This work was partly funded by the LYriCAN (INCa-DGOS-Inserm_12563). The authors acknowledge the Claude Bernard University Lyon 1 for the award of the Antonin Poncet prize for work related to this study. The authors gratefully acknowledge the willing participation of the patients involved, EDAP-TMS, the role of the Steering committee (Dominique Elias, Olivier Rouviere, Alain Sezeur, Fr&#233;d&#233;ric Marchal), which consisted of responsibility for deciding on the continuation of the study after an interim analysis, as well as Jean-Yves Scoazec for pathological examination.

The protocol was reviewed and validated by a national ethics committee according to French and European directives, with written consent from the patients.
1. Inclusion
<ol>
	<li>Include patients with resectable CLM and a good clinical condition (Eastern Cooperative Oncology Group ECOG/World Health Organization WHO performance status 0-1) who are at least 18 years old. Obtain written consent from the patients.</li>
	<li>Exclude patients with previous liver, biliary, or major abdominal surgery, pregnancy, or an inability to be followed for the duration of the study.</li>
</ol>
2. HIFU Treatment
<ol>
	<li>Perform the anesthesia of the patient with an induction composed of propofol (2 to 3 mg/kg), sufentanil (0.2 to 0.3 &#181;g/kg), cisatracurium (0.15 mg/kg) and ketamine (0.15 mg/kg).</li>
	<li>Maintain anesthesia with sevoflurane to target a bispectral index between 40 and 60.</li>
	<li>Perform tracheal intubation, with the lungs mechanically ventilated with inspired oxygen fraction of 80%.</li>
	<li>Prepare the HIFU device in the operating room by covering the sterilized HIFU probe with the dedicated sterile envelope containing a sterile ultrasound coupling liquid (Figure 1). 
	NOTE: The HIFU probe was sterilized using a low-temperature gas plasma sterilizer.</li>
	<li>Perform laparotomy using a monopolar knife after classic surgical asepsis.</li>
	<li>Place the retractors under the rib cage and between the edges of the surgical incision.</li>
	<li>Confirm liver resection by exploring the liver using intraoperative diagnostic ultrasound images with a 7.5 MHz T-type transducer and palpation to confirm that no additional metastasis compromising the resection was missed with preoperative imaging22.</li>
	<li>Free the liver from its peritoneal and vascular attachments with scissors.</li>
	<li>Place a protective abdominal gauze sponge (20 x 20 cm2) with X-ray detectable threads under the targeted area to inhibit ultrasound propagation and unintended HIFU damage to adjacent organs.</li>
	<li>Bring the HIFU probe in contact with the liver surface (Figure 2).</li>
	<li>Move the HIFU probe on the surface of the liver until the position of the expected HIFU ablation (represented by the white cone superimposed on the sonogram) is at the right location according to the objectives of the different phases (Figure 3a).</li>
	<li>Activate the HIFU exposure by activating each emitter with the same phase for 40 s. Apply an acoustic power of 70 W (free-field acoustic power) for superficial ablations and 90 W (free-field acoustic power) for in-depth lesions.
	<ol>
		<li>Ultrasound images were not synchronized with HIFU exposures and, therefore, interferences were visible in sonograms during ablations. Observe that boiling is created in liver tissues treated during HIFU single exposures. Note that a cloud of ebullition appears momentarily for 1 min at the location of the lesion (Figure 3b). 
		NOTE: The single lesion appears on the sonogram as a hyperechoic zone at its center and a hypoechoic zone at its boundaries (Figure 4a).</li>
	</ol>
	</li>
	<li>Mark the position of the HIFU probe, mainly the acoustic axis at the surface of the liver, using a surgical marker, to know where to cut the liver for gross examination (Figure 2).</li>
