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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The contrast-enhanced ultrasound (CEUS)-guided method in combination with Sonazoid has not been clinically or experimentally evaluated with regard to its use for identifying sentinel lymph node (SLN) in the stomach. Therefore, we attempted to evaluate the usefulness of the CEUS-guided method with Sonazoid for imaging of the lymphatic channels and SLN of the stomach in a porcine model by comparing it with the conventional Evans blue dye-guided method. Twenty-eight 2 to 3-month-old swine weighing 17–30 kg were used in this experiment. Anesthesia was maintained with 2.0–3.0% isoflurane/O2 inhalation. Sonazoid was injected into the intra- and sub-mucosal layers of the stomach. The intragastric or transcutaneous CEUS-guided method was used to identify the lymphatic channels and SLN of the stomach. Contrast imaging using the CEUS-guided method with Sonazoid enabled us to produce clear images of the afferent lymph vessel and SLN of the stomach until 2 h after the injection of Sonazoid. In addition, intranodal flow of the microbubble agent could be clearly identified using tissue linear harmonic images of the SLN. The SLN detection rate was not significantly different between the CEUS- and dye-guided methods. However, the Evans blue dye flowed out quickly (∼15 min after the injection) through the true SLN into the next LN of stomach. In conclusion, the use of the CEUS-guided method with Sonazoid might be the most useful clinical procedure for producing real-time images of the SLN of the stomach, and the linear harmonic images are also useful for evaluating intranodal structure within the SLN. (Cancer Sci 2011; 102: 2073–2081)

The sentinel lymph node (SLN) is the first lymph node that receives drainage from a primary tumor. Morton et al.(1) initially demonstrated the SLN concept in a feline model and later confirmed it in a clinical study of patients with breast cancer and melanoma. The clinical impact of the SLN concept has become one of the most important topics in surgical oncology.(1–3) Recently, gastric cancer has also been identified as a target for SN navigational surgery (SNNS).(4–10)

The dye-guided or radioisotope (RI)-guided method, or a combination of both, is conventionally used for SLN mapping in gastric cancer.(4–10) The dye-guided method is convenient and safe. However, it has been reported to be associated with a high false negative node ratio because the small dye particles can readily diffuse through the true SLN and transverse multiple nodes.(4–10) The RI-guided method has several advantages over the dye-guided method for identifying SLN. However, lymph vessels cannot be visualized. The high radioactivity at the primary injection site might also interfere with the intraoperative detection of nearby lymph nodes.(11,12)

Recently, contrast-enhanced ultrasonography in combination with Sonazoid was adopted to detect the SLN in patients with breast cancer.(13) However, the clinical usefulness of the CEUS-guided method in combination with Sonazoid is still controversial, because the superiority of the CEUS-guided method compared with the conventional dye- or RI-guided method, in terms of SLN detection rate and accuracy, has not been determined in preclinical or clinical studies. In addition, few or no studies exist to confirm experimentally or clinically the usefulness of the CEUS-guided method in combination with Sonazoid for imaging the SLN of the stomach.

To address the possibility that the CEUS-guided method in combination with Sonazoid will be a useful SLN detection method in patients with gastric cancer, we have attempted to: (i) first evaluate the usefulness of the CEUS-guided method in combination with the intra- and sub-mucosal injection of Sonazoid for imaging the lymphatic channels and SLN of the stomach in a porcine animal model; and then (ii) compare quantitatively the SLN detection rate and accuracy obtained using the conventional Evans blue dye-guided method in the same animal model.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

In this experiment we imaged the lymphatic channels and SLN of the stomach in a porcine model. The procedure was designed to identify the SLN and its afferent lymph vessels using lymphosonography in combination with Sonazoid and then the usefulness of the procedure was compared with the images of the lymphatic channels and SLN obtained with the conventional Evans blue dye-guided method in the animal model.

