Lennart Wiman (photographer) is acknowledged for excellent video recording.

Patients provided&#160;written&#160;informed consent and the study was approved by the Regional Ethical Review Board at the University of Gothenburg.
1. Anesthesia
<ol>
	<li>Set up all the monitoring (ECG, blood pressure cuff) 17.</li>
	<li>Insert an epidural catheter in the lower to mid thoracic level 17.</li>
	<li>Induce anesthesia with Propofol 2 mg/kg, 150 &#181;g of Fentanyl, and Rocuronium 0.6 mg/kg through a previously inserted peripheral venous catheter.</li>
	<li>Preoxygenate and intubate the patient. Maintain anesthesia with Sevoflurane, oxygen and air 17.</li>
	<li>Insert a double lumen central venous catheter and an arterial catheter. Monitor CVP and arterial pressure 17.</li>
</ol>
2. Isolation of the Liver and Cannulation of Vessels
<ol>
	<li>Administer 100 IE/kg of heparin and give antibiotic prophylaxis (e.g., 160 mg of Trimethoprim and 800 mg of Sulfamethoxazole).</li>
	<li>Using the percutaneous Seldinger technique, place a 17 Fr catheter in the femoral vein and a 15 Fr catheter in the external jugular vein 18. Verify the positions of the catheters by fluoroscopy.</li>
	<li>Perform a laparotomy by an L-shaped incision from the xiphoid process down the midline and then extended to a right subcostal incision 19.</li>
	<li>Verify that no extra-hepatic metastases exist in the abdomen by a complete inspection and palpation.
	<ol>
		<li>If any suspicious lesions are found, verify malignancy by frozen section.</li>
		<li>Terminate the procedure if there is evidence of extrahepatic manifestation (periaortal adenopathy, abdominal metastases). Lymphadenopathy in the porta hepatis is not considered a contraindication.&#160;</li>
	</ol>
	</li>
	<li>Mobilize the liver by dividing the falciform ligament, the left and right triangular ligament and the coronary ligament 19.</li>
	<li>Dissect the caval vein beginning from the renal veins up to the diaphragm with ligation of retroperitoneal tributary veins.</li>
	<li>Identify the right adrenal vein and place a vessel loop around it.</li>
	<li>Identify the right gonadal vein. Dissect it for 5-10 cm. Place a purse-string suture around the entrance to the caval vein.
	<ol>
		<li>Place a ligature distally on the gonadal vein. Using scissors, open the gonadal vein 3-5 cm from the caval vein.</li>
		<li>Insert an 18 Fr wire-reinforced catheter through the right gonadal vein and place the tip retrohepatically above the renal veins.</li>
		<li>Place a ligature around the gonadal vein to secure the position of the catheter.</li>
		<li>Verify the return of blood through the catheter. Heparinize the catheter 18.&#160;</li>
	</ol>
	</li>
	<li>Dissect the hepatoduodenal ligament and visualize the common hepatic artery, the gastroduodenal artery and the proper hepatic artery.
	<ol>
		<li>Place vessel loops around the common hepatic artery and the gastrodoudenal artery.</li>
		<li>Dissect the portal vein and place a vessel loop around it.</li>
		<li>Isolate the rest of the ligament containing the common bile duct and include all of this in a vessel loop.&#160;</li>
	</ol>
	</li>
	<li>Lift the gastroduodenal artery with the vessel loop. After interrupting the arterial inflow, make an incision using scissors.
	<ol>
		<li>Insert an 8-12 Fr catheter with its tip into the proper hepatic artery. Secure the position with a ligature around the gastroduodenal artery.</li>
		<li>Verify blood return in the catheter and then heparinize it 18.</li>
	</ol>
	</li>
	<li>Prime the perfusion system with one unit of packed erythrocytes, 100 ml of albumin (50 g/L), 100 ml of Tribonat (sodium bicarbonate), 2500 units of heparin (5,000 IE/ml) and 250 ml of crystalloids.</li>
	<li>Connect the arterial and the caval vein catheters to the perfusion system consisting of a heart lung machine equipped with a roller pump and a heater together with a venous reservoir, oxygenator and tubing systems of disposable type.</li>
	<li>Connect the femoral and external jugular catheters to the venovenous bypass pump consisting of a centrifugal pump, along with a heater unit and start bypass.</li>
	<li>Complete the isolation of the liver by tightening a Rumel tourniquet around the caval vein infrahepatically above the renal veins and clamping the caval vein suprahepatically using a Klintmalm vascular clamp.
	<ol>
		<li>Tighten a Rumel tourniquet around the common bile duct, the portal vein and the right adrenal vein and finally clamp the hepatic artery using a vascular clamp.</li>
	</ol>
	</li>
</ol>
3. The Perfusion
<ol>
	<li>Start the perfusion and manually monitor the blood level in the venous reservoir. Adjust the flow rate until a steady-state level is reached, usually a flow rate between 500-1,200 ml/min.</li>
	<li>Place thermistor probes into the right and left liver lobes and one directly into the arterial catheter.</li>
	<li>Heat the perfusate aiming at a target liver temperature of 40 oC. Keep this temperature throughout the perfusion.</li>
	<li>Place a scintillation probe over the venovenous bypass pump head.</li>
	<li>Calibrate the leakage monitoring system, by injecting a small dose of 10 MBq 99mTc-albumin (1 ml) into the systemic circulation and register the baseline activity.
	<ol>
		<li>Inject a ten-fold higher dose, 100 MBq 99mTc-albumin (1 ml), into the hepatic perfusion unit.</li>
		<li>Record any leakage from the perfusion system to the systemic circulation as an increase in activity measured by the scintillation probe.</li>
		<li>Record any leakage from the systemic circulation to the perfusion system as an increase in volume in the venous reservoir.&#160;</li>
	</ol>
	</li>
	<li>When steady-state of the flow rate is established, no leakage is present and the liver temperature has reached 40 &#176;C, inject melphalan at a dose of 1 mg/kg bodyweight into the perfusion system divided into two doses given 30 min apart.</li>
</ol>
4. End the Perfusion and Remove Catheters
<ol>
	<li>After 60 min of perfusion, empty the perfusate from the system and irrigate the liver with 1,000 ml of crystalloid.</li>
	<li>Add one unit of packed erythrocytes to the perfusion unit, infuse into the liver and then stop the perfusion.</li>
	<li>Release the Klintmalm clamp and the Rumel tourniquet from the caval vein.</li>
	<li>Release the Rumel tourniquet from the hepatoduodenal ligament.</li>
	<li>Remove the ligature from the gastroduodenal artery. Remove the catheter and ligate the vessel.</li>
	<li>Release the clamp from the hepatic artery.</li>
	<li>Remove the catheter from the gonadal vein and ligate the vessel.</li>
	<li>Stop the veno-venous bypass pump and disconnect the tubes.</li>
	<li>Close the abdomen with an uninterrupted fascial suture and then an intracutaneous skin suture.</li>
	<li>Remove the catheters in the femoral and external jugular veins and apply local pressure for 5 min.</li>
</ol>
5. Postoperative Care
<ol>
	<li>Extubate the patient and transfer to postoperative care unit for 24 hr. Keep monitoring central venous pressure and arterial blood pressure.</li>
	<li>Transfer the patient to a surgical department for a typical of 3-7 days of post-operative care.</li>
	<li>Continue with low-molecular weight heparin (LMWH) at a dose of 5,000 IE/day for a total of 4 weeks.</li>
</ol>