	<li>Measure the dimensions of the HIFU ablations along two orthogonal planes after each HIFU exposure, using the integrated ultrasound imaging probe.</li>
	<li>Move the HIFU probe on the liver surface to confirm that all eight liver segments can be covered, irrespective of their conformation in the individual patient.</li>
</ol>
3. Macroscopical and Histological Evaluation of HIFU Lesions
<ol>
	<li>Perform the planned liver resection using a Kelly clamp to crush the liver parenchyma and a scissor to cut the liver. Perform hemostasis with titanium clips and/or irrigating bipolar forceps.</li>
	<li>Close the aponeurosis with monofilament, long-lasting absorbable N&#176; 0 or N&#176; 1 suture material.</li>
	<li>Close the skin with uncolored, short-term absorbable N&#176; 3/0 suture material.</li>
	<li>Give the patient oral acetaminophen (1 g every 6 h). 
	NOTE: Nausea and vomiting were treated with 4 mg of intravenous ondansetron. Oral fluids and feeding were started on the day after surgery. Morphine was given to obtain a pain visual analogic scale under 30/100.</li>
	<li>Separate the HIFU ablations and areas of CLM with a scalpel.</li>
	<li>Cut the ablations along the exposure axis with a scalpel.</li>
	<li>Take pictures of the HIFU ablations.</li>
	<li>Measure the dimensions of the ablated area macroscopically with a ruler and on a photograph, using appropriate image analysis software.</li>
	<li>Slice each ablation into 5 mm sections to assess macroscopic homogeneity.</li>
	<li>Fix the ablated areas in 10% phosphate-buffered formalin (pH = 7), embed them in paraffin, and stain them with hematoxylin and eosin.</li>
	<li>Histologically examine the ablated and surrounding tissues.</li>
</ol>
4. Follow-up
<ol>
	<li>Follow the patients for one month according to the institution&#8217;s guidelines for patients resected for CLM.</li>
	<li>Summarize data regarding demographic characteristics, vital signs (baseline and 5 min after HIFU), and safety observations and measurements regarding the HIFU lesions.</li>
	<li>Use Spearman&#8217;s rank correlation coefficient to test the association between variables.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+a+tumor-mimic+model+for+the+evaluation+of+the+accuracy+of+HIFU+treatments.+results+of+in+vitro+experiments+in+the+liver.">Utility of a tumor-mimic model for the evaluation of the accuracy of HIFU treatments. results of in vitro experiments in the liver.</a> Ultrasound in Medicine &amp; Biology. 34 (12), 1934-1943 (2008).</li><li>Melodelima, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+ablation+produced+using+a+surgical+toroidal+high-intensity+focused+ultrasound+device+is+independent+from+hepatic+inflow+occlusion.">Thermal ablation produced using a surgical toroidal high-intensity focused ultrasound device is independent from hepatic inflow occlusion.</a> Physics in Medicine &amp; Biology. 54 (20), (2009).</li><li>Melodelima, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+ablation+by+high-intensity-focused+ultrasound+using+a+toroid+transducer+increases+the+coagulated+volume.+Results+of+animal+experiments.">Thermal ablation by high-intensity-focused ultrasound using a toroid transducer increases the coagulated volume. Results of animal experiments.</a> Ultrasound in Medicine &amp; Biology. 35 (3), 425-435 (2009).</li><li>Parmentier, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+ablation+for+the+treatment+of+colorectal+liver+metastases+during+an+open+procedure:+study+on+the+pig.">High-intensity focused ultrasound ablation for the treatment of colorectal liver metastases during an open procedure: study on the pig.</a> Annals of Surgery. 249 (1), 129-136 (2009).</li><li>Lee, Y. J., Wesley, R. 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The authors have nothing to disclose.