Animals, anesthesia and monitoring.  This experiment was approved by the Institutional Animal Care and Use Committee of Shinshu University. Twenty-eight 2 to 3-month-old, crossbred ([Yorkshire × Landrace] × Duroc) swine (11 males and 17 females) weighing 17–30 kg (23.0 ± 0.8 kg, n = 28) were used in the animal experiments in a humane and ethical fashion. The animals were fasted overnight and pre-anesthesia sedation was intramuscularly administered with 0.1–0.2 mg/kg medetomidine hydrochloride (Orion Pharma, Helsinki, Finland) and 0.7–1.2 mg/kg midazolam (Astellas Pharma, Tokyo, Japan). Anesthesia was maintained with 2.0–3.0% isoflurane (Dainippon Sumitomo Pharma, Tokyo, Japan)/O2 inhalation, titrated to effect after endotracheal intubation. Ventilation was maintained at 10–15 mL/kg per min for 10–15 breaths per minute. Electrocardiography and heart rate were monitored (FCP-140; FUKUDA DENSI, Tokyo, Japan). Physiological saline solution (Otsuka Pharma, Tokyo, Japan) was administered at 10 mL/kg per h during the experiment. The animals were subsequently euthanized after the completion of the experiments.

Experimental procedure and protocols of CEUS-guided imaging.  The swine were placed in a supine position on the operating table. The body temperature of each animal was maintained at 36.5–37.5°C using a heating pad. The abdomen was then cut along the median line and the stomach was gently dragged out to allow the pylorus and greater curvature of the stomach to be examined. In order to perform intragastric CEUS-guided imaging of the lymphatic channels and SLN of the stomach, a 5 cm linear incision was made in the avascular region between the lesser and greater curvature of the stomach. The ultrasound probe was then inserted into the stomach through the incision and used to observe the lymphatic channels and SLN via the CEUS-guided method after the injection of Sonazoid (Daiichi-Sankyo Group, Tokyo, Japan). Thus, 0.01–0.3 mL Sonazoid was injected into the intra- and sub-mucosal layers of the stomach at approximately 9 cm from above the pylorus and approximately 2 cm away from the greater curvature of the stomach, and then the injection site was gently massaged for 10 s.

Sonazoid is a lipid-stabilized suspension of 2.4–3.5 μm perfluorobutane microbubbles that was originally developed as an ultrasound contrast agent. Several studies have been reported on the use of Sonazoid for CEUS-guided imaging of the lymphatic channels and SLN in animal models.(14,15) In these studies, the contrast agent was injected into the subject, and then transcutaneous, contrast-specific gray-scale or color-flow Doppler ultrasonography was performed. The agent can be clearly visualized on ultrasound as it passes through the lymphatic channels and the SLN, but does not pass beyond the first-echelon lymph nodes.(15) The safety of the intravenous administration of Sonazoid has been established in human studies evaluating its use for imaging of the liver and heart.(16)

After intragastric CEUS-guided imaging of the lymphatic channels and SLN of the stomach, we sutured the abdominal wall and then closed the abdomen. Next, the lymphatic channels and SLN of the stomach were also re-evaluated in the same animal using the transcutaneous CEUS-guided method in combination with Sonazoid.

Ultrasound procedure.  Conventional gray-scale ultrasound using a fundamental scanner (EUB-7500; Hitachi, Tokyo, Japan) and a 3.5 MHz convex (EUP C715; Hitachi) or 13 MHz flat linear array (EUP L74M; Hitachi) transducer was used prior to the injection of Sonazoid, with adjustment of the imaging parameters such as system and depth of field. After Sonazoid injection, gray-scale contrast harmonic or tissue linear harmonic imaging was used. This technique allows clear visualization of the contrast agent flowing through the lymph channels with good accuracy and spatial resolution. For example, using tissue linear harmonic imaging, the contrast agent can be clearly seen flowing into the lymph nodes. The mechanical index (MI) is a measure of the acoustic pressure generated within the ultrasound field.(17) The MI values of the acoustic pressure used in contrast or tissue harmonic imaging of Sonazoid range from 0.2 to 0.3 in order to reduce microbubble destruction. Flash replenishment imaging (FRI) was performed with the use of a higher MI (>1.0) to confirm the presence of Sonazoid as it caused the disappearance of the Doppler signal due to microbubble rupture. Micro-flow imaging (MFI) was also adopted to confirm the reflow of Sonazoid through the lymphatic channels and the SLN of the stomach. All precontrast and postcontrast scans were performed by the same sonographer.