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<li>Schmittel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+II+trial+of+cisplatin%2C+gemcitabine+and+treosulfan+in+patients+with+metastatic+uveal+melanoma%5BTitle%5D)+AND+%22Melanoma+Research%22%5BJournal%5D)">Phase II trial of cisplatin, gemcitabine and treosulfan in patients with metastatic uveal melanoma.</a> Melanoma Research. 15 (3), 205-207 (2005).</li>
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<li>Huppert, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transarterial+chemoembolization+of+liver+metastases+in+patients+with+uveal+melanoma%5BTitle%5D)+AND+%22European+Journal+Of+Radiology%22%5BJournal%5D)">Transarterial chemoembolization of liver metastases in patients with uveal melanoma.</a> European Journal Of Radiology. 74 (3), 38-44 (2010).</li>
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<li>Dahl, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+hyperthermia+and+chemotherapy%5BTitle%5D)+AND+%22Recent+results+in+cancer+research.+Fortschritte+der+Krebsforschung.+Progres+Dans+Les+Recherches+Sur+Le+Cancer%22%5BJournal%5D)">Interaction of hyperthermia and chemotherapy.</a> Recent results in cancer research. Fortschritte der Krebsforschung. Progres Dans Les Recherches Sur Le Cancer. 107, 157-169 (1988).</li>
<li>Barker, W. C., Andrich, M. P., Alexander, H. R., Fraker, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+intraoperative+external+monitoring+of+perfusate+leak+using+iodine-131+human+serum+albumin+during+isolated+perfusion+of+the+liver+and+limbs%5BTitle%5D)+AND+%22European+Journal+Of+Nuclear+Medicine%22%5BJournal%5D)">Continuous intraoperative external monitoring of perfusate leak using iodine-131 human serum albumin during isolated perfusion of the liver and limbs.</a> European Journal Of Nuclear Medicine. 22 (11), 1242-1248 (1995).</li>
<li>Olofsson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+hepatic+perfusion+for+ocular+melanoma+metastasis%3A+registry+data+suggests+a+survival+benefit%5BTitle%5D)+AND+%22Annals+of+Surgical+Oncology%22%5BJournal%5D)">Isolated hepatic perfusion for ocular melanoma metastasis: registry data suggests a survival benefit.</a> Annals of Surgical Oncology. 21 (2), 466-472 (2014).</li>
<li>Redai, I., Emond, J., Brentjens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anesthetic+considerations+during+liver+surgery%5BTitle%5D)+AND+%22The+Surgical+Clinics+of+North+America%22%5BJournal%5D)">Anesthetic considerations during liver surgery.</a> The Surgical Clinics of North America. 84, 401-411 (2004).</li>
<li>Sakai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+and+management+of+percutaneous+veno-venous+bypass+cannula+for+liver+transplantation%3A+a+reference+for+transplant+anesthesiologists%5BTitle%5D)+AND+%22Clinical+Transplantation%22%5BJournal%5D)">Insertion and management of percutaneous veno-venous bypass cannula for liver transplantation: a reference for transplant anesthesiologists.</a> Clinical Transplantation. 24 (5), 585-591 (2010).</li>
<li>Skandalakis, J. E., Skandalakis, L. J., Skandalakis, P. N., Mirilas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+surgical+anatomy%5BTitle%5D)+AND+%22The+Surgical+Clinics+of+North+America%22%5BJournal%5D)">Hepatic surgical anatomy.</a> The Surgical Clinics of North America. 84 (2), 413-435 (1016).</li>
<li>Alexander, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+phase+I-II+study+of+isolated+hepatic+perfusion+using+melphalan+with+or+without+tumor+necrosis+factor+for+patients+with+ocular+melanoma+metastatic+to+liver%5BTitle%5D)+AND+%22Clinical+Cancer+Research+%3A+An+Official+Journal+Of+The+American+Association+for+Cancer+Research%22%5BJournal%5D)">A phase I-II study of isolated hepatic perfusion using melphalan with or without tumor necrosis factor for patients with ocular melanoma metastatic to liver.</a> Clinical Cancer Research : An Official Journal Of The American Association for Cancer Research. 6 (8), 3062-3070 (2000).</li>
