p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-alpha and melphalan.

BACKGROUND: Recombinant tumor necrosis factor-alpha (TNF-alpha) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-alpha and loss of p53 function contributes to the resistance of tumour cells to TNF-alpha. The relationship between p53 status and response to TNF-alpha and melphalan in patients undergoing ILP is unknown. PATIENTS AND METHODS: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. RESULTS: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-alpha and melphalan showed significantly higher levels of p53-mutated protein. CONCLUSIONS: Our results might be a clue to a role of p53 protein status in TNF-alpha and melphalan response in clinical use.     

Melphalan tissue concentrations in patients treated with regional isolated perfusion for melanoma of the lower limb.

In 14 consecutive patients with recurrent melanoma of the lower limb a total of 35 biopsies were taken at the end of perfusion treatment to assess melphalan tissue concentrations in tumour, skin/subcutis and muscle tissue. In tumour tissue (n = 12) the mean melphalan concentration was 6.8 micrograms g-1, which was significantly higher than that of healthy skin/subcutis (3.2 micrograms g-1; n = 10), but equal to that of muscle tissue (6.5 micrograms g-1; n = 13). The correlation between melphalan concentration in the tissues and the concentration in the perfusate was studied. The latter was assessed in the form of melphalan peak concentration and the area under the curve (AUC0-->60) of the melphalan concentration-time curve. Tumour concentration proved to be correlated linearly with AUC0-->60 (R = 0.6, P = 0.002) and muscle concentration with melphalan peak concentration (R = 0.8, P = 0.04). There was no relation between skin/subcutis concentrations and the perfusate parameters. Further research is warranted to study the relationship between melphalan tissue concentration, tumour response and regional toxicity.     

Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion

Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-alpha) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-alpha was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-alpha in ILP.     

Hyperthermic isolated hepatic perfusion using melphalan for patients with ocular melanoma metastatic to liver.

PURPOSE: Liver metastases are the sole or life-limiting component of disease in the majority of patients with ocular melanoma who recur. Because median survival after diagnosis of liver metastases is short and no satisfactory treatment options exist, we have conducted clinical trials evaluating isolated hepatic perfusion (IHP) for patients afflicted with this condition. EXPERIMENTAL DESIGN: Twenty-nine patients (male: 14, female: 15; mean age, 49 years) with unresectable liver metastases from ocular melanoma were treated with a 60-min hyperthermic IHP using 1.5 mg/kg of melphalan (mean total dose 105 mg). Via laparotomy, perfusion inflow was established with a cannula in the gastroduodenal artery and outflow via a cannula positioned in an isolated segment of the retrohepatic inferior vena cava. Portal and infra-renal inferior vena cava blood flow was shunted externally to the axillary vein using a veno-veno bypass circuit. Patients were assessed for toxicity, radiographic response, and survival. RESULTS: There was no treatment related mortality and transient grade 3/4 hepatic toxicity was observed in 19 patients (65%). Mean length of operation and hospital stay was 8.3 h and 10 days, respectively. There were 3 (10%) complete responses (duration: 12, 14+, 15 months) and 15 partial responses (52%; mean duration: 10 months). The initial site of disease progression included liver in 17 of 25 patients (68%) who recurred. At a median follow-up of 30.7 months the median actuarial progression-free and overall survivals were 8 and 12.1 months, respectively. CONCLUSIONS: IHP with melphalan alone results in significant regression of established liver metastases for patients with ocular melanoma. However, after IHP, disease progression is most commonly observed in the liver, and survival after disease progression is short. On the basis of a pattern of tumor progression predominantly in liver, continued clinical evaluation of hepatic directed therapy in this patient population is justified.     

Regional toxicity after isolated limb perfusion with melphalan and tumour necrosis factor- alpha versus toxicity after melphalan alone.