Once the contrast agent had been identified, the injection site was gently massaged (10 s) to expedite the flow of Sonazoid into the lymphatic channels. Subsequently, lymphosonography was used to identify Sonazoid as it moved through the lymphatic channels to the SLN of the stomach. Once the SLN had been identified, the abdominal skin was marked with an indelible marker. In some cases, the contrast harmonic imaging was performed transcutaneously in the region where the skin had been marked after the abdominal skin had been stitched up.

After the ultrasound imaging recording had finished (n = 23), dissection and isolation were carried out in the abdomen to identify the SLN or SL basins in the presence of the Evans blue dye. Thus, 0.1 mL 1% Evans blue dye (Sigma, St Louis, MO, USA) containing 2% bovine serum albumin (BSA; Sigma Aldrich, St Louis, MO, USA) was injected into the same intra- and sub-mucosal layers as the Sonazoid-injected sites, and the morphologies of the excised lymphatic channels and SLN of the stomach were visualized.

Imaging analysis.  All images were recorded as videos (10 frames/s, 120 s, EUB-7500; Hitachi). All data were analyzed frame by frame.

Experimental protocols of Evans blue dye-guided imaging.  To compare the SLN detection rate and accuracy between the CEUS-guided and conventional dye-guided methods, we then investigated the lymphatic channels and SLN of the porcine stomach (n = 5) using the Evans blue dye-guided method. Thus, 0.1 mL 1% Evans blue dye containing 2% BSA was injected into the same intra- and sub-mucosal layers of the stomach as the injection sites of Sonazoid, which was approximately 9 cm from above the pylorus and approximately 2 cm away from the greater curvature of the stomach. The flow patterns of Evans blue dye through the lymphatic channels, true SLN and transverse lymph nodes were observed intra-abdominally after gentle massage of the injection site for 10 s. The flow patterns of Evans blue dye were photographed by a digital camera (CX4; RICOH, Tokyo, Japan) every 1 min until 30 min after the dye injection.

In some animals, to evaluate the corresponding SLN changes depending on the dye injection point, 0.1 mL Evans blue dye containing 2% BSA was injected at approximately 9 cm from above the pylorus and approximately 2 cm away from the lesser curvature of the stomach, and then we investigated changes in the SLN position using the Evans blue dye-guided method.

Histological studies.  To identify histologically the injected position of Sonazoid or Evans blue dye in the wall of the porcine stomach, histological analyses of the wall of the stomach pre-injected with 0.01 mL Indian ink were conducted. For light microscopy observation, specimens containing Indian ink were fixed with 10% formalin solution for 24 h. The specimens were dehydrated through graded series of ethanol and then embedded in paraffin in a routine manner. Sections of 3–4 μm were processed using hematoxylin–eosin stain. The sections were then examined using a light microscope (Leica, Wetzler, Germany) and photographed.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Figure 1 shows representative images of the operated stomach (Fig. 1A), photomicrographs of the macroscopic and microscopic sites of Sonazoid or Evans blue dye (Fig. 1B–D) and a representative image produced by the intragastric CEUS-guided method using a contrast harmonic probe (Fig. 1E). As shown in Figure 1(D), the injected Indian ink was clearly observed within the intra- and sub-mucosal layers of the stomach (Fig. 1D, arrowheads). In addition, the CEUS-guided contrast harmonic and tissue linear harmonic images were recorded in the 23 pigs examined. In all cases (n = 23), the contrast agent, Sonazoid, was easily identified as it flowed through the afferent lymph vessels and into the SLN of the stomach. Thus, the detection rate of the SLN of the stomach evaluated with the CEUS-guided method in combination with Sonazoid was 100.0% (n = 23). The locations of the lymphatic channels and SLN of the stomach were confirmed using FRI and MFI in all cases.

image

Figure 1.  (A) Representative images of the operated stomach in a porcine model. (B) Representative photomicrograph of the Sonazoid injection point (low magnification, ×1). (C) Representative photomicrograph of the Sonazoid injection point (high magnification, ×3). (D) Representative histological photomicrograph of the gastric wall pre-injected with Indian ink. The Indian ink was confirmed at the intra- and sub-mucosal layers (arrowheads) of the stomach. Bar, 100 μm. (E) Representative photomicrograph produced using the intragastric contrast-enhanced ultrasound-guided method during the identification of the lymphatic channels and sentinel lymph node of the stomach using a contrast harmonic probe.

