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Sentinel Lymph Node Imaging with Microbubble Ultrasound Contrast Material
Sentinel Lymph Node Imaging with Microbubble Ultrasound Contrast Material1 Robert F. Mattrey, MD, Yuko Kono, MD, Kris Baker, Tom Peterson
RATIONALE AND OBJECTIVES Lymphadenectomy is necessary to provide local control and staging of breast cancer patients because the degree of nodal involvement remains the most important prognostic indicator (1). Because morbidity with axillary dissection occurs in as many as 20% of patients (2,3), attempts at limiting dissection have led to the development of sentinel node resection. The technique was popularized by Morton and associates (4) for staging melanoma, and Giuliano and associates (5) applied it to breast cancer. They showed that, when the sentinel node was negative, the remainder of the downstream nodes were negative in 126 of 127 cases. When the node was positive, it was the only positive node in over 60% of cases (4) and contained five times more micrometastasis than nonsentinel nodes (6). The difficulty of the procedure lies in the localization and identification of the sentinel node. As described by Giuliano and associates (5), the procedure begins with the injection of 3 to 5 mL of isosulfan blue, a water-soluble dye, in the breast mass and surrounding tissue. Approximately 5 min later, blunt dissection is made to locate a blue lymphatic channel or a blue node. Although all blue nodes are removed, an attempt is made to follow the feeding lymphatic channel toward the mass to ensure the identification of the first and true sentinel node. The resected nodes are assessed histologically. Acad Radiol 2002; 9(suppl 1):S231–S235 1 From the Department of Radiology, University of California, San Diego, MRI Institute, 410 Dickinson Street, San Diego, CA 92103-8756. Supported in part by ROICA36799 and Alliance Pharmaceutical Corp. Equipment loan from Siemens Ultrasound. Address correspondence to R.F.M.
R.F.M. is a consultant to APC. ©
AUR, 2002
If no cancer deposits are found, dissection is terminated; otherwise, classic axillary dissection follows. Much in this technique bears refinement, however, since Giuliano, the most experienced investigator, reported that the sentinel node was detected in 58% in the first 87 cases and 78% in the next 87 cases. The failure is that nodes are indistinguishable from breast tissue unless colored blue, the dye has unpredictable and rapid clearance, and the drainage pattern varies among patients. The rapid clearance of the blue dye provides a short time window of only a few minutes between operating too early when no nodes are stained, or too late when too many nodes are stained. Radiopharmaceutical colloids are available that provide some preoperative localization as they flow through the lymphatic chain (7,8). Further, with the aid of a gamma pencil probe, pinpoint localization of radioactivity could lead to the nodes intraoperatively. Although radiolabeled colloids have a more delayed transit and provide a skin marking option, they are less than ideal. The fluid is invisible intraoperatively, many nodes are enhanced, and, more importantly, the proximity of the injection site to the nodes decreases the target-to-background ratio, decreasing sentinel node specificity (9,10). At present, most centers use both the blue dye and the radiolabeled colloid methods to gain sensitivity. Particles injected subcutaneously enter the lymph vessel through gaps between lymphatic endothelial cells or by transcellular endo- or exocytosis. On average, smaller particles (10 to 40 nm) are more likely to enter than larger particles. As particles approach 1 m, their uptake into lymphatics is very poor and must be carried away by phagocytes or reduced in size by local processes. In fact, over 95% of particles larger than 400 nm stay at the injection site, whereas 74% of particles 10 times smaller (40 nm) are absorbed (11). We hypothesized that 2- to
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MATTREY ET AL
Academic Radiology, Vol 9, Suppl 1, 2002
Figure 1. Images of the popliteal fossa obtained with real-time harmonic imaging immediately after the bolus injection of 0.5 mL AF0150 in the foot pad and during massage. A, Filling of the lymphatic duct (straight arrow) and partial filling of the lymph node (curved arrow). B, Homogeneous but sparse distribution of microbubbles within the normal portion of the node (straight arrow). No contrast material is seen in the cancerous portion (curved arrow).