The aim of this study was to confirm the feasibility, safety, and efficacy of a new intraoperative HIFU device. As this was its first use in humans, an ablate-and-resect study was designed in order to verify the ability of the device to create complete ablation and accurate targeting without compromising the chance of potentially curative therapy for patients. In total, 30 HIFU ablations were safely created in 15 patients, each within 40 s. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2. There was no secondary lesion in neighboring organs. Importantly, the shape of the HIFU ablations was not altered by the major blood vessels (up to 5 mm in diameter). This is of particular importance since it has been reported that blood flow plays a pivotal role in thermal ablation by limiting the ablation size and altering ablation shape23. Another important advantage of this intraoperative HIFU device when compared with ablations performed using radiofrequency or microwaves is that there is no need to insert a probe in the liver parenchyma.
The choice of developing a HIFU device dedicated to intraoperative use was supported by oncological necessities and technological challenges that remain unmet in HIFU technology. Extracorporeal HIFU treatments have been proved to be clinically feasible. However, the liver volume accessible to an extracorporeal HIFU treatment is about 30% unless a partial rib resection is performed24. In addition, when using HIFU extracorporeally for treating liver tumors, skin burns and gastric lesions can be created25,26. The intraoperative approach was selected to avoid these potentially important complications.More importantly, intraoperative ablations are widely employed in the management of CLM, whether they are associated with surgery or not. Despite improvements in imaging, new liver metastasis are found in approximately 15% of patients22, and new findings, such as peritoneal carcinomatosis, may preclude the need for liver-directed therapy. For this first use in humans of the intraoperative HIFU device, ablations were created close to metastases and only in areas planned for resection. This first step was regarded as essential to demonstrate the accuracy and the safety of this new HIFU device before considering its routine use to treat CLM.
The size of the HIFU probe (10 cm in diameter) does not limit its ease of use since it was possible to place the probe as such that it allowed a targeting of approximately 90% of the entire liver. This was possible even through a limited midline laparotomy. HIFU ablations were easily identified by ultrasound imaging, allowing a reliable monitoring of the treatment, which is a major advantage over other ablative techniques. The slight difference between ablation dimensions measured on ultrasound images and those measured on macroscopic examination was due to the difficulty to cut fresh liver exactly along the imaging plane.
Although this study confirmed the theoretical advantages of HIFU for liver ablation (i.e., no need for an intraparenchymal introduction of a probe and real-time monitoring, and independence from the heat-sink effect), the efficacy of intraoperative HIFU should be demonstrated on liver metastases. If the ability to target liver metastases is confirmed during phase IIb, the next step will be to design a multicenter prospective cohort of patients treated with curative-intent with intraoperative HIFU.