AIMS: To determine whether the addition of high-dose tumour necrosis factor-alpha (TNF alpha) to isolated limb perfusion (ILP) with melphalan increases acute regional tissue toxicity compared to ILP with melphalan alone. METHODS: A retrospective, multivariate analysis of toxicity after normothermic (37--38 degrees C) and 'mild' hyperthermic (38--40 degrees C) ILPs for melanoma was undertaken. Normothermic ILP with melphalan was performed in 294 patients (70.8%), 'mild' hyperthermic ILP with melphalan in 71 patients (17.1%) and 'mild' hyperthermic ILP with melphalan combined with TNF alpha in 50 patients (12.0%). Toxicity was nil or mild (grades I--II according to Wieberdink et al.) in 339 patients (81.7%), and more severe acute regional toxicity (grades III--V) developed in 76 patients (18.3%). A stepwise logistic regression procedure was performed for the multivariate analysis of prognostic factors for more severe toxicity. RESULTS: On univariate analysis, 'mild' hyperthermic ILP with melphalan plus TNF alpha significantly increased the incidence of more severe acute regional toxicity compared to normothermic and 'mild' hyperthermic ILP with melphalan alone (36% vs 16% and 17%; P=0.0038). However, after ILP using TNF alpha no grade IV (compartment compression syndrome) or grade V (toxicity necessitating amputation) reactions were seen. Significantly more severe toxicity was seen after ILPs performed between 1991 and 1994 compared with earlier ILPs (33%vs 14%P=0.0001). Also, women had a higher risk of more severe toxicity than men (22% vs 7%; P=0.0007). After multivariate analysis, prognostic factors which remained significant were: sex (P=0.0013) and either ILP schedule (P=0.013) or treatment period (P=0.0003). CONCLUSIONS: Regional toxicity after 'mild' hyperthermic ILP with melphalan and TNF alpha was significantly increased compared to ILP with melphalan alone. This may be caused by increased thermal enhancement of melphalan due to the higher tissue temperatures (39--40 degrees C) at which the melphalan in the TNF alpha-ILPs was administered or by an interaction between high-dose TNF alpha and melphalan. Copyright Harcourt Publishers Limited.     

Isolated hepatic perfusion in the pig with TNF-alpha with and without melphalan.

Isolated limb perfusion with tumour necrosis factor alpha (TNF-alpha) and melphalan is well tolerated and highly effective in irresectable sarcoma and melanoma. No data are available on isolated hepatic perfusion (IHP) with these drugs for irresectable hepatic malignancies. This study was undertaken to assess the feasibility of such an approach by analysing hepatic and systemic toxicity of IHP with TNF-alpha with and without melphalan in pigs. Ten healthy pigs underwent IHP. After vascular isolation of the liver, inflow catheters were placed in the hepatic artery and portal vein, and an outflow catheter was placed in the inferior vena cava (IVC). An extracorporeal veno-venous bypass was used to shunt blood from the lower body and intestines to the heart. The liver was perfused for 60 min with (1) 50 microg kg(-1) TNF-alpha (n = 5), (2) 50 microg kg(-1) TNF-alpha plus 1 mg kg(-1) melphalan (n = 3) or (3) no drugs (n = 2). The liver was washed with macrodex before restoring vascular continuity. All but one pigs tolerated the procedure well. Stable perfusion was achieved in all animals with median perfusate TNF-alpha levels of 5.1 +/- 0.78 x 10(6) pg ml(-1) (+/- s.e.m). Systemic leakage of TNF-alpha from the perfusate was consistently < 0.02%. Following IHP, a transient elevation of systemic TNF-alpha levels was observed in groups 1 and 2 with a median peak level of 23 +/- 3 x 10(3) pg ml(-1) at 10 min after washout, which normalized within 6 h. No significant systemic toxicity was observed. Mild transient hepatotoxicity was seen to a similar extent in all animals, including controls. IHP with TNF-alpha with(out) melphalan in pigs is technically feasible, results in minimal systemic drug exposure and causes minor transient disturbances of liver biochemistry and histology.     

