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In this paper we describe 4 new monoclonal antibodies to be applied in rat models for cancer. The monoclonal antibodies were obtained by immunizing Balb/c mice with CC531 rat colon adenocarcinoma cells. Hybridomas were produced and 4 were selected for their reactivity with CC531 in vitro (MG1, 2, 3 and 4). All 4 antibodies recognized other rat tumour cell lines and showed limited cross-reactivity with normal rat tissues. Intraperitoneally injected MG1, 2 and 4 homed to in vivo growing, artificially induced CC531 liver metastases. In these in vivo experiments, limited cross-reactivity with normal rat tissues, predominantly of the gastro-intestinal tract, was found. MG4 was found to enhance lysis of CC531 tumour cells mediated by IL-2 activated, cultured natural killer cells. These antibodies are potentially useful for antibody-based laboratory techniques and for investigation of antibody-based immunotherapy of cancer in a rat model.

In this study, cultured T cells, pre-incubated with the bispecific monoclonal antibody (BiMAb) R73IgG1 x CC52IgG1 were adoptively transferred, via systemic and regional routes, to rats bearing day 10 hepatic metastases of the CC531 adenocarcinoma of the colon to investigate the role of the route of administration in tumour infiltration by these BiMAb-retargeted effector cells. The BiMAb, directed against the T-cell receptor and the tumour-associated antigen CC52, were used to crosslink CC531 tumour cells and T cells to induce tumour cell lysis. Retargeted T cells were administered via the jugular vein, hepatic artery or the portal vein. The number of BiMAb-retargeted T cells that reached the liver tumours was independent of the route of administration. There was also no difference between the number of T cells that reached the portal tracts, central veins of parenchyma of the liver, after loco-regional or systemic administration. These findings are in contrast to the interleukin (IL)-2 activated NK (A-NK) cells biodistribution studies earlier performed in the same animal model in our laboratory. Compared with A-NK cells, a lower number of BiMAb-retargeted T cells reached the tumours, irrespective of their route of administration while for A-NK cells, there was an advantage of administration via the hepatic artery.

The colon adenocarcinoma cell line CC531 was adopted as a model for immunotherapeutical treatment of experimental colorectal metastases in a syngeneic rat model. We studied the presence and localization of T and natural killer cells, vessels and matrix proteins in in vivo growing CC531 tumours by immunohistochemistry. CC531 tumours were induced either in the lungs by injecting CC531 tumour cells into a tail vein or in the liver by injection of CC531 tumour cells under the liver capsule or into a mesenteric vein. All 3 tumour types were composed of islets of tightly apposed tumour cells surrounded by abundantly present tumour-stroma which contained tumour vessels and matrix proteins. Some of these matrix proteins, especially laminin and collagen IV formed a basal membrane-like structure around the tumour nodules. This structure was most pronounced in mesenteric vein-induced liver tumours and less prominent in subcapsular-induced liver tumours and tail vein-induced lung tumours. Tumour-infiltrating lymphocytes of both T and natural killer cell origin were found in the tumours, but predominantly in the tumour stroma, separated from the islets of tumour cells by the basal membrane-like structure. We hypothesize that the matrix proteins of these tumours play an ambivalent role: they may provide a substratum for migration of effector cells into the tumour stroma but may also provide a barrier preventing direct contact between tumour target cells and immune effector cells.

With the recent availability of novel antibodies against melanoma antigens tyrosinase and MART-1, it is important to validate their usefulness in pathology practice and in screening patients for immunotherapy treatment. In the present study conducted by the Melanoma Cooperative Group of the European Organization for Research and Treatment of Cancer (EORTC-MCG), immunohistochemical staining for gp100 (antibodies NKI-beteb and HMB-45), MART-1 (A103), tyrosinase (T311), and S100 (S100) was compared on formalin-fixed and paraffin-embedded tumour lesions from 80 patients with 130 malignant melanoma lesions, comprising 44 primary tumours, 18 locoregional metastases, 41 lymph node metastases, and 27 visceral metastases from the lung, liver, and brain. A score between 0 and 5 was allocated to each immunohistochemically stained section. These scores were evaluated in a statistical analysis. S100 was by far the most sensitive marker in all four types of lesions tested. Apart from a significantly better performance for T311 in primary melanomas compared with HMB-45, no significant differences were observed between the four remaining antigens tested. Three settings were next investigated to determine whether the expression of melanoma antigens decreases with tumour progression. First, within the primary melanomas, only NKI-beteb and A103 staining showed a nearly significant negative correlation with Clark's level of invasion and a similar tendency was observed for these antibodies with Breslow thickness. Second, when comparing primary melanoma-metastasis pairs from the same patient, lymph node metastases showed less staining with NKI-beteb, HMB-45, A103, and T311, at a level near significance. This difference was not significant when comparing the primary tumour with visceral metastases, probably due to the lower numbers of pairs. Third, regarding tumour progression from primary melanoma to locoregional, to lymph node, to visceral metastasis, a significant decrease with progression was found only for T311. The apparently stable expression of most of the melanoma antigens, and the small contribution of decreased expression in melanoma tumour progression, supports the rationale for immunotherapy based on the melanoma immunogens gp100, MART-1, and tyrosinase.