Colorectal liver metastases (CLMs) represent a major public health issue. When resection and/or ablation of CLM can be achieved, a cure is possible, and five-year survival rates of up to 51% have been reported1. Despite progress in surgery (complex surgical techniques)2 and downsizing obtained with chemotherapy3, only a minority of patients are eligible for surgery (less than 20%). When metastases are confined to the liver, surgery is precluded by the size and/or location of metastases, their number, or because the necessary resection will leave an insufficient volume of functional liver4.
Radiofrequency ablation (RFA) and microwave ablation (MWA) have been widely used in association with surgery to increase the number of patients treated with a curative intent. However, these techniques require the intraparenchymal introduction of a probe. Moreover, these techniques are inadequate due to the heat-sink effect of blood flow, and real-time monitoring is not possible with these techniques.
Medical ultrasound is mainly known as a diagnostic imaging technique. However, ultrasound can be used as a modality to deliberately cause tissue damage. The energy of the HIFU beam is several orders of magnitude greater than those of a standard diagnostic ultrasound beam. The high energy levels carried in a HIFU beam are magnified further by focusing the beam deep in tissues with precision to a small volume while sparing surrounding tissues. The tissue temperature at the focal point quickly rises to 70&#8211;90 &#176;C, resulting in an irreversible destruction by coagulative necrosis5. HIFU has been proven effective in a wide range of clinical applications, especially prostate cancer6,7. Current medical devices using HIFU are based on an extracorporeal approach, and the single ablation achieved is small and ellipsoidal. The dimensions of every single lesion vary according to the transducer characteristics but are typically 1&#8211;3 mm (transverse) and 8&#8211;15 mm (along the beam axis)8. To treat tumors, hundreds of lesions must be placed systematically side by side to ablate the target tumor and some of the surrounding normal tissue margin. The placement of small lesions side by side requires precision that is difficult to ensure in moving organs and results in long-lasting treatments (in the order of 1&#8211;2 h). Whereas numerous publications have reported about MWA and RFA for the treatment of CLM9,10,11,12,13, few studies have been published about HIFU for CLM14,15. HIFU treatment presents several potential advantages when compared to MWA or RFA as follows: there is no need to puncture the parenchyma, the treatment is independent of perfusion8, and real-time monitoring is possible. Extracorporeal treatment of the liver is difficult because the focusing effect is altered by the respiratory motion of the liver and the presence of the ribcage, which may stop the propagation of ultrasound waves.
As focal destruction is often associated with open liver surgery in the management of patients with CLM, we aimed to design a new and powerful HIFU. Such a device would be able to perform large ablations in a short amount of time. Thus, we developed a new technology based on toroidal transducers16 with encouraging results in preliminary in vitro and preclinical work17,18,19. We previously demonstrated in a porcine model that this HIFU device can create reproducible ablations in 40 s20. The average volume of these ablations was 7 cm3, corresponding to an average diameter of 20 mm and an average long axis of 25 mm. These ablations were created by activating each emitter with the same phase and by applying an acoustic power of 70 W (free-field acoustic power) for 40 s18. To confirm these results, this HIFU device was used in a controlled trial in patients undergoing hepatectomy for CLM. The objective of the study was to assess the safety and the feasibility of the procedure. Data regarding the efficacy and the accuracy of the procedure were also collected. This HIFU device was not developed to replace the resection part of surgery. The long-term objective is to provide a complementary tool to surgeons in order to expand the number of patients treated with a curative intent by treating otherwise unresectable patients, using HIFU.
The study participants could not expect any personal benefit from their participation, and the priority was to avoid any loss of chance, which is why an ablate-and-resect study was designed. The surgical procedure had to be identical to the one patients would have undergone if not included in the study. For this first use of the HIFU device in humans, ablations were created on the part of the liver scheduled for resection, in order to protect participating patients from any adverse effects related to the use of this new device.
Study Design:
The protocol was a prospective, single-center phase I/II study designed to assess specific objectives in three different phases (I, IIa, and IIb). In phases I and IIa, the HIFU treatment was performed in parts of the liver planned for resection but at a distance from metastases. Liver metastases were targeted during phase IIb. Data of phase IIb are not presented as this part of the protocol is still ongoing.
The primary objectives of phase I were to evaluate the safety, feasibility, and tolerability of HIFU ablation for patients with CLM. The safety and feasibility were assessed by the ability to use the HIFU device in aseptic conditions. The ability to perform two HIFU ablations without injuring organs adjacent to the liver was also assessed. In all patients, one HIFU lesion was superficial and one was created with the focal zone placed at least 1 cm deeper. Vital signs were monitored during and for 5 min after the HIFU ablations in order to assess the tolerability of the procedure.
Secondary objectives were to determine the reliability of the ultrasound images and the ease of use of the device. The reliability of the ultrasound images was assessed by using the imaging probe integrated with the HIFU device to visualize and target the region to be treated. Ease of use was assessed by the ability to place the HIFU device such that ablations could be performed in at least 80% of the whole liver. The entire HIFU procedure has to be performed in less than 30 min.
This two-step phase was designed following Lee&#39;s sequential criteria21, allowing an early termination of enrollment in the case of an unacceptable global failure rate. The definition of failure was a period longer than 30 min to perform the HIFU procedure, the inability to place the focal zone at least 1 cm from Glisson&#39;s capsule, asepsis failure when using the device, HIFU lesions in adjacent organs, changes in vital signs superior to 10% from the baseline. The enrollment was considered to be closed if a failure was observed in at least 2/2 patients (first step) and 3/6 patients (second step).
In phase IIa, a major anatomical structure was mimicked by a metallic marker with a diameter of 5 mm, implanted in the liver parenchyma. The primary objective of phase IIa was to assess the accuracy of the HIFU device. In step 1, the ability to target the metallic marker with safety margins was assessed. In step 2, the ability to ablate the liver at a predetermined distance from the metallic marker was assessed. The ideal distance between the metallic marker and the closest ablation boundary was fixed at 7.5 mm, with a tolerated range of 1&#8211;15 mm. The secondary objective was to assess the safety of the procedure by creating HIFU lesions in the liver without injury to adjacent organs. The feasibility to image HIFU ablations using the integrated ultrasound imaging probe was a common secondary endpoint with phase I. Three to six patients were included in both steps of phase IIa. Binomial probabilities in 3 and 6 patients were used. If a failure occurred in at least 54% and 32% of the patients, respectively, there was a 90% probability of observing that failure.