<li>Alexander, H. R., Jr,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperthermic+isolated+hepatic+perfusion+using+melphalan+for+patients+with+ocular+melanoma+metastatic+to+liver%5BTitle%5D)+AND+%22Clinical+Cancer+Research+%3A+An+Official+Journal+Of+The+American+Association+For+Cancer+Research%22%5BJournal%5D)">Hyperthermic isolated hepatic perfusion using melphalan for patients with ocular melanoma metastatic to liver.</a> Clinical Cancer Research : An Official Journal Of The American Association For Cancer Research. 9 (17), 6343-6349 (2003).</li>
<li>Rizell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+hepatic+perfusion+for+liver+metastases+of+malignant+melanoma%5BTitle%5D)+AND+%22Melanoma+research%22%5BJournal%5D)">Isolated hepatic perfusion for liver metastases of malignant melanoma.</a> Melanoma research. 18 (2), 120-126 (2008).</li>
<li>Iersel, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+hepatic+perfusion+with+200+mg+melphalan+for+advanced+noncolorectal+liver+metastases%5BTitle%5D)+AND+%22Annals+of+Surgical+Oncology%22%5BJournal%5D)">Isolated hepatic perfusion with 200 mg melphalan for advanced noncolorectal liver metastases.</a> Annals of Surgical Oncology. 15 (7), 1891-1898 (2008).</li>
<li>Carvajal, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+selumetinib+vs+chemotherapy+on+progression-free+survival+in+uveal+melanoma%3A+a+randomized+clinical+trial%5BTitle%5D)+AND+%22JAMA+%3A+the+Journal+Of+The+American+Medical+Association%22%5BJournal%5D)">Effect of selumetinib vs chemotherapy on progression-free survival in uveal melanoma: a randomized clinical trial.</a> JAMA : the Journal Of The American Medical Association. 311 (23), 2397-2405 (2014).</li>
<li>Sharma, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+arterial+chemoembolization+for+management+of+metastatic+melanoma%5BTitle%5D)+AND+%22AJR.+American+Journal+Of+Roentgenology%22%5BJournal%5D)">Hepatic arterial chemoembolization for management of metastatic melanoma.</a> AJR. American Journal Of Roentgenology. 190 (1), 99-104 (2008).</li>
<li>Eldredge-Hindy, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yttrium-90+Microsphere+Brachytherapy+for+Liver+Metastases+From+Uveal+Melanoma%3A+Clinical+Outcomes+and+the+Predictive+Value+of+Fluorodeoxyglucose+Positron+Emission+Tomography%5BTitle%5D)+AND+%22American+Journal+Of+Clinical+Oncology%22%5BJournal%5D)">Yttrium-90 Microsphere Brachytherapy for Liver Metastases From Uveal Melanoma: Clinical Outcomes and the Predictive Value of Fluorodeoxyglucose Positron Emission Tomography.</a> American Journal Of Clinical Oncology. , (2014).</li>
<li>Etten, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+hypoxic+hepatic+perfusion+with+orthograde+or+retrograde+flow+in+patients+with+irresectable+liver+metastases+using+percutaneous+balloon+catheter+techniques%3A+a+phase+I+and+II+study%5BTitle%5D)+AND+%22Annals+of+Surgical+Oncology%22%5BJournal%5D)">Isolated hypoxic hepatic perfusion with orthograde or retrograde flow in patients with irresectable liver metastases using percutaneous balloon catheter techniques: a phase I and II study.</a> Annals of Surgical Oncology. 11 (6), 598-605 (2004).</li>
<li>Ku, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracorporeal+adriamycin-removal+following+hepatic+artery+infusion%3A+use+of+direct+hemoperfusion+combined+with+veno-venous+bypass%5BTitle%5D)+AND+%22Nihon+Geka+Gakkai+Zasshi%22%5BJournal%5D)">Extracorporeal adriamycin-removal following hepatic artery infusion: use of direct hemoperfusion combined with veno-venous bypass.</a> Nihon Geka Gakkai Zasshi. 90 (10), 1758-1764 (1989).</li>
<li>Curley, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+arterial+infusion+chemotherapy+with+complete+hepatic+venous+isolation+and+extracorporeal+chemofiltration%3A+a+feasibility+study+of+a+novel+system%5BTitle%5D)+AND+%22Anti-Cancer+Drugs%22%5BJournal%5D)">Hepatic arterial infusion chemotherapy with complete hepatic venous isolation and extracorporeal chemofiltration: a feasibility study of a novel system.</a> Anti-Cancer Drugs. 2 (2), 175-183 (1991).</li>
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</ol>

