Regional perfusion treatment with melphalan for melanoma in a limb: an evaluation of drug kinetics

The time course of tissue levels of melphalan during normothermic isolated limb perfusion, and the overall tissue levels per 60 min of perfusion, were estimated from the known pharmacokinetic parameters for a fixed dose of drug per liter of tissue (Benckhuijsen et al., J. Pharmacol Exp Ther 1986; 237: 583-8). The application of differing total doses of drug resulted in varying concentrations in the perfusate plasma. Above a certain plasma level, uptake into the bulk of the tissues did not increase with the area under the plasma concentration vs time curve or its beta-phase. Similar tissue levels were found after perfusion of regions of less than three and a half liter with 13 mg/l as in regions of 5 to 16 liter after perfusion with 10 mg of melphalan per liter. It cannot be predicted from the available data whether the extent of uptake of melphalan into the tumour tissue is equal to or greater than that into the bulk of the tissues. The estimated uptake of drug into the tissues confirms the validity of the dose calculation per liter of tissue. On the basis of the present results, a refined dosimetric formula will be obtainable that includes the desired area under the plasma concentration vs time curve as a determinant for an optimal dose.     

Pharmacokinetics of isolated lung perfusion with melphalan for resectable pulmonary metastases, a phase I and extension trial

BACKGROUND: Isolated lung perfusion (ILuP) with melphalan was performed under normo- and hyperthermic conditions combined with pulmonary metastasectomy for patients with resectable lung metastases. We present the results of pharmacokinetic analysis of a phase I and extension trial. METHODS: Twenty-one procedures of ILuP with melphalan were performed in the phase I trial according to a dose-escalation schedule under normothermic and hyperthermic conditions followed by surgical resection of pulmonary metastases. In an extension trial 8 additional procedures with 15 and 45 mg melphalan were performed under hyperthermic conditions. Samples of serum, perfusate, lung, and tumor tissue were obtained. RESULTS: High perfusate concentrations of melphalan were recorded in contrast to low systemic concentrations. Marked interindividual variability was observed in melphalan concentrations in perfusate, tumor, and lung tissue. Statistically significant correlation between melphalan dose, and perfusate area under the concentration-time curve (R(2) = 0.578, P = 0.001) and lung tissue concentrations (R(2) = 0.459, P = 0.028) were noted. No significant correlation between melphalan dose and tumor tissue concentrations could be established. CONCLUSION: Isolated lung perfusion effectively delivers high doses of melphalan to the lung and tumor tissues with minimal systemic levels. Significant correlation between perfused melphalan dose, perfusate area under the concentration-time curve and lung tissue melphalan concentrations were observed.     

The effects of perfusion conditions on melphalan distribution in the isolated perfused rat hindlimb bearing a human melanoma xenograft.

An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann''s solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 ml min(-1)) resulted in higher tissue blood flow (determined with 51Cr-labelled microspheres) and melphalan uptake by tumour and normal tissues. The distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann''s solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding (no albumin) is preferred for maximum uptake of drug by the tumour.     

Pharmacokinetics of buthionine sulfoximine (NSC 326231) and its effect on melphalan-induced toxicity in mice

Intravenous doses of buthionine sulfoximine (BSO, NSC 326231), an inhibitor of glutathione synthesis, were eliminated rapidly from mouse plasma in a biexponential manner. The initial phase of the plasma concentration versus time curve had a half-life of 4.9 min and accounted for 94% of the total area under the curve. The half-life of the terminal phase of the curve was 36.7 min and the area accounted for only 6% of the total area under the curve. Plasma clearance of BSO was 28.1 ml/min/kg and the steady state volume of distribution was 280 ml/kg. The oral bioavailability of BSO, based on plasma BSO levels, was extremely low. However, comparable glutathione depletion was apparent after i.v. and p.o. doses of BSO, suggesting a rapid tissue uptake and/or metabolism of BSO. Therefore, due to the rapid elimination of BSO from mouse plasma, plasma drug levels do not directly correlate with BSO-induced tissue glutathione depletion. Administration of multiple i.v. doses of BSO to male and female mice resulted in a marked 88% depletion of liver glutathione at doses of 400-1600 mg/kg/dose. Toxicity of i.v. administered BSO was limited to a transient depression of peripheral WBC levels in female mice given six doses of 1600 mg/kg. Multiple i.v. doses of BSO of up to 800 mg/kg/dose (every 4 h for a total of six doses) did not alter the toxicity of i.v. administered melphalan. However, multiple doses of 1600 mg/kg/dose of BSO did potentiate histopathological evidence of melphalan-induced bone marrow toxicity in 30% of the mice and, additionally, the combination of BSO and melphalan produced renal tubular necrosis in 80% of the male mice. The potentiation of melphalan induced toxicity did not appear to be related to GSH depletion, since: quantitatively similar amount of GSH depletion occurred at lower dose of BSO without any increase in melphalan toxicity.     