High mitomycin C concentration in tumour tissue can be achieved by isolated liver perfusion in rats

Summary To enable the treatment of hepatic metastasis with higher, theoretically more effective, doses of systemically toxic anticancer drugs, an isolated liver perfusion (ILP) technique was developed in WAG/Ola rats. First, in a toxicity study the maximally tolerated dose (MTD) of mitomycin C (MMC) was determined for a 25-min ILP and for hepatic artery infusion (HAI) after the administration of a bolus dose. The MTD in the ILP setting (4.8 mg/kg) was 4 times that using HAI (1.2 mg/kg). Subsequently, in a rat colorectal hepatic-metastasis model, concentrations of MMC in tumour, liver, plasma and perfusate were measured during a 25-min ILP to investigate the expected pharmacokinetic advantage of ILP. The mean plasma level determined after ILP (1.2 as well as 4.8 mg/kg MMC) was significantly lower (P<0.001) than that obtained following HAI. This may explain both the absence of severe systemic toxicity and the higher MTD in ILP-treated groups. No significant difference in mean tumour and liver tissue concentrations of MMC were found when the groups treated with 1.2 mg/kg drug via HAI vs ILP were compared. The mean MMC concentration in tumour tissue was significantly higher (almost 5 times;P<0.05) in rats treated by ILP with the MTD (4.8 mg/kg) than in those treated via HAI with the MTD (1.2 mg/kg). ILP of MMC can be safely performed using a dose 4 times higher than the MTD in the HAI setting, leading to an almost 5-fold concentration of MMC in hepatic metastasis. ILP of MMC may therefore represent a promising therapy for metastasis confined to the liver.Abbreviations HAI hepatic artery infusion - HPLC high-performance liquid chromatography - ILP isolated liver perfusion - MMC mitomycin C - MTD maximally tolerated dose Supported by grant IKW 88-07 from the Dutch Cancer Foundation     

Prerequisites for effective isolated limb perfusion using tumour necrosis factor alpha and melphalan in rats.

An isolated limb perfusion (ILP) model using soft tissue sarcoma-bearing rats was used to study prerequisites for an effective ILP, such as oxygenation of the perfusate, temperature of the limb, duration of the perfusion and concentration of tumour necrosis factor (TNF). Combination of 50 microg TNF and 40 microg melphalan demonstrated synergistic activity leading to a partial and complete response rate of 71%. In comparison to oxygenated ILP, hypoxia was shown to enhance anti-tumour activity of melphalan alone and TNF alone but not of their combined use. Shorter perfusion times decreased anti-tumour responses. At a temperature of 24-26 degrees C, anti-tumour effects were lost, whereas temperatures of 38-39 degrees C or 42-43 degrees C resulted in higher response rates. However, at 42-43 degrees C, local toxicity impaired limb function dramatically. Synergy between TNF and melphalan was lost at a dose of TNF below 10 microg in 5 ml perfusate. We conclude that the combination of TNF and melphalan has strong synergistic anti-tumour effects in our model, just as in the clinical setting. Hypoxia enhanced activity of melphalan and TNF alone but not the efficacy of their combined use. For an optimal ILP, minimal perfusion time of 30 min and minimal temperature of 38 degrees C was mandatory. Moreover, the dose of TNF could be lowered to 10 microg per 5 ml perfusate, which might allow the use of TNF in less leakage-free or less inert perfusion settings.     

Limb salvage by neoadjuvant isolated perfusion with TNFalpha and melphalan for non-resectable soft tissue sarcoma of the extremities.

AIMS: Patients with non-resectable soft tissue sarcomas of the extremities do not live longer if they are treated by amputation or disarticulation. In order to avoid major amputations, we tested isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF)+melphalan+/-interferon-gamma (IFN) as a pre-operative, neoadjuvant limb salvage treatment. METHODS: Twenty-two patients were included (six men and 16 women; three upper limb and 19 lower limb tumours). The AJCC stage was IIA in four patients, III in seven and IV in 11. Thirteen cases were recurrent or progressive after previous therapy; five tumours had a diameter >/=20 cm, and four were multiple or regionally metastatic. There were six malignant fibrous histiocytomas, five liposarcomas, four malignant peripheral nerve sheath tumours, three rhabdomyosarcomas, two leiomyosarcomas, one recurrent extraskeletal osteosarcoma and one angiosarcoma. RESULTS: Twenty-four ILPs were performed in the 22 patients, and 18 (82%) experienced an objective response: this was complete in four (18%) and partial in 14 (64%). Three patients had a minimal or no response and the tumour progressed in one case. All patients had fever for 24 hours but only one developed a reversible grade 3 distributive shock syndrome with no sequelae. There was no grade 4 toxicity. Seventeen patients (77%) underwent limb-sparing resection of the tumour remnants after a median time of 3.4 months: 10 resections were intracompartmental and seven extracompartmental. Surgery included flaps or skin grafts in five patients, arterial replacement in two and knee arthrodesis in one. Adjuvant chemotherapy was given to eight patients and radiotherapy to six. In one patient amputation was necessary after a second ILP. Secondary amputations were performed for recurrence in two patients, resulting in an overall limb salvage rate of 19/22 (86%). After a median follow-up of 18.7 months, 10 recurrences were recorded: seven were both local and systemic and three were only local. The median disease free and overall survival times have been >12.5 and 18.7 months respectively: this is similar to the outcome after primary amputations for similar cases. CONCLUSION: ILP with TNF and chemotherapy is an efficient limb sparing neoadjuvant therapy for a priori non-resectable limb soft tissue sarcomas.     