The authors have nothing to disclose.

Patients with metastatic uveal melanoma have few therapeutic options. Chemotherapy 6, immunotherapy 23 and targeted therapy 24 have not been able to show improved OS. For patients with few liver metastases, liver resection is an option 12. Other treatments include hepatic intra-arterial chemotherapy (HIA) 9, transarterial chemoembolization (TACE) 25 and selective internal radiation therapy (SIRT) 26.
IHP is a major and complex surgical intervention and since the first report by Ausman there have been many developments in the surgical technique. In 2004, van Etten and colleagues reported the first clinical results using the isolated hypoxic hepatic perfusion (IHHP) technique 27. In the early 1990s, three independent groups developed a novel percutaneous hepatic perfusion (PHP) system using extracorporeal chemofiltration 28-30. No studies comparing these techniques to IHP have been performed. The reports so far do not seem to be in favor of any specific technique.
Patient selection is of high importance for this treatment. Since it is a local treatment, there should be no evidence of extrahepatic disease. Due to the risk of developing hepatic failure, no more than 50 % of the liver volume should be replaced by tumour 22. Patients should have a good performance status (ECOG 0-1) and no heart-, lung-, kidney- or liver disease that could increase the risk of severe morbidity after surgery.
In a recent phase II trial from our group, a median survival of 26 months was shown, implying a potential survival benefit of 14 months compared with a control group 16. To verify this finding, a multicenter randomized phase III trial between IHP and Best Alternative Care (BAC) has recently started in Scandinavia (the SCANDIUM trial, ClinicalTrials number: NCT01785316). The BAC treatment may include all other treatment options, either existing or experimental, with the notion that cross-over is not allowed from the BAC to IHP group. Inclusion criteria include biopsy verified liver metastases and no evidence of extra-hepatic tumor manifestations by PET-CT. The primary endpoint is overall survival at 24 months with secondary endpoints including response rate, progression-free survival and quality of life. The planned inclusion is 78 patients during 5 years.
In summary, there still exists no treatment that in phase III trials has shown any prolonged overall survival for patients with uveal melanoma liver metastases. IHP is an interesting and promising regional therapy where potential benefits in survival are currently being investigated in a randomized trial (the SCANDIUM trial).