Can tissue drug concentrations be monitored by microdialysis during or after isolated limb perfusion for melanoma treatment?

Isolated limb perfusion (ILP) with melphalan is used to treat recurrent melanoma. This study aimed to develop a microdialysis technique for melphalan tissue concentration measurement during ILP. The effects of melphalan concentration (50-600 microg/ml), microdIalysis flow rate (0.55-17.5 microl/min), probe length (5-50 mm) and temperature (25-41.5 degrees C) on in vitro recovery were studied. In addition, in vivo recovery was measured in rat hindlimbs perfused with melphalan using 50 mm microdialysis probes implanted subcutaneously and into muscle. Both dialysate and tissue sample melphalan concentrations were determined by high performance liquid chromatography. The in vitro recovery of melphalan was not affected by melphalan concentration or temperature, but increased with probe length and decreased with flow rate. The melphalan concentrations in subcutaneous and muscle dialysates were not significantly different. A linear relationship was found between tissue dialysate concentrations and actual tissue concentrations of melphalan (r2 = 0.97). Microdialysis is a potential method for tissue drug monitoring which may assist in the efficacious use of cytotoxics in human ILP.     

Pharmacokinetics of high-dose melphalan (200 mg/m2) in a case of total renal insufficiency

The pharmacokinetics of the alkylating agent melphalan was determined in a dialysed patient, 30 years old, who underwent unilateral orchidectomy for a malignant testicular tumor. Melphalan was included in a polychemotherapy treatment with eight 1-h infusions of 230 mg of etoposide (VP 16), then one 5-min infusion of 370 mg of melphalan (200 mg/m2) followed by autologous bone marrow grafting (ABMG). In this patient, melphalan pharmacokinetics was different from that of patients without important renal dysfunction for the area under the concentration curve (1,324 mg.l-1.min). However, with a melphalan elimination half-life of 80 min, ABMG could be performed, as usually, 24 h after melphalan administration. Plasma alpha-fetoprotein (AFP) concentrations showed that chemotherapy was efficient. Furthermore, we observed a modification of etoposide kinetics due to melphalan.     

Dosimetry of cytostatics in hyperthermic regional isolated perfusion

During the period from February to October 1983, 21 patients with malignant melanoma of the extremities were treated by hyperthermic regional isolated perfusion with L-phenylalanine mustard (melphalan). The melphalan dose for each patient was determined by the tissue volume of the perfused region, using a dose of 10 mg/l perfused tissue. Despite an average increase of melphalan dosage of 18% above the maximum for iliac perfusions recommended in the literature, no increase in toxic tissue reactions was observed after hyperthermic iliac perfusions. The same dose of 10 mg/l perfused tissue was used in hyperthermic axillary perfusions, resulting in an average decrease of melphalan dosage of 14% below the minimum recommended in the literature. By applying a constant dose per unit tissue volume, a standardization of treatment is achieved. This excludes variations like body weight, age, type of complexion, and hair color, which so far have determined dosimetry.     