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Effects of the injected dose of Sonazoid on contrast harmonic imaging of the lymphatic channels and SLN of the stomach. Figure 2 demonstrates representative tracings of the effects of the injected dose of Sonazoid, which ranged from 0.01 to 0.1 mL, on contrast images of the lymphatic channels and SLN of the stomach. In all cases (n = 23), the afferent lymph vessels of the SLN were clearly identified within 30 s of the intra- and sub-mucosal injection of more than 0.03 mL Sonazoid. The SLN of the stomach were also identified using contrast harmonic imaging within 30 s of the Sonazoid injection in all cases (Fig. 2B,C). In contrast, the injection of Sonazoid at a dose of 0.01 mL produced no contrast imaging of the afferent lymph vessel or SLN of the stomach within 1 min of its injection. However, approximately 5 min after the injection of Sonazoid, a small SLN was dimly observed in the stomach (Fig. 2A).

image

Figure 2.  Representative tracings of the effects of Sonazoid injected at doses 0.01 mL (A), 0.03 mL (B) and 0.1 mL (C) on contrast imaging of the lymphatic channels and sentinel lymph node (SLN) of the stomach in a porcine model. Solid arrows, SLN of the stomach; dotted arrows, afferent lymph vessel. IVC, inferior vena cava; m, minutes; s, seconds; PV, portal vein.

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Figure 3 shows representative tracings of the FRI and MFI of the afferent lymph vessels and SLN of the stomach produced using the CEUS-guided method at an MI of more than 1.0. The intra- and sub-mucosal injection of Sonazoid at a dose of 0.3 mL allowed rapid acquisition of clear contrast harmonic images of the afferent lymph vessels and SLN of the stomach (Fig. 3A). Thus, just after (<20 s) the injection of 0.3 mL Sonazoid, the afferent lymph vessels of the stomach were clearly identified (Fig. 3A, left-most panel). The contrast images of the afferent lymph vessels and SLN of the stomach became more and more clear in a time-dependent manner. This finding was confirmed in all cases (n = 23). To confirm the flow of Sonazoid through the lymphatic channels and SLN of the stomach, we conducted an experiment in which we ruptured the perfluorobutane microbubbles using the CEUS-guided method at a higher acoustic pressure (MI more than 1.0). In the same animal, we obtained FRI of the afferent lymph vessel and SLN of the stomach using ultrasound at an MI of 1.2, which resulted in significant sharpening of the images of the afferent lymph vessels and SLN of the stomach (Fig. 3B, left-most panel). Approximately 2 s after the FRI, no contrast images of the lymph vessel or SLN of the stomach were observed (Fig. 3B, 2 s after the FRI). At more than 20 s after stimulation, the afferent lymph vessels and SLN of the stomach reappeared on the contrast harmonic images in a time-dependent manner (Fig. 3B, 20 s after the FRI).

image

Figure 3.  (A) Representative tracings of contrast harmonic images of the afferent lymph vessels and sentinel lymph node (SLN) of the stomach obtained using the intragastric contrast-enhanced ultrasound (CEUS)-guided method in combination with the intra- and sub-mucosal injection of 0.3 mL Sonazoid. (B) Representative tracings of flash replenishment images (FRI) and contrast harmonic images obtained at 2, 10 and 20 s after FRI stimulation in the same animal. (C) Representative tracings of contrast harmonic images of the SLN of the stomach obtained using the intragastric CEUS-guided method at 10, 20, 40, 60, 90 and 120 min after the intra- and sub-mucosal injection of 0.3 mL Sonazoid. Solid arrows, SLN of the stomach; dotted arrows, afferent lymph vessel. IVC, inferior vena cava; m, minutes; s, seconds; PV, portal vein.

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Next, to evaluate the time-dependent changes in the contrast harmonic images of the lymphatic channels and SLN of the stomach, we investigated the changes in the images recorded every 10, 20 or 30 min until 2 h after the intra- and sub-mucosal injection of 0.3 mL Sonazoid.