3-m bubbles, particularly those with nonrigid shells, can deform to enter lymphatic vessels. Further, because ultrasound is extremely sensitive to small quantities of microbubbles, it may be able to detect any absorption that occurs (12). Therefore, the purpose of this study was to demonstrate the feasibility that microbubbles can enter the lymphatic channels in sufficient quantity to enhance local
S232
lymph nodes and provide real-time observation of microbubble filling of the draining nodes. MATERIALS AND METHODS Vx2 cancer cells were harvested from carrier rabbits and inoculated in the thigh of 15 healthy New Zealand
Academic Radiology, Vol 9, Suppl 1, 2002
SENTINEL LYMPH NODE IMAGING
Figure 2. Images of the iliac chain obtained with intermittent imaging (5-sec interframe delay) are shown (A) before, (B) early, and (C) late during filling. A, Enlarged nodes (straight arrows) along the iliac vessel (curved arrow). B, Shortly after massage, the lymph duct (arrowhead) is seen leading to the enhanced node (straight arrow). The vessel (curved arrow) remains unenhanced. C, Image obtained after 20-sec delay shows complete filling of the nodes seen in A (arrows). Nodes filled to a degree that shadowing is observed (arrowheads). The nodes were normal on direct lymphography (not shown).
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Academic Radiology, Vol 9, Suppl 1, 2002
Figure 3. (A) The standard two-dimensional (2D) image and (B) the Burst mode overlay were obtained after a 30-sec delay over the popliteal node of the same rabbit shown in Figure 1. A, Normal and abnormal portions of the popliteal nodal structure. B, The Burst image assigns signal to regions containing microbubbles and severely suppresses tissue signal. The tracing outlining the node in A is shown again in B to demonstrate that bubbles are limited to the normal half of the node as seen in Figure 1b.
White (NZW) rabbits. Implants were allowed to grow for 14 to 18 days before imaging, which was done under anesthesia (50 mg/kg ketamine and 8.8 mg/kg xylazine given subcutaneously) using a Siemens Sonoline Elegra
S234
scanner (Siemens Ultrasound, Issaquah, Wash). This instrument is equipped with intermittent imaging capability as well as wideband harmonic imaging (13) that was operated at 7.2 MHz.
SENTINEL LYMPH NODE IMAGING
Academic Radiology, Vol 9, Suppl 1, 2002
Injections of AF0150 (Imagent; Alliance Pharmaceutical Corp., San Diego, Calif) were made in the foot pad, popliteal fossa, thigh, and/or around the tumor for a total dose of 1 mL per rabbit. Popliteal as well as iliac lymph nodes were imaged as the injection sites were massaged. Real-time scanning was used first, and then intermittent imaging was used with the interframe delay increased from 1 to 45 sec. After imaging, direct x-ray lymphography of the pelvic nodes was performed to assess whether nodes were normal or contained filling defects. This was performed by dissecting the popliteal fossa of the affected leg to locate the popliteal node and slowly infusing 0.5 mL of Ethiodol (Savage Laboratories, Melville, NY) directly into the node. The injection site in the thigh was also dissected for gross assessment.