Kinetics of melphalan leakage during hyperthermic isolation perfusion in melanoma of the limb

Summary The kinetics of melphalan leakage into the peripheral blood were studied in 21 patients undergoing hyperthermic isolation perfusion of the upper or lower limb as an adjuvant treatment in high-risk melanoma; in 5 patients cisplatin was added. The melphalan concentrations in the peripheral blood rose predominantly during the first 20 min of perfusion and levelled out to an apparent steady state of about 0.28 g/ml in upper extremity perfusions, and 0.34 (without cisplatin) and 0.37 g/ml (with cisplatin) in lower extremity perfusions. Erythrocytes labelled with technetium Tc 99m, which were added concomitantly with melphalan to the perfusion medium, appeared in the systemic circulation of the patients at an almost constant rate of 0.32% (lower and upper limb perfusions without cisplatin and 0.37% (with cisplatin) of total tracer/min. This perfusate flow rate indicated by labelled erythrocytes completely explained the leakage of melphalan from the perfusion circuit into the peripheral blood. Peak concentrations of melphalan in the peripheral blood were observed immediately after reconstitution of normal hemodynamic conditions once isolation perfusion had been teminated. This fraction of melphalan might originate from tissue-binding sites, but also from vascular compartments; therefore, a thorough washing-out procedure might minimize this effect.This study was supported by the Deutsche Krebshilfe/Dr. Mildred Scheel Stiftung, Bonn-Bad Godesberg, FRG     

Complete remission of previously intractable peripheral cutaneous T-cell lymphoma of the lower extremity using isolated hyperthermic limb perfusion with melphalan (1-phenylalanine mustard)

The patient is a 74-year-old woman first diagnosed with a peripheral cutaneous T-cell lymphoma (PCTCL) in April of 1994. Initially she presented with subcutaneous indurated areas in the right forearm, scapula, and submadibular region. After chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), she went into remission for 2 years before relapse of her PCTCL localized to the right lower extremity. Persistent isolated disease in the extremity since then led to numerous chemotherapy regimens and localized radiation therapy. Due to dramatic limb threatening progression of the disease in 2001, she underwent isolated hyperthermic limb perfusion with melphalan (1-phenylalanine mustard). Although limb preservation could not be achieved, this treatment resulted in complete clinical and pathological regression of the lesions of the perfused extremity.     

Technology insight: Utility of TNF-alpha-based isolated limb perfusion to avoid amputation of irresectable tumors of the extremities

Isolated limb perfusion (ILP) with melphalan is effective in the treatment of small multiple melanoma intransit metastases and is utilized widely for this indication. The treatment is much less effective against bulky melanoma metastases and has uniformly failed in the treatment of irresectable extremity soft tissue sarcomas. The addition of tumor-necrosis factor-alpha (TNF-alpha) to this treatment approach has changed the situation dramatically. High response rates and limb-salvage rates have been reported in multicenter trials that combined ILP with TNF-alpha plus melphalan; these trials resulted in the approval of TNF-alpha for bulky melanoma metastases and soft tissue sarcomas in Europe in 1998. Subsequently, many doctors working in European centers have been trained, and a series of confirmatory reports from single institutions have now been published regarding the efficacy of the procedure. TNF-alpha has an early and a late effect; it enhances tumor-selective drug uptake during the perfusion, and plays an essential role in the subsequent selective destruction of the tumor vasculature. These effects result in a high response rate in bulky tumors, soft tissue sarcomas, bulky melanomas, and various other tumor types. This induction therapy therefore allows tumor remnants to be resected some 3 months after ILP thus avoiding limb amputation. TNF-alpha-based ILP is a well-established treatment that aims to avoid amputations regardless of the tumor size and type. It represents an important example of combination therapy that modulates the tumor vasculature and should be offered in high-volume tertiary referral centers.     

Intractible cutaneous non-Hodgkin's lymphoma of the lower limb. Complete remission after sequential regional isolated hyperthermic perfusion and perfusion with 1-phenylalanine-mustard (melphalan, L-Pam)

A 61-year-old man with an intractible and progressively disabling cutaneous non-Hodgkin's lymphoma (NHL) in the lower limb was treated with sequential regional isolated perfusion at 10-day intervals. The first perfusion was hyperthermic (40.2-43.1 degrees C tumor-temperature); the second was at controlled normothermia with high-dose 1-phenylalanine mustard (melphalan, L-Pam; 11 mg/l perfused tissue). This treatment resulted in a complete remission in the perfused area of significant duration and has prevented amputation.     