Uveal melanoma is the most common primary intraocular malignancy in adults, with the incidence being highest in Caucasian populations 1. Local treatment consists of enucleation or local plaque radiation therapy 2,3. Irrespective of local treatment, metastatic disease will ultimately develop in about 35-50% of the patients 4,5. The liver is the most common site of metastases and approximately 50% of the patients present with isolated liver metastases. The median overall survival (OS) for patients with liver metastases is 6-12 months 4. Several different systemic and regional treatment strategies have been explored, but survival rates have not improved 2.
Treatment with systemic chemotherapy, both single substance (Dacarbazine or Temozolomide6) or combination treatments (BOLD regimen7, gemcitabine with treosulfan 8), shows response rates of less then 10 %. Hepatic intra-arterial chemotherapy (HIA) has shown a slightly higher overall response rate compared to systemic chemotherapy, but no increase in overall survival 9. Transarterial chemoembolization (TACE) with cisplatin and carboplatin showed a partial response in 57% of patients with a median survival of 9 months10. Selective internal radiation therapy (SIRT) has shown a partial response in 62% of patients with a median survival of 7 months 11. In a study on liver resection, including 255 patients, an overall survival of 14 months was reported; in 76 of the patients where a microscopically complete resection was possible, the survival was 27 months 12.
Isolated hepatic perfusion (IHP) is a regional treatment option, originally developed by Ryan and Ausman. The rationale behind the technique is to surgically isolate and perfuse the liver with a high dose of a chemotherapeutic agent while avoiding systemic toxicity 13. In addition, concurrent hyperthermia mediates an increased uptake of the chemotherapeutic agent by changes in tumor blood flow and cellular membrane permeability 14. Complete vascular isolation is confirmed with a radioactive tracer technique using Tc-99 labeled human serum albumin 15. A retrospective phase II study has shown a potential survival benefit of 14 months (26 vs. 12 months) for patients that underwent IHP compared to the longest survivors with uveal melanoma liver metastases in Sweden 16.
Here we present the technique of IHP with a short review of current results and future perspectives.