Pharmacokinetics of melphalan in isolated limb perfusion

The pharmacokinetics of melphalan was studied by sampling of tissue and plasma in 72 rats that␣underwent isolated hyperthermic limb perfusion under different conditions. A miniaturized extracorporeal circulation system for small animals was used for␣perfusion of the rat hindlimb. Melphalan levels (l-phenylalanine mustard, L-PAM) were determined by high-performance liquid chromatography (HPLC). The temperature of the perfusate plasma and tissue, pH, administration method, and flow rate were modified and compared with regard to their influence on pharmacokinetic parameters. The highest tissue penetration of melphalan was observed under the following conditions: (a) pH range of the perfusate plasma between 7.3 and 7.7 (physiological environment), (b) temperature range of the perfusate from 40° to 41.5 °C (destruction of cellular carrier systems at higher temperatures and increased inactivation by hydrolysis of melphalan above 41.5 °C), (c) application of melphalan as a single dose into the reservoir of the extracorporeal circuit (optimal tissue penetration), and (d) reduced perfusate flow (prolonged contact time between perfusate and tissue). Received: 23 February 1998 / Accepted: 2 June 1998     

TNF-alpha levels in patients with malignant tumors after hyperthermic isolated regional perfusion

Hyperthermic isolated regional perfusion is an alternative method for the treatment of malignancies especially sited in the pelvic region and extremities. The perfusion is performed via the extracorporeal system with a pump flow and chemotherapeutic agents and some cytokines, like tumor necrosis factor, may be added. It's well known that in the ischemia-reperfusion injury, hemorrhagic necrosis and cell death occurs and these cytokines are produced endogenously. In this study the endogenous TNF-alpha levels before, during and after hyperthermic isolated regional perfusion were compared in 16 patients with malignant tumors. The TNF-alpha levels were determined from the blood samples taken systemically before and after perfusion, and from perfusate at the 15th, 30th, 45th minute. The 15th minute samples were the ones where vascular clamps were applied to the vessels before starting the perfusion. TNF-alpha levels between the 15th minute samples and the blood samples that were taken systemically before and after perfusion were found statistically significant. The cause of this was suggested to be the ischemia-reperfusion injury in this period. It was shown in this study that high endogenous TNF-alpha levels and good clinical results could be achieved with hyperthermic isolated regional perfusion in the treatment of malignant tumors.     

In vivo isolated kidney perfusion with tumour necrosis factor alpha (TNF-alpha) in tumour-bearing rats

Isolated perfusion of the extremities with high-dose tumour necrosis factor alpha (TNF-alpha) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-alpha and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 microg TNF-alpha. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-alpha and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-alpha alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-alpha, the minimal threshold concentration of TNF-alpha to exert its anti-tumour effects was not reached. The applicability of TNF-alpha in isolated kidney perfusion for human tumours seems, therefore, questionable.     

Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-alpha has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma-bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2- and IL-2 plus melphalan-treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan-treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2-based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.     

Long-term functional morbidity after mild hyperthermic isolated limb perfusion with melphalan.