Figure 3(C) demonstrates representative tracings of the contrast images of the afferent lymph vessels and SLN of the stomach recorded every 10–30 min. The SLN of the stomach was clearly identified at 10 min after the intra- and sub-mucosal injection of 0.3 mL Sonazoid (Fig. 3C, left-most panel at the upper tracing). Very similar contrast images of the lymph vessels and SLN of the stomach were produced at 20, 40, 60, 90 and 120 min after the injection of 0.3 mL Sonazoid (Fig. 3C, upper and lower tracings). In addition, we confirmed that the contrast agent did not pass beyond the first-echelon lymph node.

Tissue linear harmonic imaging of the lymphatic channels and SLN of the stomach.  To evaluate the lymph flow within the SLN of the stomach in detail, we produced tissue linear harmonic images of the SLN of the stomach using a linear transducer (EUP L74M, 13MHz; Hitachi).

Figure 4(A) shows representative tracings of the distribution of Sonazoid within the SLN of the stomach. The microbubbles were clearly identified in the marginal and trabecular sinuses within the SLN of the stomach. Thus, the video composed of tissue linear harmonic images enabled us to visualize the Sonazoid flowing within the SLN of the stomach.

image

Figure 4.  (A) Representative tracings of tissue linear harmonic images of the afferent lymph vessels and sentinel lymph node (SLN) of the stomach obtained with the intragastric contrast-enhanced ultrasound (CEUS)-guided method at 500, 510, 520, 530 and 540 s after the intra- and sub-mucosal injection of 0.3 mL Sonazoid. (B) Representative tracings of contrast harmonic (B-1) and tissue linear harmonic (B-2) images of the SLN of the stomach in the same animal. (B-3) Representative photomicrograph of the SLN of the stomach identified by injecting Evans blue dye into the same location as the Sonazoid. Solid arrows, SLN of the stomach; dotted arrows, afferent lymph vessel. m, minutes; s, seconds; PV, portal vein.

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Confirmation of the SLN of the stomach using the Evans blue dye-guided method.  To evaluate whether the CEUS-guided images of the SLN of the stomach agreed with those produced after injection of Evans blue dye are as the same position as Sonazoid, we compared the CEUS-guided images with the photomicrographs of the SLN of the stomach produced using Evans blue dye.

Figure 4(B) shows representative CEUS-guided images and a photomicrograph of the SLN of the stomach produced by the Evans blue dye injection. Parts of the SLN of the stomach were clearly stained by Evans blue dye (Fig. 4B-3). Very similar areas of the SLN of the stomach were identified by the contrast harmonic (Fig. 4B-1) and tissue linear harmonic images (Fig. 4B-2) of the SLN of the stomach.

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Figure 5.  (A) Representative tracing of contrast imaging of the SLN of the stomach obtained using the intragastric contrast-enhanced ultrasound (CEUS)-guided method in combination with the intra- and sub-mucosal injection of 0.3 mL Sonazoid. (B) Representative photomicrograph of the transcutaneous CEUS-guided method in the same animal. (C) Representative tracings of the contrast harmonic and flash replenishment images (FRI) of the sentinel lymph node (SLN) of the stomach obtained using the transcutaneous CEUS-guided method after the intra- and sub-mucosal injection of 0.3 mL Sonazoid in the same animal. Solid arrows, SLN of the stomach. s, seconds.

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image

Figure 6.  (A) Representative photomicrographs of the afferent lymph vessel (LV), the sentinel lymph node (SLN), the efferent lymph vessel, the next lymph node and its efferent LV obtained using the conventional Evans blue dye-guided method at 3, 4, 8 and 15 min after the intra- and sub-mucosal injection of the dye. (B) Left panel (8 min): representative photomicrograph of the afferent LV, the SLN and the efferent LV obtained using the Evans blue dye-guided method at 8 min after the Evans blue dye was injected 2 cm away from the greater curvature of the stomach; right panel (15 min): representative photomicrograph of another SLN of the stomach obtained at just 2 min after the additional injection of Evans blue dye 2 cm away from the lesser curvature of the stomach.