<table>
	<tbody>
		<tr>
			<td>MFOCUS</td>
			<td></td>
			<td>N/A</td>
			<td>The transducer has a toroidal geometry and is divided into eight radial ultrasound emitters of 4.16 cm2 each. The working frequency of the transducer is 3 MHz. Each of the eight emitters is divided into 32 individual transducers of equal surface (13 mm2). The radius of curvature of the transducer was 7 cm and the diameter was 7 cm. A curved array ultrasound imaging probe working at a frequency of 7.5 MHz (Vermon, Tours, France) was placed in the center of the device and connected to an ultrasound imaging scanner. The imaging transducer is composed of 128 elements with a 63% bandwidth. The ultrasound imaging plane was aligned with the HIFU acoustic axis in order to position the region to be treated and visualize the ablation with the same device. Due to the geometrical characteristics of a torus, the ultrasound beams coming from each of the eight emitters intersect beyond the principal focal ring and form a secondary focal zone, which contributes to reinforce the homogeneity of the lesion at its center. The location of the focal zones could be changed by electronic focusing or by adjusting the quantity of liquid between the device and the tissue.</td>
		</tr>
		<tr>
			<td>Curved array ultrasound imaging probe working at a frequency of 7.5 MHz</td>
			<td>Vermon</td>
			<td>6,5/R11,5/192</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound imaging scanner BK HAWK</td>
			<td>B-K Medical</td>
			<td>2102 EXL</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallic marker</td>
			<td>BIP GmbH</td>
			<td>O-twist-marker</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J Image analysis software</td>
			<td>Wayne Rasband</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile envelope</td>
			<td>CIVCO</td>
			<td>CIV-Flex Transducer Cover</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound coupling liquid</td>
			<td>EDAP-TMS</td>
			<td>Liv-kit</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the feasibility, safety, and accuracy of an intraoperative high-intensity focused ultrasound device for treating liver metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Fifteen patients were included between March 2010 and November 2011. Six patients were included in phase I. Six patients were included in step 1 of phase IIa. Three patients were included in step 2 of phase IIa. A right or extended right hepatectomy was performed in all patients.Phase IIb is currently ongoing, and results are not described. Complete results of phases I and IIa have been previously published14.
Phase I:
In total, twelve HIFU ablations were successfully created in the six phase-I patients (Figure 5). One superficial HIFU ablation and one deep HIFU ablation were created in each of the 6 patients. Deep HIFU lesions were placed at a mean depth of 12 &#177; 2 (10&#8211;20) mm corresponding to a focal distance of 22 &#177; 2 (20&#8211;40) mm (Figure 3a). The resulting mean distance between Glisson&#39;s capsule and the HIFU ablation was, on average, 7 &#177; 4 (2&#8211;13) mm. Asepsis was not comprised in all patients during the preparation and utilization of the device. There was no lesion in neighboring tissues after HIFU ablation. There were no any substantial changes in respiratory and hemodynamic parameters. The duration of the HIFU procedure was, on average, 15 &#177; 7 (9&#8211;27) min. The ultrasound imaging probe integrated with the HIFU device allows visualizing, on average, 88% (95% CI: 67 to 100%) of the 15 predefined areas.
Phase IIa:
For the first three patients of step 1, the metallic mark had not been included in the HIFU lesions in one of six ablations. After reviewing the record of the procedure, it was established that a Glissonian pedicle was confused with the metallic marker (Figure 6). As required by the study design, three more patients were included, and all six additional HIFU lesions were correctly created around all six metallic markers. Since the primary objective of step 1 was achieved, the steering committee approved the continuation of the study.
The aim of step 2 was to achieve ablations distant from a metallic marker by 7.5 mm. Six ablations with a distance of 7.0 &#177; 2.3 (4.3&#8211;9.8) mm were achieved, demonstrating that it was possible to preserve an area from damage. The procedure was safe, asepsis was not compromised, and there was no lesion in the neighboring organs and no rupture of Glisson&#39;s capsule.
Common Secondary Objectives of Phases I and IIa:
All 30 HIFU ablations created in 15 patients were slightly cone-shaped. The average length of the HIFU ablations was 27.5 &#177; 6 mm. The average superior width of the HIFU ablations was 21 &#177; 3.9 mm. In ultrasound images, the average length of HIFU ablations was 28.9 &#177; 4.6 mm and the average superior width was 23.9 &#177; 3.8 mm. Figure 4b shows the rank correlation coefficient (r = 0.88; p &#60; 0.001; 95% CI, 0.82 to 0.91) between macroscopic measurements and measurements performed using ultrasound images.
Figure 7 shows the capability of the imaging system to visualize metastases. Figure 7a and 7c show examples of two metastases imaged using the ultrasound imaging probe integrated with the HIFU device. Figure 7b and 7d show the corresponding metastases observed on gross pathology after surgery. The HIFU focal region was superimposed on the sonogram and displayed in the user interface, making it possible to place the location of the ablation with precision (Figure 7a).
As illustrated in Figure 8, the delimitation between treated and untreated liver is very sharp (about 200 &#181;m). There was a halo of congestive tissues around all HIFU ablations. The microscopic examination confirmed homogeneous necrosis with all HIFU ablations. Since there were only 1 to 2 h between HIFU ablation and hepatectomy, it was not possible to observe evidence of apoptosis. However, pronounced alterations in hepatocytes were observed in all HIFU ablations. The microscopic analysis confirmed homogeneous ablations with all cases, notably around vessels (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57964/57964fig1.jpg" /> 
Figure 1: Preparation of the HIFU device. The HIFU device was prepared by covering the sterilized HIFU probe with a sterile envelope containing an ultrasound coupling liquid. (a) This panel shows the HIFU transducer covered with a sterile envelope. (b) This panel shows the tube for the cooling circuit. (c) This panel shows the connector for the electrical power. (d) This panel shows the connector for the ultrasound imaging probe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57964/57964fig2.jpg" /> 
Figure 2: Use of the HIFU probe. The HIFU probe was held by hand and brought into contact with the liver surface. (a) This panel shows the HIFU probe. (b) The position of the HIFU probe at the time of ablation was marked on the liver. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57964/57964fig3.jpg" /> 
Figure 3: Targeting process. (a) The user interface displayed the position of the HIFU focal region superimposed on the sonogram, making it possible to place the location of the ablation in the tissues. (b) Immediately after the HIFU exposure, a cloud of ebullition appeared momentarily (for approximately 1 min) at the location of the lesion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57964/57964fig4.jpg" /> 
Figure 4: Monitoring. After boiling, the single lesion appeared on the sonogram as a hyperechoic zone at its center and a hypoechoic zone at its boundaries. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57964/57964fig5.jpg" /> 
Figure 5: Macroscopic visualization of two HIFU ablations. HIFU ablations were clearly visible by gross pathology. (a) This panel shows a typical HIFU ablation. (b) This panel shows an example of a HIFU lesion created during step 1 of phase IIa. The aim was to target the metallic marker. The shape and homogeneity of the ablation were not altered by the presence of hepatic veins. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57964/57964fig6.jpg" /> 
Figure 6: Macroscopic visualization of a HIFU ablation created in phase IIa. In one case, the metallic marker had not been included in the HIFU. After reviewing the record of the procedure, it was established that a Glissonian pedicle was confused with the metallic marker. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57964/57964fig7.jpg" /> 
Figure 7: Visualization of metastases using the integrated ultrasound imaging probe. Panels (a) and (c) show examples of two metastases imaged using the ultrasound imaging probe integrated with the HIFU device. Panels (b) and (d) show the corresponding metastases observed by gross pathology after surgery. (a) The user interface displayed the position of the HIFU focal region superimposed on the sonogram, making it possible to place the location of the ablation in the tissues and to visualize if the metastasis will be treated with safety margins. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57964/57964fig8.jpg" /> 
Figure 8: Histopathological examination after HIFU ablation. This panel shows a representative liver examination using H&#38;E staining. There was a sharp demarcation between treated and untreated liver with pronounced alterations in hepatocytes. Here, the magnification is 200x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57964/57964fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases at JoVE.com