CONCLUSION This study demonstrated the feasibility of lymphatic imaging after the subcutaneous injection of AF0150, a microbubble contrast agent. The advantage of this technique is that it can be used immediately after injection to localize and mark regional nodes preoperatively and can be used again intraoperatively. Further, because microbubbles were visible in the lymphatic duct with real-time and were also destroyed by sound when they accumulated in nodes, the combination of massage and imaging techniques (real-time and intermittent) can be used to map out the drainage field and to recognize the true sentinel lymph node. REFERENCES
RESULTS While the injection site was massaged, contrast material filled the lymphatic duct and was seen flowing toward the lymph node (Fig 1). The microbubbles in the lymph node were sparsely but homogeneously distributed throughout the node (see Fig 1). Some microbubbles could be seen exiting the node, suggesting that they were carried by lymph. With intermittent imaging, greater filling of the node was achieved particularly with longer delay times (Fig 2). When the delay time was greater than 20 sec, some nodes filled with sufficient amount of contrast material to produce shadowing (see Fig 2). When nodes had a cancerous deposit, the cancer within the node did not fill with contrast material, leaving a filling defect (Figs 1, 3). Further work is required to confirm that this technique can be used. Although the agent could be seen when in motion with standard ultrasound, visibility was dramatically improved with the wideband harmonic imaging technique. Imaging eliminated the enhancement of the node, likely secondary to bubble destruction (14,15). Remassaging the injection site refilled the node. However, this was not possible after the second or third filling despite further massaging and the continued presence of the agent at the injection site at postmortem inspection. This suggests that only a subpopulation of microbubbles injected were able to enter the lymphatic vessel during the immediate postinjection period. It also suggests that optimization of the agent could promote additional uptake. Degree of nodal enhancement was variable from rabbit to rabbit, likely due to variations in the injection and imaging techniques.
1. Fisher ER, Costantino J, Fisher B, Redmond C. Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol 4). Discriminants for 15-year survival. National Surgical Adjuvant Breast and Bowel Project Investigators. Cancer 1993; 71(6 suppl):2141–2150. 2. Coburn MC, Bland KI. Surgery for early and minimally invasive breast cancer. Curr Opin Oncol 1995; 7:506 –510. 3. Petrek JA, Blackwood MM. Axillary dissection: current practice and technique. Curr Probl Surg 1995; 32:257–323 4. Morton DL, Wen D-R, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127:392–399. 5. Giuliano AE, Kirgan DM, Geunther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994; 220: 391– 401. 6. Giuliano AE, Dale PS, Turner RR, et al. Improved axillary staging of breast cancer with sentinel lymphadenectomy. Ann Surg 1995; 222: 394 –399. 7. Uren RF, Howman-Giles R, Thompson JF, et al. Mammary lymphoscintigraphy in breast cancer. J Nucl Med 1995; 36:1775–1780. 8. Krag DN, Weaver DL, Alex JC, Fairbank JT. Surgical resection and radiolocalization of the sentinel lymph node in breast cancer using a gamma probe. Surg Oncol 1993; 2:335–339. 9. Ege GN, Warbick A. Lymphoscintigraphy: a comparison of 99Tc(m) antimony sulphide colloid and 99Tc(m) stannous phytate. Br J Radiol 1979; 52:124 –129. 10. Kapteijn BAE, Baidjnath Panday RKL, Liem IH. Lymphoscintigraphy and intraoperative gamma probe detection of the first draining lymph node in clinically localized melanoma (abstr). J Nucl Med 1995; 35:222P. 11. Oussoren C, Zuidema J, Crommelin DJ, Storm G. Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid composition and lipid dose. Biochim Biophys Acta 1997; 1328:261–272. 12. Mattrey RF, Wrigley R, Steinbach GC, Schutt EG, Evitts DP. Gas emulsion as ultrasound contrast agents: preliminary results in rabbits and dogs. Invest Radiol 1994; 29:S139 –S141. 13. Mattrey RF, Steinbach G, Lee Y, Wilkenning W, Lazenby J. High resolution harmonic grayscale (GS) imaging of normal and abnormal vessels and tissues in animals. Acad Radiol 1998; 5(suppl 1):S63–S65. 14. Sirlin CB, Girard MS, Steinbach GC, et al. Effect of ultrasound transmit power on liver enhancement with Imagent® US, a PFC-stabilized microbubble contrast agent. Int J Imaging Systems Technol 1997; 8:82– 88. 15. Sirlin CB, Girard MS, Baker KG, Steinbach GC, Deiranieh LH, Mattrey RF. Effect of acquisition rate on liver and portal vein enhancement with microbubble contrast. Ultrasound Med Biol 1999; 25:331–338.