<table><tbody><tr><td>Wire re-inforced catethers</td><td>Medtronic Inc, Minnesota, USA</td><td></td><td>12-18Fr</td></tr><tr><td>St&amp;ouml;ckert S5</td><td>Sorin Group, Mirandola, Italy</td><td></td><td>Heart-lung machine</td></tr><tr><td>Rotaflow</td><td>Maquet, Jostra Medizintechnik AG, Hirrlingen, Germany</td><td></td><td>Centrifugal pump</td></tr><tr><td>HU 35</td><td>Maquet cardiopulmonary AG, Hirrlingen, Germany</td><td></td><td>Heater unit</td></tr><tr><td>Flexbumin 200 g/l</td><td>Baxter Medical AB, Kista, Sweden</td><td></td><td></td></tr><tr><td>Tribonat</td><td>Fresenius Kabi AB, Uppsala, Sweden</td><td></td><td></td></tr><tr><td>Heparin LEO, 5000 IE/ml</td><td>LEO Pharma AB, Sweden</td><td></td><td></td></tr><tr><td>Ringer Acetate</td><td>Baxter Medical AB, Kista, Sweden</td><td></td><td></td></tr><tr><td>Vasculosis</td><td>Cis-Bio International, Gif-Sur-Yvette, France</td><td></td><td>99mTc-albumin</td></tr><tr><td>MedicView</td><td>SystemData AB, Gothenburg, Sweden</td><td></td><td>Leakage monitoring system</td></tr><tr><td>Alkeran</td><td>Aspen Europe GmbH, Bad Oldesloe, Germany</td><td></td><td>melphalan</td></tr></tbody></table>

isolated hepatic perfusion as a treatment for liver metastases of uveal melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