AIMS: To assess long-term functional morbidity in patients entered in the prospective randomized EORTC trial investigating the role of adjuvant isolated limb perfusion (ILP) with melphalan for high-risk primary melanoma. METHODS: In 65 patients (ILP 36, wide excision only 29), limb circumference and joint mobility measurements were performed on the treated and the contralateral limb after a mean interval of 48 months after primary treatment. The two treatment groups were comparable regarding age, sex distribution, percentage of skin grafts or regional lymph-node dissections, and interval between primary treatment and physical measurements. RESULTS: None of the patients had severe complaints of the treated limb at the time of analysis. The ankle suffered most from ILP, with a statistical significant restricted extension in approximately 40% of the perfused patients. Abduction of the shoulder was minimally affected in treated upper limbs, probably as a result from the formation of scar tissue after axillary lymph-node dissection. Although no significant differences could be demonstrated in the circumference of upper or lower limbs, atrophy was seen in 24% of perfused lower limbs. Of the five perfused patients who developed oedema, four had also undergone a regional lymph-node dissection. CONCLUSION: This risk of long-term functional morbidity should be weighed against the possible advantages of ILP in patients with limb melanoma or sarcoma.     

The pharmacokinetic advantages of isolated limb perfusion with melphalan for malignant melanoma.

We describe melphalan pharmacokinetics in 26 patients treated by isolated limb perfusion (ILP). Group A (n = 11) were treated with a bolus of melphalan (1.5 mg kg-1), and in a phase I study the dose was increased to 1.75 mg kg-1. The higher dose was given as a bolus to Group B (n = 9), and by divided dose to Group C (n = 6). Using high performance liquid chromatography (HPLC) the concentrations of melphalan in the arterial and venous perfusate (during ILP) and in the systemic circulation (during and after ILP) were measured. Areas under the concentration time curves for perfusate (AUCa, AUCv) and systemic (AUCs) data were calculated. In all three groups the peak concentrations of melphalan were much higher in the perfusate than in the systemic circulation. The pharmacokinetic advantages of ILP can be quantified by the ratio of AUCa/AUCs, median value 37.8 (2.1-131). AUCa and AUCv were both significantly greater in Group B than in Group A (P values less than 0.01, Mann-Whitney). In Groups B and C acceptable 'toxic' reactions occurred but were not simply related to melphalan levels. Our phase I study has allowed us to increase the dose of melphalan to 1.75 mg kg-1, but we found no pharmacokinetic advantage from divided dose administration.     

Adjuvant regional isolated perfusion with melphalan for patients with clark V melanoma of the extremities

From 1978 to 1990, 32 patients with Clark V melanoma were treated by wide excision of the primary and adjuvant regional isolated perfusion with melphalan. M.D. Anderson stage of disease was stage I in 22 and stage IIIb in 10 patients. Five-year survival rates were 58% and 27%, respectively. Seven patients developed a recurrence in the perfused limb [stage I, 2, stage IIIb, 5 patients (P = 0.03)], and 4 of 17 patients developed regional lymph node metastases. Of the well-known prognostic variables, only ulceration of the primary tumor significantly influenced survival (P = 0.03). We did not see any improvement in survival rates compared with literature data on nonperfused patients. In the absence of data on locoregional recurrence rates in nonperfused Clark V melanoma patients, we cannot say whether adjuvant perfusion diminished this risk. Therefore, the results of the prospective randomized EORTC/WHO trial in primary high-risk extremity melanoma have to be awaited. © 1993 Wiley-Liss, Inc.     

Repeat isolated limb perfusion with TNFalpha and melphalan for recurrent limb melanoma after failure of previous perfusion.