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RATIONALE AND OBJECTIVES Lymphadenectomy is necessary to provide local control and staging of breast cancer patients because the degree of nodal involvement remains the most important prognostic indicator (1). Because morbidity with axillary dissection occurs in as many as 20% of patients (2,3), attempts at limiting dissection have led to the development of sentinel node resection. The technique was popularized by Morton and associates (4) for staging melanoma, and Giuliano and associates (5) applied it to breast cancer. They showed that, when the sentinel node was negative, the remainder of the downstream nodes were negative in 126 of 127 cases. When the node was positive, it was the only positive node in over 60% of cases (4) and contained five times more micrometastasis than nonsentinel nodes (6). The difficulty of the procedure lies in the localization and identification of the sentinel node. As described by Giuliano and associates (5), the procedure begins with the injection of 3 to 5 mL of isosulfan blue, a water-soluble dye, in the breast mass and surrounding tissue. Approximately 5 min later, blunt dissection is made to locate a blue lymphatic channel or a blue node. Although all blue nodes are removed, an attempt is made to follow the feeding lymphatic channel toward the mass to ensure the identification of the first and true sentinel node. The resected nodes are assessed histologically. Acad Radiol 2002; 9(suppl 1):S231–S235 1 From the Department of Radiology, University of California, San Diego, MRI Institute, 410 Dickinson Street, San Diego, CA 92103-8756. Supported in part by ROICA36799 and Alliance Pharmaceutical Corp. Equipment loan from Siemens Ultrasound. Address correspondence to R.F.M.
R.F.M. is a consultant to APC. ©
AUR, 2002
If no cancer deposits are found, dissection is terminated; otherwise, classic axillary dissection follows. Much in this technique bears refinement, however, since Giuliano, the most experienced investigator, reported that the sentinel node was detected in 58% in the first 87 cases and 78% in the next 87 cases. The failure is that nodes are indistinguishable from breast tissue unless colored blue, the dye has unpredictable and rapid clearance, and the drainage pattern varies among patients. The rapid clearance of the blue dye provides a short time window of only a few minutes between operating too early when no nodes are stained, or too late when too many nodes are stained. Radiopharmaceutical colloids are available that provide some preoperative localization as they flow through the lymphatic chain (7,8). Further, with the aid of a gamma pencil probe, pinpoint localization of radioactivity could lead to the nodes intraoperatively. Although radiolabeled colloids have a more delayed transit and provide a skin marking option, they are less than ideal. The fluid is invisible intraoperatively, many nodes are enhanced, and, more importantly, the proximity of the injection site to the nodes decreases the target-to-background ratio, decreasing sentinel node specificity (9,10). At present, most centers use both the blue dye and the radiolabeled colloid methods to gain sensitivity. Particles injected subcutaneously enter the lymph vessel through gaps between lymphatic endothelial cells or by transcellular endo- or exocytosis. On average, smaller particles (10 to 40 nm) are more likely to enter than larger particles. As particles approach 1 m, their uptake into lymphatics is very poor and must be carried away by phagocytes or reduced in size by local processes. In fact, over 95% of particles larger than 400 nm stay at the injection site, whereas 74% of particles 10 times smaller (40 nm) are absorbed (11). We hypothesized that 2- to
S231
MATTREY ET AL
Academic Radiology, Vol 9, Suppl 1, 2002
Figure 1. Images of the popliteal fossa obtained with real-time harmonic imaging immediately after the bolus injection of 0.5 mL AF0150 in the foot pad and during massage. A, Filling of the lymphatic duct (straight arrow) and partial filling of the lymph node (curved arrow). B, Homogeneous but sparse distribution of microbubbles within the normal portion of the node (straight arrow). No contrast material is seen in the cancerous portion (curved arrow).