Item does not contain fulltext With the recent availability of novel antibodies against melanoma antigens tyrosinase and MART-1, it is important to validate their usefulness in pathology practice and in screening patients for immunotherapy treatment. In the present study conducted by the Melanoma Cooperative Group of the European Organization for Research and Treatment of Cancer (EORTC-MCG), immunohistochemical staining for gp100 (antibodies NKI-beteb and HMB-45), MART-1 (A103), tyrosinase (T311), and S100 (S100) was compared on formalin-fixed and paraffin-embedded tumour lesions from 80 patients with 130 malignant melanoma lesions, comprising 44 primary tumours, 18 locoregional metastases, 41 lymph node metastases, and 27 visceral metastases from the lung, liver, and brain. A score between 0 and 5 was allocated to each immunohistochemically stained section. These scores were evaluated in a statistical analysis. S100 was by far the most sensitive marker in all four types of lesions tested. Apart from a significantly better performance for T311 in primary melanomas compared with HMB-45, no significant differences were observed between the four remaining antigens tested. Three settings were next investigated to determine whether the expression of melanoma antigens decreases with tumour progression. First, within the primary melanomas, only NKI-beteb and A103 staining showed a nearly significant negative correlation with Clark's level of invasion and a similar tendency was observed for these antibodies with Breslow thickness. Second, when comparing primary melanoma-metastasis pairs from the same patient, lymph node metastases showed less staining with NKI-beteb, HMB-45, A103, and T311, at a level near significance. This difference was not significant when comparing the primary tumour with visceral metastases, probably due to the lower numbers of pairs. Third, regarding tumour progression from primary melanoma to locoregional, to lymph node, to visceral metastasis, a significant decrease with progression was found only for T311. The apparently stable expression of most of the melanoma antigens, and the small contribution of decreased expression in melanoma tumour progression, supports the rationale for immunotherapy based on the melanoma immunogens gp100, MART-1, and tyrosinase.

Aims To determine whether the addition of high-dose tumour necrosis factor- α (TNF α ) 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°C) and «mild» hyperthermic (38–40°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 α 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 α 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 α 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 α 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°C) at which the melphalan in the TNF α -ILPs was administered or by an interaction between high-dose TNF α and melphalan.

Tumor necrosis factor-alpha is a multifunctional cytokine and its potential as antitumor agent has been extensively investigated for the treatment of cancer: Initial enthusiasm was tempered when systemic treatment with TNF-alpha was found to cause severe toxic side effects in phase I/II studies. Other applications were sought and a revival of the drug was its application in combination with the cytotoxic drug melphalan in isolated limb perfusion (ILP). Here we discuss the pre-clinical and clinical studies which led to the success in treatment of patients with irresectable extremity soft tissue sarcoma and multiple melanoma in-transit metastases confined to the limb. This achievement may herald the development of new applications of TNF-alpha in isolated organ perfusion settings. An extension of its use may be found in the application of less toxic TNF-mutants or encapsulated in sterically stabilized liposomes. The review concludes with the possible application of low dose TNF-alpha, which can be given systemically, to enhance the anti-tumor potency of formulated drugs (such a liposomal doxorubicin) by increasing solid tumor targeting.

An isolated limb perfusion model (ILP) using soft tissue sarcoma bearing rats (BN175) was used to study antitumour activity of a tumour necrosis factor alpha mutant (TNF-SAM2) in combination with melphalan and doxorubicin. Progressive disease was demonstrated after ILP without agents (sham) or with 50 micrograms TNF-SAM2. ILP with 40 micrograms melphalan or 400 micrograms doxorubicin resulted in no change of tumour volume or progressive disease five days after perfusion. Partial and complete response rates were demonstrated in 76% of rats when the combination of TNF-SAM2 and melphalan was used. TNF-SAM2 in combination with doxorubicin was synergistic as well with a 70% response rate. Histopathologically these responses consisted of hemorrhagic necrosis of the coagulative type. 2 In conclusion, TNF-SAM2 has similar antitumour activity in combination with melphalan or doxorubicin as rHuTNF in sarcoma-bearing rats and is eligible to be tested in clinical ILP or organ perfusion settings because of its potential decreased toxicity.