AIM: To assess the effectiveness of isolated limb perfusion (ILP) with tumour necrosis factor-alpha (TNFalpha) and melphalan for recurrent or persistent melanoma lesions after previous ILP. METHODS: Between 1978 and 2001, 21 patients (mean age 65, range 29-83 years) underwent repeat ILP for recurrent or persistent melanoma after a previous ILP. First ILPs had been performed with melphalan alone in 13 patients and with addition of TNFalpha in eight, for a median of nine lesions (interquartile (IQ) range 2-23 lesions). Repeat ILP was performed with TNFalpha and melphalan in all 21 patients for a median of nine lesions (IQ range 5-25 lesions). Median follow-up after repeat ILP was 18 months (IQ range 6-36 months). RESULTS: Thirteen patients attained a complete response (CR) after repeat ILP compared to 11 of 17 with measurable lesions after the first ILP. Nine patients relapsed after CR. Median limb recurrence-free survival was 13 months. Fourteen patients had mild acute regional toxicity after repeat ILP compared to 18 after the first ILP (n.s.). One patient underwent amputation for critical limb ischemia 10 months following repeat ILP. The limb salvage rate was 95%. Overall median survival was 62 months after CR compared to 13 months for those without CR (P=0.05). CONCLUSION: Repeat ILP with TNFalpha and melphalan is feasible after previous ILP with mild regional toxicity. The CR rate is relatively high and comparable to the first procedure with good limb recurrence-free survival and high limb salvage rate.     

Increased local cytostatic drug exposure by isolated hepatic perfusion: a phase I clinical and pharmacologic evaluation of treatment with high dose melphalan in patients with colorectal cancer confined to the liver

A phase I dose-escalation study was performed to determine whether isolated hepatic perfusion (IHP) with melphalan (L-PAM) allows exposure of the liver to much higher drug concentrations than clinically achievable after systemic administration and leads to higher tumour concentrations of L-PAM. Twenty-four patients with colorectal cancer confined to the liver were treated with L-PAM dosages escalating from 0.5 to 4.0 mg kg(-1). During all IHP procedures, leakage of perfusate was monitored. Duration of IHP was aimed at 60 min, but was shortened in eight cases as a result of leakage from the isolated circuit. From these, three patients developed WHO grade 3-4 leukopenia and two patients died due to sepsis. A reversible elevation of liver enzymes and bilirubin was seen in the majority of patients. Only one patient was treated with 4.0 mg kg(-1) L-PAM, who died 8 days after IHP as a result of multiple-organ failure. A statistically significant correlation was found between the dose of L-PAM, peak L-PAM concentrations in perfusate (R = 0.86, P< or =0.001), perfusate area under the concentration-time curve (AUC; R = 0.82, P<0.001), tumour tissue concentrations of L-PAM (R = 0.83, P = 0.011) and patient survival (R = 0.52, P = 0.02). The peak L-PAM concentration and AUC of L-PAM in perfusate at dose level 3.0 mg kg(-1) (n = 5) were respectively 35- and 13-fold higher than in the systemic circulation, and respectively 30- and 5-fold higher than reported for high dose oral L-PAM (80-157 mg m(-2)) and autologous bone marrow transplantation. Median survival after IHP (n = 21) was 19 months and the overall response rate was 29% (17 assessable patients; one complete and four partial remissions). Thus, the maximally tolerated dose of L-PAM delivered via IHP is approximately 3.0 mg kg(-1), leading to high L-PAM concentrations at the target side. Because of the complexity of this treatment modality, IHP has at present no place in routine clinical practice.     

TNF-alpha and melphalan-based isolated limb perfusion: no evidence supporting the early destruction of tumour vasculature

Background:

Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a highly effective treatment for locally advanced tumours of the extremities. Previous research suggests an almost immediate disintegration of the blood supply of the tumour. The aim of the present study was to verify this hypothesis using non-invasive measurements of microvascular perfusion and tissue oxygenation.

Methods:

A total of 11 patients were included in the study. TM-ILP was performed under mildly hyperthermic conditions (39 °C) in the extremities via proximal vascular access. Capillary-venous microvascular blood flow, haemoglobin level (Hb) and oxygen saturation (SO₂) were determined using laser Doppler and white-light spectroscopy, respectively, before TM-ILP and at 30 min, 4 h, 1 day, 4 days, 1 week, 2 weeks and 6 weeks after TM-ILP from tumour and healthy muscle tissues.

Results:

Blood flow and Hb were mostly higher, whereas SO₂ was lower, in tumour tissue compared with muscle tissue. In both tumour and muscle tissues, blood flow significantly increased immediately after TM-ILP and remained elevated for at least 2 weeks, followed by a return to the initial values 6 weeks after the procedure.

Conclusion:

No signs were found of early destruction of the tumour vasculature. The observations suggest that an inflammatory reaction is one of the key elements of TM-ILP.