3-m bubbles, particularly those with nonrigid shells, can deform to enter lymphatic vessels. Further, because ultrasound is extremely sensitive to small quantities of microbubbles, it may be able to detect any absorption that occurs (12). Therefore, the purpose of this study was to demonstrate the feasibility that microbubbles can enter the lymphatic channels in sufficient quantity to enhance local
S232
lymph nodes and provide real-time observation of microbubble filling of the draining nodes. MATERIALS AND METHODS Vx2 cancer cells were harvested from carrier rabbits and inoculated in the thigh of 15 healthy New Zealand
Academic Radiology, Vol 9, Suppl 1, 2002
SENTINEL LYMPH NODE IMAGING
Figure 2. Images of the iliac chain obtained with intermittent imaging (5-sec interframe delay) are shown (A) before, (B) early, and (C) late during filling. A, Enlarged nodes (straight arrows) along the iliac vessel (curved arrow). B, Shortly after massage, the lymph duct (arrowhead) is seen leading to the enhanced node (straight arrow). The vessel (curved arrow) remains unenhanced. C, Image obtained after 20-sec delay shows complete filling of the nodes seen in A (arrows). Nodes filled to a degree that shadowing is observed (arrowheads). The nodes were normal on direct lymphography (not shown).
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MATTREY ET AL
Academic Radiology, Vol 9, Suppl 1, 2002
Figure 3. (A) The standard two-dimensional (2D) image and (B) the Burst mode overlay were obtained after a 30-sec delay over the popliteal node of the same rabbit shown in Figure 1. A, Normal and abnormal portions of the popliteal nodal structure. B, The Burst image assigns signal to regions containing microbubbles and severely suppresses tissue signal. The tracing outlining the node in A is shown again in B to demonstrate that bubbles are limited to the normal half of the node as seen in Figure 1b.
White (NZW) rabbits. Implants were allowed to grow for 14 to 18 days before imaging, which was done under anesthesia (50 mg/kg ketamine and 8.8 mg/kg xylazine given subcutaneously) using a Siemens Sonoline Elegra
S234
scanner (Siemens Ultrasound, Issaquah, Wash). This instrument is equipped with intermittent imaging capability as well as wideband harmonic imaging (13) that was operated at 7.2 MHz.
SENTINEL LYMPH NODE IMAGING
Academic Radiology, Vol 9, Suppl 1, 2002
Injections of AF0150 (Imagent; Alliance Pharmaceutical Corp., San Diego, Calif) were made in the foot pad, popliteal fossa, thigh, and/or around the tumor for a total dose of 1 mL per rabbit. Popliteal as well as iliac lymph nodes were imaged as the injection sites were massaged. Real-time scanning was used first, and then intermittent imaging was used with the interframe delay increased from 1 to 45 sec. After imaging, direct x-ray lymphography of the pelvic nodes was performed to assess whether nodes were normal or contained filling defects. This was performed by dissecting the popliteal fossa of the affected leg to locate the popliteal node and slowly infusing 0.5 mL of Ethiodol (Savage Laboratories, Melville, NY) directly into the node. The injection site in the thigh was also dissected for gross assessment.