To investigate whether the course of primary melanoma disease correlates with expression of the various components of the proteolytic plasminogen activation (PA) system, immunohistochemical stainings for activators of plasminogen (tissue type (tPA) and urokinase type (uPA)), inhibitors of plasminogen activation (type 1 (PAI-I) and type 2 (PAI-2)) and the receptor for uPA (uPAR) were performed on 214 routinely processed melanoma lesions. All lesions were primary cutaneous melanomas, minimally 1.5 mm thick, and derived from patients with only local disease at the moment of diagnosis (clinically stage II (T3-4 NoMo), American Joint Committee on Cancer). Median patient follow-up was 6.1 years. Single variables as immunohistochemical staining results (extent of tumour cell staining, pattern of tumour cell staining and for some components also staining of stromal cells), histopathological and clinical parameters as well as treatment variables were analysed in order to assess their prognostic importance, in terms of time to recurrence, time to distant metastasis and duration of survival. The extent of tPA tumour cell positivity, categorized as 0-5%, 6-50% and 51-100%, appeared to be of importance for these endpoints. Lesions with 51-100% tPA-positive tumour cells were found to have the best prognosis, whereas lesions with 6-50% tPA-positive tumour cells had the worst. Moreover, the prognostic significance of Breslow thickness, microscopic ulceration and sex was confirmed in this study. Multivariate analyses, incorporating these relevant factors, showed that the extent of tPA tumour cell positivity was an independent prognostic factor for distant metastasis-free interval (P = 0.012) and for the duration of survival (P = 0.043). (C) 2000 Cancer Research Campaign.

The success of and our extensive experience with TNF alpha-based isolated limb perfusions in patients with unresectable extremity soft tissue sarcomas made us explore the possibilities for a similar approach for the treatment of hepatic metastases. After experience with the classic surgical isolated hepatic perfusion in pigs and in patients, we concluded that the classic surgical approach was associated with serious drawbacks i.e., magnitude of the procedure with morbidity, lack of repeatability of the procedure, complexity and costs. These problems were addressed in a program aimed at developing a repeatable method of isolated perfusion of the liver with minimally invasive techniques.We validated the methodology of isolated hypoxic hepatic perfusion using balloon catheter techniques in pigs.The excellent pharmacokinetic profile of the procedure, resulting in very high regional drug concentrations and negligible systemic drug concentrations, allowed us to move on to the clinic and start to study and further develop this method in a phase I-II study using the drug melphalan, in patients with irresectable hepatic metastases.We aim to develop step by step a fully percutaneous approach for isolated hypoxic hepatic perfusion.

Isolated hepatic perfusion (IHP) is an attractive approach to treating nonresectable liver tumors, because the effects of systemic chemotherapy are poor and its application is hampered by severe general toxicity. In clinical and experimental settings, the efficacy of isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF alpha) in combination with melphalan to treat melanoma in transit and soft-tissue sarcoma has been well established. In an ILP model in rats, the authors previously observed synergistic anti-tumor effects of TNF and melphalan on BN 175 soft-tissue sarcoma extremity tumors. The aim of the current study was to determine whether similar synergy in anti-tumor effects could be achieved by treating experimental BN 175 soft-tissue sarcoma liver tumors by IHP using these agents. The authors found that IHP with TNF and melphalan resulted in a dramatic increase in regional concentrations of perfused agents with virtually no concomitant systemic leakage. Isolated hepatic perfusion with only carrier solution resulted in a significantly diminished growth rate of BN 175 liver tumors compared with the growth rate of tumors in nonperfused rats. Perfusion with melphalan alone resulted in minimal anti-tumor effects. Perfusion with only TNF had a slight growth-stimulatory effect on the BN 175 liver tumors, but no negative effects on tumor growth were observed. When TNF was added to melphalan, a dramatic anti-tumor effect was observed. Thus, as in the rat ILP setting, the anti-tumor effect is augmented when TNF is added to IHP with melphalan to treat BN 175 soft-tissue sarcoma tumor-bearing rats. Strikingly, the tumor response was potentiated at relatively low concentrations of TNF compared with concentrations that elicited synergy with melphalan in ILP.

Controversy still exists about standard management of a primary melanoma. Over the last decades randomized phase III trials have addressed questions about the width of margin in relation to the Breslow thickness of the primary lesion, the role of prophylactic isolated limb perfusion, and the role of elective lymph node dissection. Overall these trials have demonstrated that less extensive surgery is as good as more extensive surgery. Wide excision margins, prophylactic isolated limb perfusions, or the elective lymph node dissection did not improve overall survival significantly in any of the phase III trials conducted.No standard systemic adjuvant therapy with confirmed impact on overall survival has been identified thus far for clinically node negative stage I-II (TxN0M0) patients after excision of the primary, nor for clinically node positive stage III (TxN1-2M0) patients after lymph node dissection for metastatic regional node involvement. Poor Staging in the Past. One of the main problems associated with the trials assessing systemic adjuvant treatments in management of high risk primary melanoma is the fact that in general patients were poorly staged. About 25%-30% of patients with primaries thicker than 1.5 mm have micro-metastatic disease in the regional lymph nodes and beyond. This population was usually submerged by the other 70%-75% of the patients with excellent prognosis, obscuring the potential benefit of the adjuvant surgical procedure (ELND) or a systemic adjuvant treatment.Sentinel lymph node (SLN) mapping is resolving many of the inadequacies of the past and has completely changed the management of primary melanoma. As a small procedure with low morbidity it identifies that part of the population which has microscopic involvement of regional lymph nodes with greater precision than an elective lymph node dissection. SLN-mapping allows for a detailed histopathologic evaluation involving multiple sections, HE staining in combination with IHC (immunohistochemical staining) of the node with the highest chance of containing metastatic foci. Moreover in the near future it is most likely that RT-PCR on negative nodes will complete the diagnostic workup as a promising last step in the procedure to determine whether tumor cells are present in the sentinel node. Sentinel lymph node status has been shown recently to be by far the strongest independent prognostic factor of melanoma stage I-II patients. SLN-status is a much stronger prognostic factor than tumor thickness, which looses its prognostic relevance in SLN-positive patients.Thus we now have a procedure by which the melanoma stage I-II population can be dissected in a group at truly high risk for recurrence and a group with truly low risk of recurrence. The high risk group with a greater than 75% chance for systemic disease can then be selected for trial participation of various systemic adjuvant therapy regimens that may be allowed to be toxic, considering the very high risk for relapse in these patients. The node negative group of patients can be selected for participation in trials evaluating systemic adjuvant treatment of low toxicity considering the low chance for distant metastatic disease.