An initial report included 22 patients treated with melphalan with or without TNF-alpha. The results showed an overall response rate of 62%, including two patients with CR (10%), and a median overall survival of 11 months 20. A subsequent study reported on the outcome of 29 consecutive patients treated using melphalan alone. The outcome was very similar with a 62% overall response including 10% complete response and a median survival of 12 months 21.
In a study by Rizell et al., 20 patients with uveal melanoma were included. The patients were divided into three different time eras, and the results showed a decrease in mortality from 27% to 0% due to improved patient selection and technical development 22.
The knowledge about IHP is primarily based on smaller retrospective single institution series, and the published results are listed in Table 1. In summary, the overall response rate is 33-100%, including a 0-20% complete response rate. The post-operative mortality is 6% and the median survival is 12 months.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Author</td>
			<td>n</td>
			<td>Type</td>
			<td>ORR</td>
			<td>CR</td>
			<td>Median survival</td>
			<td>Mortality</td>
		</tr>
		<tr>
			<td>Alexander et al. 2000</td>
			<td>22</td>
			<td>IHP</td>
			<td>62%</td>
			<td>10%</td>
			<td>11 months</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Alexander et al. 2003</td>
			<td>29</td>
			<td>IHP</td>
			<td>62%</td>
			<td>10%</td>
			<td>12 months</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>Noter et al. 2003</td>
			<td>8</td>
			<td>IHP</td>
			<td>50%</td>
			<td>0%</td>
			<td>10 months</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>Pingpank et al. 2005</td>
			<td>10</td>
			<td>PHP</td>
			<td>50%</td>
			<td>20%</td>
			<td>N/A</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>Rizell et al. 2008</td>
			<td>27</td>
			<td>IHP</td>
			<td>70%</td>
			<td>7%</td>
			<td>13 months</td>
			<td>22%</td>
		</tr>
		<tr>
			<td>Verhoef et al. 2008</td>
			<td>4</td>
			<td>IHHP</td>
			<td>100%</td>
			<td>0%</td>
			<td>9 months</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>van Iersel et al. 2008</td>
			<td>12</td>
			<td>IHP</td>
			<td>33%</td>
			<td>0%</td>
			<td>10 months</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>van Etten et al. 2009</td>
			<td>8</td>
			<td>IHHP</td>
			<td>37%</td>
			<td>0%</td>
			<td>11 months</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>Weighted mean</td>
			<td>120</td>
			<td></td>
			<td>59%</td>
			<td>7%</td>
			<td>12 months</td>
			<td>6%</td>
		</tr>
		<tr>
			<td>Olofsson et al.</td>
			<td>34</td>
			<td>IHP</td>
			<td>68%</td>
			<td>12%</td>
			<td>24 months&#160;</td>
			<td>0%</td>
		</tr>
	</tbody>
</table>
Table 1: Results of isolated hepatic perfusion. Publications with clinical outcomes reported for patients with isolated liver metastases treated with different modalities of isolated hepatic perfusion. ORR = overall response rate; CR = complete response; IHP = isolated hepatic perfusion; IHHP = isolated hypoxic hepatic perfusion; PHP = percutaneous hepatic perfusion; N/A = Not available.
<img alt="Figure 1" src="/files/ftp_upload/52490/52490fig1.jpg" /> 
Figure 1: Schematic picture of the veno-venous bypass system and the perfusion system. The red line represents the catheter in the hepatic artery and the blue one represents the catheter in the caval vein. They are connected to a perfusion system consisting of an oxygenator, a roller pump and a heating unit. The black lines represent the veno-venous bypass system connected to a roller pump.

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Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

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13.4K Views.  Institute of Clinical Sciences. Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Video: Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Cancer Research

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

Engineering

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Development and Validation of a Porcine Ex Situ Isolated Liver Perfusion Model 

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

Minimally Invasive Isolated Limb Perfusion (MI-ILP) for Locally Advanced Melanomas and Sarcomas of the Extremity

Minimally invasive isolated limb perfusion (MI-ILP) is a treatment option for patients with locally advanced melanomas and sarcomas of the extremities. Briefly, the procedure starts with percutaneous access of the femoral or brachial vessels in the diseased extremity. It is then isolated from the rest of the body with a tourniquet. The catheters are connected to a heart-lung machine, and the extremity is perfused with a high dose of melphalan. In the literature, the reported overall and complete response rates for ILP are approximately 80% and 60%, respectively. Our previously reported results using MI-ILP, showed similar response rates. The objective of this manuscript is to provide a step-by-step guide on how to perform an MI-ILP. The purpose of this protocol is to enable local perfusion of the extremities with a high dose of chemotherapy without systemic leakage in a minimally invasive manner.

This protocol presents a step-by-step guide for performing minimally invasive isolated limb perfusion (MI-ILP), a treatment option for patients with locally advanced melanomas and sarcomas of the extremities.

Minimally invasive isolated limb perfusion (MI-ILP) is a treatment option for patients with locally advanced melanomas and sarcomas of the extremities. ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

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