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Transcutaneous contrast-enhanced ultrasonography of the SLN of the stomach.  Next, to evaluate the similarity of intragastric CEUS-guided images of the SLN of the stomach with those obtained by the transcutaneous CEUS-guided method, we investigated the SLN of the stomach using the transcutaneous CEUS-guided method in the same animal after we had identified the SLN of the stomach using the intragastric CEUS-guided method and then sutured the abdominal wall. Figure 5 demonstrates representative tracings of intragastric and transcutaneous CEUS-guided images (Fig. 5A,C) and a photomicrograph of the operated animal (Fig. 5B). The contrast harmonic images of the SLN of the stomach produced after the intra- and sub-mucosal injection of 0.3 mL Sonazoid were clear (Fig. 5A). In the same animal, the abdominal wall was sutured (Fig. 5B), and then the SLN of the stomach was confirmed to be located in the same position using the transcutaneous CEUS-guided method (Fig. 5C). In addition, in the case of the transcutaneous method, the contrast imaging of the SLN of the stomach was augmented by ultrasound stimulation at an MI of more than 1.2 (Fig. 5C, 170 s). At 2 s after stimulation, the image of the SLN of the stomach had completely disappeared (Fig. 5C, 2 s after FRI). The image reappeared 20 s after stimulation with FRI (Fig. 5C, 20 s after FRI).

Imaging of the lymphatic channels and SLN of the stomach using the conventional dye-guided method.  To compare the SLN detection rate and accuracy in the animal experiments between the CEUS-guided and conventional dye-guided methods, we evaluated the lymphatic channels and SLN of the stomach by the intra- and sub-mucosal injection of 0.1 mL 1% Evans blue dye into the stomach (n = 5). Figure 6(A) shows representative photomicrographs of the Evans blue dye-guided images of the afferent lymph vessel, true SLN, efferent lymph vessel, next lymph node and its efferent lymph vessel of the stomach photographed every 1 min until 15 min after injection. The true SLN, next lymph node and each efferent lymph vessel were clearly identified at 3 min after the injection of Evans blue dye. In addition, the Evans blue dye within the SLN of the stomach was stained heterogeneously and then disappeared in a time-dependent manner. At 15 min after the Evans blue injection, the dye within the SLN was diluted and then the dye within the next lymph node gradually became condensed. Thus, the phenomenon of overflow of Evans blue dye through the true SLN, which might produce a high false negative node, was confirmed within 3 min after the Evans blue dye injection. The detection rate of the SLN of the stomach evaluated by the dye-guided method at 3 min after the injection of Evans blue was 100.0% (n = 5). However, the accuracy of the Evans blue dye-guided method was confirmed in the present experiments to be significantly lower than the CEUS-guided method, because the dye-guided method was associated with a high false negative node ratio due to overflow out of the SLN.

To evaluate the SLN changes depending on the injection point, we conducted another preliminary experiment to identify the SLN of the stomach using 0.1 mL 1% Evans blue dye intra- and sub-mucosal injection, which was approximately 9 cm from above the pylorus and approximately 2 cm away from the lesser curvature of the stomach. Figure 6(B) demonstrates representative photomicrographs of the Evans blue dye images of true SLN before (Fig. 6B, 8 min) and after (Fig. 6B, 15 min) the injection of Evans blue dye at the lesser curvature of the stomach. The additional injection of Evans blue dye at the lesser curvature of the stomach produced the new image of corresponding SLN of the stomach. Thus, the SLN of the stomach was clearly different from the SLN of the stomach identified by the injection of Evans blue dye at the greater curvature of the stomach.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Gastric SLN identification using the CEUS-guided method with Sonazoid.  The identification of the SLN, that is, the LN that tumor cells reach first, is important for deciding whether axillary LN dissection should be performed in breast cancer patients. The current standard methods for SLN detection are the dye-guided and RI-guided methods. Although the dye is inexpensive, some skill is required to use it for detecting the node. Moreover, since anaphylactic reactions to the dye, although rare, have been reported,(18) care is necessary during the use of the dye. In contrast, the RI-guided method requires many hours to detect the SLN after the radioactive colloid has been injected. Another disadvantage of this method is that it must be performed in a hospital that can handle radioactive materials. To overcome these problems, the CEUS-guided method of SLN detection has recently been developed.