Tumour necrosis factor-alpha (TNFalpha) has been found to be very effective in the isolated limb perfusion setting for advanced extremity tumours. In a phase I study of intrapleural administration of TNFalpha 5 patients were followed for inflammatory response patterns.Malignant mesothelioma patients were treated with repeated intrapleural administration of 0. 1-0.2 mg recombinant TNFalpha. Samples of serum and pleural fluid were taken at different time-points before and after TNFalpha-administration. Levels of TNFalpha, interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and secretory phospholipase A2 (sPLA2) were measured using enzyme-linked immunosorbent assays (ELISAs). Alpha 1-acid glycoprotein (alpha1-AG) was measured by nephelometry.In pleural fluid TNFalpha and IL-8 reached peak levels, up to 50-700 ng mL-1 and 6-60 ng mL-1, respectively, 24 h after administration of TNFalpha. IL-6 (peak levels up to 250 ng mL-1) and sPLA2 peaked after 48 h. A slower and less dramatic pattern was observed for the levels of CRP and alpha1-AG. In serum no detectable levels of TNFalpha and no IL-8 were observed, whereas serum levels of IL-6, sPLA2 and CRP showed a clear increase after intrapleural administration of TNFalpha. Cytokines and acute-phase proteins showed the same pattern during subsequent cycles even up to 12 cycles. Tumour regression was not observed.In the setting of a phase I study of repetitive intrapleural administration of TNFalpha in mesothelioma patients, we studied the characteristics of the inflammatory response. Intrapleural administration was followed by a clear inflammatory response locoregionally. In spite of TNFalpha peak levels as high as 700 ng mL-1 systemic levels were never detectable. The secondary cytokine response led to very high intrapleural IL-6 and IL-8 levels. Systemically IL-8 levels were never detectable whereas high IL-6 levels were induced systemically initially, with a decreased response to each intrapleural TNFalpha administration over time. The acute-phase response in contrast remained remarkably constant throughout the course of repeated intrapleural administrations of TNFalpha. Intrapleural administration of TNFalpha is well tolerated but associated with inconsistent and rather moderate impact on production of pleural fluid. This can be achieved by other simpler and cheaper treatment, thus we see no justification for further studies.

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. (C) 2000 Cancer Research Campaign.

Cytokine-based treatment regimens have been evaluated for advanced melanoma in a number of phase I and phase II trials within the last decade. Treatment with interleukin-2 (IL-2) as a single agent has resulted in response rates of approximately 15%, if a high dose of IL-2 is administered. Combination of interferon-alpha (IFNalpha) and high dose IL-2 yields response rates ranging from 10 to 41%. Response rates exceeding 50% have been reported with chemoimmunotherapy, if the treatment regimens included at least the three agents IL-2, IFNalpha and cisplatin. Recent randomized trials have evaluated the impact of these drugs on the survival of patients with advanced melanoma. The current 'state of the art' is discussed in this review.

To review the current information available from the European Organization for Research and Treatment of Cancer (EORTC) programs on the use of interleukin (IL)-2 in stage IV melanoma patients.A database from 631 patients treated within 27 trials with high-dose IL-2-based regimens was compiled to develop hypotheses and valid stratification factors for randomized trials. Subsequently, 126 patients were enrolled in a trial evaluating interferon alfa (IFN-alpha) and IL-2 with or without cisplatin, and 325 patients were enrolled in an ongoing EORTC trial (18951) to evaluate dacarbazine, cisplatin, and IFN-alpha, with or without IL-2.The database suggests long-term survival rates of 23% and a 5-year survival rate of 13% for patients receiving a combination of IFN-alpha and IL-2 with or without chemotherapy. The addition of chemotherapy improved response rate but not survival. The first randomized trial testing the role of cisplatin in a chemoimmunotherapy regimen for advanced melanoma revealed a palliative effect for cisplatin but no survival benefit. The current trial (EORTC 18951), which is testing the impact of IL-2 on survival, is still immature. In the translational research program, we have evidence that patients in continuous complete remission after IL-2-based treatment have evidence of residual disease by polymerase chain reaction assay and, at the same time, melanoma-reactive T cells are present in the peripheral blood.Mature results defining the role and, to some extent, the mechanism of IL-2 in advanced melanoma are emerging.

