in SLNs in the rabbit model(. In addition, partial enhancement of SLN indicating the proliferation of lymphatic follicles or lymphatic sinus was reported in a dog model(.

Fig. 3

Malignant infiltration of a lymph node using CEUS intravenously. Note atypical vessels also peripheral located

Moreover, it is reported( that the usefulness of lymphatic CEUS for the detection of SLNs in patients with melanoma was disappointing, though it has been proven successful in the detection of SLN in breast cancer patients and in melanoma swine models. However, since so far there has been only one report about the visualization of SLN in patients with melanoma using CEUS, more studies with alternative setup and/or UCA are warranted.

Furthermore, there are several UCAs available on the market, but which agent is the most effective for the detection and evaluation of SLNs is unclear so far. A multicenter trial approach including comparison of UCAs is needed. Moreover, there is a problem concerning short half-life of some microbubbles, which might be solved by prolonged or repeated injections.

In conclusion the application of CEUS for the investigation of SLN is at an initial stage in animal models and has not been sufficiently evaluated in humans, therefore, this technique is not recommended for a routine application.