Early gastric cancer is the most suitable target of SLN mapping in gastrointestinal cancer because individualized and minimally invasive surgery based on SLN biopsy might be applicable,(19–21) However, in cases of gastric cancer, 5–10% of SLN are located in the second compartment that might be accounted for by aberrant drainage routes from the primary lesion. No suitable technique for identify lymphatic networks and SLN located in the second compartment is available. In addition, the benefit of using the CEUS-guided method in combination with Sonazoid to identify the lymphatic channels and SLN of the stomach has not been evaluated in a clinical setting. Therefore, in the present experiments, we attempted to perform real-time imaging of the lymphatic channels and SLN of the stomach in a porcine model using the CEUS-guided method in combination with Sonazoid, and evaluate the effectiveness of the CEUS-guided method in terms of the SLN detection rate and accuracy in comparison with the conventional dye-guided method. Thus, the present study is the first demonstrating that using the CEUS-guided method in combination with intra- and sub-mucosal injection of Sonazoid (0.05–0.3 mL) enables the production of clear contrast harmonic images of the afferent lymph vessels and SLN approximately 20 s after the injection. The images of the lymph vessels and SLN last 120 min after the injection of 0.3 mL Sonazoid. In addition, SLN mapping of the perigastric region can be performed using the transcutaneous CEUS-guided method. In conclusion, the use of the CEUS-guided method in combination with the intra- and sub-mucosal injection of Sonazoid could become the most useful clinical procedure for producing real-time images of the lymphatic channels and SLN of the stomach, and also for evaluating the SLN located in the second compartment. Thus, the SLN concept, but not sentinel basins, is applicable to gastric cancers when we use the real-time CEUS-guided method in combination with Sonazoid. In addition, the SLN detection rate was confirmed to be not significantly different between the CEUS-guided and conventional dye-guided methods. However, in relation to accuracy in the detection of the SLN of the stomach, the CEUS-guided method in combination with Sonazoid was confirmed to be better than the dye-guided method. Therefore, the SLN might be a good target for selective lymphadenectomy for early gastric cancer associated with a risk of micrometastasis. However, further investigations are necessary to evaluate the molecular and functional mechanisms of the flow of intra- and sub-mucosally injected Sonazoid into the initial lymphatics, but not the blood capillaries or venules. In addition, further studies will be needed in the future to evaluate the corresponding SLN changes depending on the injection point of Sonazoid using the CEUS-guided method.

Evaluation of intranodal structure within the SLN.  Another important aspect of the present study is that intranodal flowing of Sonazoid can be clearly identified on videos composed of tissue linear harmonic images within the SLN of the stomach. Thus, we concluded that the CEUS-guided method in combination with Sonazoid enabled us to visualize the intranodal structure and real-time flow of Sonazoid within the SLN of the stomach.

The SLN is the most common and earliest site of malignant tumor metastasis. Lymph nodes act as a mechanical barrier to prevent the passage of tumor cells through the node and also act as a biological barrier to inhibit tumor growth in the node.(22–26) In contrast, it is also known that primary tumors alter the tumor tissue microenvironment prior to the formation of metastases.(27,28) Recently, we(29) demonstrated that intracellular adhesion molecule-1 (ICAM-1) in the premetastatic regional lymph node is involved in producing a suitable environment for micro-metastasis within the lymph nodes.

According to the results of our present and previous studies, we would like to propose the new clinical possibility that microenvironmental changes within premetastatic or micrometastatic SLN could be identified on tissue linear harmonic images within the SLN of the stomach using Sonazoid associated with molecular markers such as ICAM-1. If such contrast agents are developed, changes in the intranodal structure depending on flow patterns of Sonazoid or the distribution of ICAM-1 expression within the SLN might be identified clinically. Further investigation will be needed in the future to evaluate the relationship between these changes within the SLN and the micrometastases of carcinoma cells.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The study was financially supported, in part, by Grants-in-Aid for Scientific Research (19209044, 22249052) from the Japanese Ministry of Education, Science, Sports and Culture, and by the Intelligent Surgical Instruments Project of METI (Japan) (2007–2012).

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The authors declare that no competing financial interests exist.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References