A multicentre study of IL2 and IFN alpha has been performed in 58 patients with metastatic melanoma. The scheme consisted of IL2 3.0 BRMP MU/m2/d as a continuous infusion for 4 d combined with subcutaneous administration of IFN alpha 6 MU/m2/d, day 1 + 4. The cycle was repeated every 2 weeks for a maximum duration of 26 weeks. 54 patients were evaluable for response. One (2%) achieved a complete and 10 (19%) a partial response. 19 (35%) patients were stable and 24 (44%) showed progressive disease. Common side-effects included fever, chills, fatigue, skin rash, anorexia, nausea and diarrhoea. Hypothyroidism was noted in 10% of the patients. These results show that this regimen of IL2 and IFN alpha is active but, in contrast to what could be expected, not superior to IL2 alone possibly due to suboptimal dosing. In an ongoing study in Rotterdam and Nijmegen, a more intense schedule was chosen, consisting of three daily i.v. doses of IL2 4.5 BRMP MU/m2 and IFN alpha 3.0 MU/m2 for 5 d. This regimen is repeated at intervals of 3 weeks for a total of three cycles. Presently, nine patients have been entered. One patient achieved a complete response, four a partial response (overall 56%), three had stable disease and one progressed. Toxicity was severe and treatment was prematurely stopped in five patients: myocardial infarction (one patient), atrial fibrillation (one patient), negative T waves and myocardial hypokinesia (one patient) and psychosis (two patients). This regimen can only be justified if the therapeutic results are superb, which has yet to be awaited.

In epidemiologic studies, sunscreen use is associated with increased risk of cutaneous melanoma, basal cell skin cancer, and higher numbers of nevi. It has been proposed that sunscreens may encourage prolonged sun exposure because they delay sunburn occurrence. We examined whether, under habitual conditions of sunscreen use, the sun-protection factor (SPF) had an influence on sun-exposure duration.Before the 1997 summer holidays, we randomly assigned 87 French and Swiss participants who were 18-24 years of age to receive an SPF 10 or an SPF 30 sunscreen. Neither medical personnel nor study participants were aware of their sunscreen assignment. Participants were asked to complete daily records of their sun exposure. To avoid influencing the recreational sun-exposure habits of the study participants, no recommendation was made about sun exposure or sun protection. Furthermore, participants were told that the trial end point was the number of pigmented skin lesions before and after the holidays. One subject was lost to follow-up. All statistical tests were two-sided.The SPF 10 (n = 44) and SPF 30 (n = 42) groups had equivalent mean holiday durations (19.4 days versus 20.2 days) and mean quantities of sunscreen used (72.3 g versus 71.6 g). The mean cumulative sun exposures for the two groups were 58.2 hours and 72.6 hours, respectively (P =.011). The mean daily durations of sunbathing were 2.6 and 3.1 hours, respectively (P =.0013), and, for outdoor activities, they were 3.6 and 3.8 hours, respectively (P =.62). There was no difference in sunburn experience between the two groups.Use of higher SPF sunscreen seems to increase the duration of recreational sun exposure of young white Europeans.

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.

In this study, we have evaluated hepatotoxicity, secondary cytokine production and hepatic acute-phase response (APR) in patients who underwent isolated hepatic perfusion (IHP) with tumour necrosis factor (TNF) alpha and melphalan for irresectable colorectal liver metastases.An extracorporeal veno-venous bypass was used to shunt blood from the lower body and intestines to the heart. Inflow catheters were placed in the hepatic artery and portal vein, and an outflow catheter in the inferior caval vein. The liver was perfused for 60 min with 0.4 mg of TNF-alpha plus 1 mg kg-1 melphalan (IHPTM group, n = 6) or 1 mg kg-1 melphalan (IHPM group, n = 3). The liver was washed with macrodex before restoring vascular continuity.After the washout procedure, a TNF-alpha peak (169 +/- 38 pg mL-1) was demonstrated in the IHPTM group only. Both groups demonstrated peak levels of interleukin 6 (IL-6) in the perfusate as well as systemically. These were significantly higher in the IHPTM group. Acute-phase protein (APP) levels followed a similar pattern as has been demonstrated after major surgery, with no significant differences between both groups. The addition of TNF-alpha to the perfusate did not lead to a significant difference in APP levels and the time course between groups.IHP with TNF and melphalan is followed by a transient systemic peak of TNF directly after liver washout. Secondary IL-6 induction was seen in the present study after IHP with and without TNF, which was highest when TNF was added. This phenomenon cannot be extrapolated to APP induction, which appeared unaffected by the addition of TNF, presumably because the surgical procedure itself already causes maximal stimulation of APP production.