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

evaluation-feasibility-safety-accuracy-an-intraoperative-high

Nanyang Technological University

Research

JoVE Journal

Cancer Research

7.7K Views.  Centre Léon Bérard. Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Video: Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of Interventional Radiology. This model employs an anaplastic squamous cell carcinoma that is easily and reliably propagated in the skeletal muscle of donor rabbits for eventual harvest and allograft implantation into the liver of na&#239;ve recipients. This tumor graft rapidly grows within the liver of recipient rabbits into an angiographically identifiable tumor characterized by a necrotic core surrounded by a viable hypervascular capsule. The physical size of the rabbit anatomy is sufficient to facilitate vascular instrumentation allowing for the application and testing of various interventional techniques. Despite these benefits, there exists a paucity of technical resources to act as a concrete reference for researchers working with the model. Herein, we present a comprehensive visual outline for the technical aspects of development, growth, propagation, and angiographic utilization of the rabbit VX2 tumor model for use by novice and experienced researchers alike.

The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer.

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy exceeding its constituents. This protocol describes the development of an ML algorithm to predict the growth of breast cancer bone macrometastases in a rat model before any abnormalities are observable with standard imaging methods. Such an algorithm can facilitate the detection of early metastatic disease (i.e., micrometastasis) that is regularly missed during staging examinations.
The applied metastasis model is site-specific, meaning that the rats develop metastases exclusively in their right hind leg. The model&#8217;s tumor-take rate is 60%&#8211;80%, with macrometastases becoming visible in magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a subset of animals 30 days after induction, whereas a second subset of animals exhibit no tumor growth.
Starting from image examinations acquired at an earlier time point, this protocol describes the extraction of features that indicate tissue vascularization detected by MRI, glucose metabolism by PET/CT, and the subsequent determination of the most relevant features for the prediction of macrometastatic disease. These features are then fed into a model-averaged neural network (avNNet) to classify the animals into one of two groups: one that will develop metastases and the other that will not develop any tumors. The protocol also describes the calculation of standard diagnostic parameters, such as overall accuracy, sensitivity, specificity, negative/positive predictive values, likelihood ratios, and the development of a receiver operating characteristic. An advantage of the proposed protocol is its flexibility, as it can be easily adapted to train a plethora of different ML algorithms with adjustable combinations of an unlimited number of features. Moreover, it can be used to analyze different problems in oncology, infection, and inflammation.

This protocol was designed to train a machine learning algorithm to use a combination of imaging parameters derived from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a rat model of breast cancer bone metastases to detect early metastatic disease and predict subsequent progression to macrometastases.

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...