Results of regional isolation perfusion with cytostatics in patients with soft tissue tumors of the extremities

From 1975 to 1986, 26 patients with soft tissue tumors of the extremities underwent a total of 29 perfusions. The cytostatics used were doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH) (19 perfusions), melphalan (two perfusions), and a combination of these agents (eight perfusions). Before perfusion most patients had been treated by surgical excision(s), radiotherapy, or systemic chemotherapy. Of 17 patients perfused because of local inoperable tumor, four showed prolonged complete remission of the tumor mass, stable disease was seen in three, and ten showed progression. The complete remissions observed in three patients with aggressive fibromatosis and in one with lymphangiosarcoma occurred after perfusion with doxorubicin combined with melphalan. Doxorubicin added to the perfusate as the sole cytostatic was not effective. Local recurrence was observed in five of nine patients treated by adjuvant perfusion, always after dubiously radical tumor excision. Toxicity was high, especially in the first few years. Tissue necrosis necessitated amputation in three cases (in two after perfusion with doxorubicin and melphalan and in one after repeated perfusion with doxorubicin only). This complication was no longer seen after adjustment of the dosage and dose distribution of doxorubicin, but the morbidity after perfusion with doxorubicin remained considerable.     

Isolated limb perfusion with melphalan,tumour necrosis factor‐alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in‐transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer‐selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour‐targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor‐alpha (TNF‐α) and Lister strain vaccinia virus (GLV‐1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF‐α, melphalan and GLV‐1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV‐1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF‐α/GLV‐1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF‐α/melphalan‐based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.     

Isolated limb perfusion with actinomycin D and TNF-alpha results in improved tumour response in soft-tissue sarcoma-bearing rats but is accompanied by severe local toxicity

Previously we demonstrated that addition of Tumour Necrosis Factor-alpha to melphalan or doxorubicin in a so-called isolated limb perfusion results in synergistic antitumour responses of sarcomas in both animal models and patients. Yet, 20 to 30% of the treated tumours do not respond. Therefore agents that synergise with tumour necrosis factor alpha must be investigated. Actinomycin D is used in combination with melphalan in isolated limb perfusion in the treatment of patients with melanoma in-transit metastases and is well known to augment tumour cell sensitivity towards tumour necrosis factor alpha in vitro. Both agents are very toxic, which limits their systemic use. Their applicability may therefore be tested in the isolated limb perfusion setting, by which the tumours can be exposed to high concentrations in the absence of systemic exposure. To study the beneficial effect of the combination in vivo, BN-175 soft tissue sarcoma-bearing rats were perfused with various concentrations of actinomycin D and tumour necrosis factor alpha. When used alone the drugs had only little effect on the tumour. Only when actinomycin D and tumour necrosis factor alpha were combined a tumour response was achieved. However, these responses were accompanied by severe, dose limiting, local toxicity such as destruction of the muscle tissue and massive oedema. Our results show that isolated limb perfusion with actinomycin D in combination with tumour necrosis factor alpha leads to a synergistic anti-tumour response but also to idiosyncratic locoregional toxicity to the normal tissues. Actinomycin D, in combination with tumour necrosis factor alpha, should not be explored in the clinical setting because of this. The standard approach in the clinic remains isolated limb perfusion with tumour necrosis factor alpha in combination with melphalan.     

Isolated Limb Perfusion with Melphalan and TNF-α in the Treatment of Extremity Sarcoma

Opinion statement Isolated limb perfusion (ILP) with chemotherapy alone has uniformly failed in the treatment of irresectable extremity soft tissue sarcomas. The addition of tumor necrosis factor-alpha (TNF-α) to this treatment approach contributed to a major step forward in the treatment of locally advanced extremity soft tissue sarcoma (STS). High response rates and limb salvage rates have been reported in multicenter trials, which combined ILP with TNF-α plus melphalan, which resulted in the approval of TNF-α for this indication in Europe in 1998. Subsequently a series of confirmatory single institution reports on the efficacy of the procedure have now been published. TNF-α has an early and a late effect; it enhances tumor-selective drug uptake during the perfusion and plays an essential role in the subsequent selective destruction of the tumor vasculature. These effects result in a high response rate in high-grade soft tissue sarcomas. This induction therapy thus allows for resection of tumor remnants some 3 months after ILP and thus avoidance of limb amputation. TNF-α-based ILP is a well-established treatment to avoid amputations. It represents an important example of tumor vasculatory-modulating combination therapy and should be offered in large volume tertiary referral centers.     