The objective of the present study was to investigate the effects of isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan on circulating T cells from cancer patients using two different methods.Eight patients undergoing an ILP entered the study. At first, the number of T cells at several time points was determined using FACScan. Subsequently, production of interferon gamma (IFN-gamma) (T-helper 1) and interleukin (IL) 4 (T-helper 2) was measured at the intracytoplasmic level after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. IFN-gamma, IL-4 and IL-2 (also T-helper 1) production in the whole-blood cell culture system was then determined after stimulation with a combination of anti-CD3/anti-CD28 monoclonal antibodies.An enormous decrease in the number of circulating T cells was observed. In the remaining T-cell population cytokine production (IL-2, IL-4, IFN-gamma) was depressed, showing the same pattern in both methods. No difference could be detected between the effect of TNF-alpha and melphalan on Th1 cells and Th2 cells.The results demonstrate that TNF-alpha and melphalan reduce the number of circulating T cells and at the single-cell level decrease cytokine production in the remaining circulating T cells. No selective effect of TNF-alpha on Th1 or Th2 cells could be detected. If the impaired T-cell function is representative of all T cells remaining in the systemic circulation, this could help to explain the tolerability of high TNF concentrations after ILP, perhaps by decreasing the synthesis and production of T-cell-derived cytokines.

Isolated limb perfusion (ILP) with TNFalpha, melphalan (M), and IFNgamma results in high tumor response rates in patients with soft tissue sarcomas, melanomas and other tumors. IFNgamma can act synergistically in combination with TNFalpha but in clinical studies this has not been fully investigated. In the BN175 rat sarcoma limb perfusion model we investigated the role of IFNgamma. There were 8 different treatment groups: (I) sham ILP (n=9); (II) IFNgamma alone (n=10); (III) TNFalpha 50 microg (n=9); (IV) TNFalpha + IFNgamma (n=6); (V) melphalan (M) 40 microg (n=11); (VI) M + IFNgamma (n=6); (VII) TNFalpha + M (n=27); (VIII) TNFalpha + M + IFNgamma (n=9). Tumor response and hindlimb function were analyzed. In group I-VI no tumor regressions were observed at 5 days after ILP. ILP with TNFalpha + M had highly effective response rate (RR) of 73%; complete response (CR) rate 55%), very similar to RR in patients. Addition of IFNgamma increased the RR by 16% to 89% and the CR rate by 23% to 78%. This difference was not statistically significant. When IFNgamma was added to TNFalpha or TNFalpha + M it increased limb toxicity significantly (p

The clinical applicability of Tumor Necrosis Factor-a pi (TNF) is under renewed investigation because of its successful use in the isolated limb perfusion in patients with irresectable soft tissue extremity sarcomas. The high response rate of80% with a similarly successful limb salvage rate in this patient population has led to the submission of TNF for registration for this indication in Europe. Similarly, the agent has been shown to be successful in the isolated limb perfusion setting for tumors other than sarcomas, e.g. melanoma, carcinomas. This has caused renewed interest in TNF alpha and in its application in isolated organ perfusions, such as isolated hepatic perfusion. At the Rotterdam Cancer Center a preclinical-clinical interactive development program has been established dedicated to isolated limb, kidney, liver and lung perfusions and the application of new drugs such as TNF and TNF-mutants in these systems. Moreover a program dedicated to reduce the magnitude of surgical procedures by using occlusion balloon catheters is investigating the techniques and pharmacokinetics of procedures such as balloon catheter mediated hypoxic pelvic perfusions (HPP) and isolated hypoxic hepatic perfusions (IHHP). Here we present an overview of these developments.

Interleukin-2-activated, cultured NK cells (A-NK) cells were adoptively transferred into a syngeneic rat liver-tumor model. The kinetics of tumor infiltration by NK cells, originating either from adoptively transferred or from endogenous sources, the localization of these cells in the tumor, and their interactions with extracellular-matrix proteins were studied by immunohistochemistry and transmission-electron microscopy. The adoptive transfer of A-NK cells via the hepatic artery and s.c. injections of IL-2 into rats bearing subcapsularly induced CC531 liver tumors, but also IL-2 monotherapy, resulted in a significant increase of the number of NK cells both at the tumor border and in the tumor center. The majority of tumor-infiltrating NK cells was present in the tumor stroma and only occasionally was an NK cell observed in a tumor nodule in direct contact with tumor cells. Observations by electron microscopy suggested that matrix proteins, abundantly present in the tumor stroma but absent in the tumor nodules, provide a substrate for migration of infiltrating cells, whereas tight structures of matrix proteins surrounding tumor nodules provide a barrier for establishment of direct NK-cell-to-tumor-cell-contact. Our results suggest that direct NK-cell-to-target-cell-contact-mediated lysis is of minor importance for attaining an anti-tumor effect in this model. We hypothesize that treatment of tumor-bearing rats with A-NK cells and/or IL-2 initiates a cascade of events (e.g., secretion of tumor-killing cytokines and/or infiltration of other immune cells) ultimately leading to tumor regression.