CONCLUSION This study demonstrated the feasibility of lymphatic imaging after the subcutaneous injection of AF0150, a microbubble contrast agent. The advantage of this technique is that it can be used immediately after injection to localize and mark regional nodes preoperatively and can be used again intraoperatively. Further, because microbubbles were visible in the lymphatic duct with real-time and were also destroyed by sound when they accumulated in nodes, the combination of massage and imaging techniques (real-time and intermittent) can be used to map out the drainage field and to recognize the true sentinel lymph node. REFERENCES
RESULTS While the injection site was massaged, contrast material filled the lymphatic duct and was seen flowing toward the lymph node (Fig 1). The microbubbles in the lymph node were sparsely but homogeneously distributed throughout the node (see Fig 1). Some microbubbles could be seen exiting the node, suggesting that they were carried by lymph. With intermittent imaging, greater filling of the node was achieved particularly with longer delay times (Fig 2). When the delay time was greater than 20 sec, some nodes filled with sufficient amount of contrast material to produce shadowing (see Fig 2). When nodes had a cancerous deposit, the cancer within the node did not fill with contrast material, leaving a filling defect (Figs 1, 3). Further work is required to confirm that this technique can be used. Although the agent could be seen when in motion with standard ultrasound, visibility was dramatically improved with the wideband harmonic imaging technique. Imaging eliminated the enhancement of the node, likely secondary to bubble destruction (14,15). Remassaging the injection site refilled the node. However, this was not possible after the second or third filling despite further massaging and the continued presence of the agent at the injection site at postmortem inspection. This suggests that only a subpopulation of microbubbles injected were able to enter the lymphatic vessel during the immediate postinjection period. It also suggests that optimization of the agent could promote additional uptake. Degree of nodal enhancement was variable from rabbit to rabbit, likely due to variations in the injection and imaging techniques.
1. Fisher ER, Costantino J, Fisher B, Redmond C. Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol 4). Discriminants for 15-year survival. National Surgical Adjuvant Breast and Bowel Project Investigators. Cancer 1993; 71(6 suppl):2141–2150. 2. Coburn MC, Bland KI. Surgery for early and minimally invasive breast cancer. Curr Opin Oncol 1995; 7:506 –510. 3. Petrek JA, Blackwood MM. Axillary dissection: current practice and technique. Curr Probl Surg 1995; 32:257–323 4. Morton DL, Wen D-R, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127:392–399. 5. Giuliano AE, Kirgan DM, Geunther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994; 220: 391– 401. 6. Giuliano AE, Dale PS, Turner RR, et al. Improved axillary staging of breast cancer with sentinel lymphadenectomy. Ann Surg 1995; 222: 394 –399. 7. Uren RF, Howman-Giles R, Thompson JF, et al. Mammary lymphoscintigraphy in breast cancer. J Nucl Med 1995; 36:1775–1780. 8. Krag DN, Weaver DL, Alex JC, Fairbank JT. Surgical resection and radiolocalization of the sentinel lymph node in breast cancer using a gamma probe. Surg Oncol 1993; 2:335–339. 9. Ege GN, Warbick A. Lymphoscintigraphy: a comparison of 99Tc(m) antimony sulphide colloid and 99Tc(m) stannous phytate. Br J Radiol 1979; 52:124 –129. 10. Kapteijn BAE, Baidjnath Panday RKL, Liem IH. Lymphoscintigraphy and intraoperative gamma probe detection of the first draining lymph node in clinically localized melanoma (abstr). J Nucl Med 1995; 35:222P. 11. Oussoren C, Zuidema J, Crommelin DJ, Storm G. Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid composition and lipid dose. Biochim Biophys Acta 1997; 1328:261–272. 12. Mattrey RF, Wrigley R, Steinbach GC, Schutt EG, Evitts DP. Gas emulsion as ultrasound contrast agents: preliminary results in rabbits and dogs. Invest Radiol 1994; 29:S139 –S141. 13. Mattrey RF, Steinbach G, Lee Y, Wilkenning W, Lazenby J. High resolution harmonic grayscale (GS) imaging of normal and abnormal vessels and tissues in animals. Acad Radiol 1998; 5(suppl 1):S63–S65. 14. Sirlin CB, Girard MS, Steinbach GC, et al. Effect of ultrasound transmit power on liver enhancement with Imagent® US, a PFC-stabilized microbubble contrast agent. Int J Imaging Systems Technol 1997; 8:82– 88. 15. Sirlin CB, Girard MS, Baker KG, Steinbach GC, Deiranieh LH, Mattrey RF. Effect of acquisition rate on liver and portal vein enhancement with microbubble contrast. Ultrasound Med Biol 1999; 25:331–338.
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