Current clinical and research approaches to optimizing regional chemotherapy: novel strategies generated through a better understanding of drug pharmacokinetics, drug resistance, and the development of clinically relevant animal models

A common treatment modality for in transit melanoma of the extremity has been hyperthermic isolated limb perfusion (HILP) with melphalan and more recently, isolated limb infusion (ILI) with melphalan with or without dactinomycin. Research in the area has primarily focused on maximizing drug delivery through a better understanding of pharmacokinetics while maintaining acceptable levels of toxicity. Although other agents continue to be explored as regional chemotherapy agents, melphalan is currently the drug of choice, although temozolomide has demonstrated promising preclinical results. Resistance mechanisms to melphalan and melphalan pharmacokinetics are studied using animal models of HILP and ILI. The addition of modulators to overcome resistance to the traditional chemotherapy regimen may ultimately improve the clinical response in patients with in-transit melanoma of the extremity.     

Comparative brain and plasma pharmacokinetics and anticancer activities of chlorambucil and melphalan in the rat

Summary Equimolar doses of chlorambucil and melphalan (both 10 mg/kg) were administered i.v. to anesthetized rats, and the plasma and brain concentrations of chlorambucil, its metabolites 3,4-dehydrochlorambucil and phenylacetic mustard, and melphalan were determined by high-performance liquid chromatography from 5 to 240 min therafter. Chlorambucil demonstrated a monophasic disappearance from plasma, with a half-life of 26 min. The compound was 99.6% plasma-protein-bound. Chlorambucil underwent -oxidation to yield detectable concentrations of 3,4-dehydrochlorambucil and substantial amounts of phenylacetic mustard in the plasma. Low concentrations of chlorambucil and phenylacetic mustard were detected in the brain. Calculated from the areas under the concentration-time curves, the brain: plasma concentration integral ratios of chlorambucil and phenylacetic mustard were 0.021 and 0.013, respectively. Melphalan demonstrated a biphasic disappearance from plasma, with half-lives of 1.9 and 78 min. The compound was approximately 86% plasma protein-bound. Low concentrations of melphalan were detected in the brain, and its brain: plasma ratio was 0.13. These data demonstrate that following the administration of chlorambucil and melphalan, only low concentrations of active drug are able to enter the brain. As a consequence, concentrations of both drugs that cause the complete inhibition of extracerebrally located tumor have no effect on those located within the brain. Further, the brain uptake of melphalan, although low, is greater than that of chlorambucil and its active metabolites, which coincides with its slightly greater intracerebral activity following the systemic administration of very high doses.     

Isolated limb perfusion for extremity soft-tissue sarcomas, in-transit metastases, and other unresectable tumors: Credits, debits, and future perspectives

Isolated limb perfusion (ILP) with melphalan is effective against melanoma in-transit metastases but has failed in the treatment of limb-threatening extremity sarcomas. Tumor necrosis factor-α (TNF) has changed this situation completely. Now, ILP with TNF + melphalan is a very successful treatment to prevent amputation. In a multicenter European trial, ILP with TNF + melphalan resulted in a 76% response rate and a 71% limb salvage rate in patients with limb-threatening soft-tissue sarcomas, deemed unresectable by independent review committees, leading to approval of TNF in Europe. We have also reported on the success of this regimen against bulky melanomas, multifocal skin cancers, and drug-resistant bony sarcomas. High-dose TNF destructs tumor vasculature, and, most importantly, it enhances tumor-selective drug uptake (ie, melphalan and doxorubicin) by threefold to sixfold. Similar synergy is observed in well-vascularized liver metastases after isolated hepatic perfusion with TNF and melphalan. New (vasoactive) drugs and mechanisms of action and interaction with chemotherapy are in development. ILP is also a promising treatment modality for adenoviral vector-mediated gene therapy. Many clinical phase I/II evaluations in ILP are now underway.     

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.     

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.