Treatment of stage IIIA/B melanoma patients by isolated limb perfusion (ILP) with a combination of tumor necrosis factor-alpha (TNF-alpha) and melphalan induces a complete response in 80-90% of the cases. The mechanism of tumor regression induced by the combination of TNF-alpha and melphalan is not precisely understood. Previous studies focused on the immediate (ie., within a few days) clinico-pathological changes after perfusion involving hemorrhagic necrosis. However, clinical data clearly indicate that complete tumor remission frequently requires a period of a few weeks to as much as months after ILP. Because the mechanism underlying this delayed-type reaction is completely unknown, we studied the clinico-pathological events in patients with such slowly regressing melanoma lesions. For this purpose, 94 biopsies of in-transit melanoma metastasis that were taken sequentially from 11 patients between 1 week and 9 months after ILP were analyzed by light and electron microscopy and immunohistochemistry. Clinical data included patient sex, age, anatomical localization and size of the tumor, and follow-up. All of the 11 patients ultimately responded to perfusion treatment (9 complete, 1 partial, 1 stable disease). Serial biopsies showed scattered individual tumor cell necrosis without hemorrhage. Most of the lesions with this delayed-type reaction pattern were less than 0.5 cm in diameter. They contained varying amounts of histologically viable-looking tumor cells and tumor-infiltrating melanophages. In addition, a marked but transient infiltrate of peritumoral eosinophils and moderate interstitial edema and dermal fibrosis were encountered. Only small numbers of lymphocytes were present. In comparison with the reaction pattern after treatment with melphalan alone, the delayed-type reaction pattern was similar but more intense. The scattered tumor cell necrosis in the latter type may be explained by a TNF-alpha-induced increase in permeability of the tumor vascular bed, which results in higher intratumoral concentrations of melphalan or in a prolongation of its effect. Subsequently, degenerated tumor cells are cleared by macrophages, and, finally, repair by fibrosis occurs. Because the immediate reaction type is evoked by hyperpermeability of the tumor vessels as well, quantitative differences seem to determine which reaction type ensues. We suggest that the extent of tumor vasculature that is sensitive to TNF-alpha determines the onset and histopathological pattern of tumor regression after ILP.

PURPOSE Patients with primary cutaneous melanoma > or = 1.5 mm in thickness are at high risk of having regional micrometastases at the time of initial surgical treatment. A phase III international study was designed to evaluate whether prophylactic isolated limb perfusion (ILP) could prevent regional recurrence and influence survival. PATIENTS AND METHODS A total of 832 assessable patients from 16 centers entered the study; 412 were randomized to wide excision (WE) only and 420 to WE plus ILP with melphalan and mild hyperthermia. Median age was 50 years, 68% of patients were female, 79% of melanomas were located on a lower limb, and 47% had a thickness > or = 3 mm. RESULTS Median follow-up duration is 6.4 years. There was a trend for a longer disease-free interval (DFI) after ILP. The difference was significant for patients who did not undergo elective lymph node dissection (ELND). The impact of ILP was clearly on the occurrence-as first site of progression - of in-transit metastases (ITM), which were reduced from 6.6% to 3.3%, and of regional lymph node (RLN) metastases, with a reduction from 16.7% to 12.6%. There was no benefit from ILP in terms of time to distant metastasis or survival. Side effects were higher after ILP, but transient in most patients. There were two amputations for limb toxicity after ILP. CONCLUSION Prophylactic ILP with melphalan cannot be recommended as an adjunct to standard surgery in high-risk primary limb melanoma.

The acute-phase response and anaemia of chronic disease are characterized by hypoferraemia associated with an increased ferritin synthesis, which might be mediated by the activated cytokine cascade.We examined the prolonged effects of isolated limb perfusion (ILP) with recombinant human tumour necrosis factor alpha (rTNF), recombinant human interferon gamma (rIFN-gamma) and melphalan on interleukin (IL) 6 and acute-phase protein levels, iron status and serum transferrin receptor (sTfR) levels in 12 patients with melanoma or sarcoma. Patients were treated with ILP during 90 min after pretreatment with rIFN-gamma during 2 days.After ILP, leakage of TNF resulted in systemic peak levels at 3 min followed by an increase in IL-6 with maximum levels at 4h. C-reactive protein (CRP) rose at 4 h to peak levels at day 2, whereas alpha 1-antitrypsin and alpha 1-acid glycoprotein increased to maximum levels at day 3. Albumin and transferrin levels decreased after ILP and recovered after day 2. Serum iron and sTfR levels decreased during pretreatment and after ILP to minimum levels at 8 h and day 1 respectively. This was associated with an increase in serum ferritin levels, which paralleled CRP values.Our data point to a central role for the cytokine network in the modulation of iron metabolism in the acute-phase response and anaemia of chronic disease. TNF, possibly via induction of IL-6, and IFN-gamma induce hypoferraemia, which may in part result from a decrease in tissue iron release based on a primary stimulation of ferritin synthesis. The fall in sTfR levels may reflect an impaired erythroid growth and/or TfR expression mediated by TNF and IFN-gamma.

The blood residence half-life and organ distribution of recombinant human tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically stabilized liposomes, were investigated in rats bearing a soft tissue sarcoma in the hind leg. We studied the decay in blood concentration of "empty" liposomes using the aqueous marker 67gallium-desferal, as well as the blood concentration of soluble TNF-alpha and liposome encapsulated TNF-alpha using l25I. Encapsulation efficacy of TNF-alpha was 24%. The pharmacokinetics of TNF-alpha were markedly altered after encapsulation in liposomes, with a 33-fold increase in mean residence time of TNF-alpha in the blood, and a concomitant 14-fold increase in the area under the plasma concentration vs. time curve for liposomal TNF-alpha. Although the liposomes exhibit Stealth characteristics, uptake by mononuclear phagocyte-rich organs (e.g., liver and spleen) was noticeable, especially at later time points. Encapsulation of TNF-alpha in sterically stabilized liposomes resulted in a marked increase in localization of the cytokine in tumor measured as total uptake over time. However, peak TNF-alpha concentration levels in tumor were not significantly enhanced compared with free TNF-alpha. Besides the augmented localization of TNF-alpha after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed.

We report our experience with isolated hepatic perfusion (IHP) with tumor necrosis factor (TNF) and melphalan in an experimental pig study and of a phase I study in humans. IHP was performed with inflow catheters in the hepatic artery and portal vein and an outflow catheter in eh caval vein. An extracorporeal venovenous bypass was used. IHP consisted of a 60-min perfusion with hyperthermia (41 degrees C). For the pig protocol rhTNF alpha 50 micrograms/kg alone (n = 5) or rhTNF alpha 50 micrograms/kg plus melphalan 1 mg/kg (n = 3) were added. In two control pigs no drugs were added. In the phase I study, patients received melphalan 1 mg/kg with 0.4 mg rhTNF alpha (n = 8) or 0.8 mg rhTNF alpha (n = 1). After the perfusion the liver was washed with Macrodex before vascular continuity was restored. All pigs but one survived the procedure. Systemic leakage was less than 0.02%. Transient, mild liver toxicity was seen in all pigs, including controls, as demonstrated by liver enzyme assays and histology. There was no significant hemodynamic, cardiopulmonary hematological, or renal toxicity. In the phase I clinical study there was leakage in one patient (cumulative leakage 20%). There were three perioperative deaths (one possibly drug-related). All patients demonstrated significant hepatotoxicity. Survival ranged from 6 to 26 months (median 10.3 months). All patients demonstrated a tumor response (partial response 5/6, 1/6 stable disease) with a median duration of 18 weeks. In contrast to our pig program, many problems were encountered in the phase I study. By using both the hepatic artery and portal vein for IHP we encountered more toxicity than expected based on data from the pig program, resulting in fatal coagulative disturbances in one patient who received the second rhTNF alpha dose. Furthermore, local control after one IHP with TNF alpha and melphalan is only temporary.

In advanced melanoma of the limbs with in-transit metastasis, melphalan with isolated limb perfusion (M-ILP) produces around 50% complete remissions (CR). The combination of melphalan with tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in isolated limb perfusion (TIM-ILP) gives around 80% CR. A prospective randomised phase II study compared 32 patients who received TIM-ILP with 32 patients who received TM-ILP (without IFNgamma). The overall remission rate (ORR) and the CR rate were superior with TIM-ILP as compared to TM-ILP, 100% vs. 91% and 78% vs. 69% respectively, but the differences are not significant. Given the efficacy of M-ILP on in-transit metastasis, the procedure was tested as an adjunct to surgery in high-risk (Breslowor = 1.5 mm) primary melanoma of the limbs. Through the combined effort of the melanoma groups of the European Organization for Research and Treatment of Cancer (EORTC), the World Health Organization (WHO), and the North American Perfusion Group, 832 evaluable patients from 16 centres were entered in a phase III study. Median followup is 6.4 years. There was a trend for a longer disease-free interval after M-ILP. The difference is significant if the patients without elective lymph node dissection (ELND) are separately analysed, with a high significance in the 1.5 to 3 mm thickness subgroup. The occurrence of in-transit metastases was reduced from 6.6% to 3.3% by M-ILP. There was, however, no benefit of M-ILP in terms of survival. Prophylactic M-ILP cannot be recommended as a standard adjunct to surgery in high-risk primary limb melanoma. TIM-ILP or TM-ILP is a regional therapy with a very high regional response rate on melanoma in-transit metastasis.

The clinical success of the application of tumor necrosis factor-alpha (TNFalpha) in isolated limb perfusions in patients with advanced sarcomas, melanomas and other tumors has renewed the interest in this agent as an anticancer drug. At the Rotterdam Cancer Center, we have developed an interactive preclinical-clinical TNFalpha program that explores new methods to use TNFalpha in various settings. Regional organ perfusion models were developed and the effectivity of targeting of TNFalpha to the tumor by means of systemic administration of liposomes are tested. Furthermore various drugs and mechanisms that may enhance the activity of TNFalpha are under investigation. A summary of this comprehensive program is presented here.

Hyperthermic isolated limb perfusion (HILP) with various chemotherapeutic agents has been used for the local treatment of high-grade soft tissue sarcomas (STS) of the extremities, but in most cases, with a disappointing result. Most such regimens certainly should not be considered superior to surgery plus radiotherapy. Although the majority of extremity STS can be resected locally, some are very large and are in close proximity to bone, nerve or blood vessels. In these cases, amputation is the only means of resecting the tumour. A new combination of drugs used in the set-up of HILP with tumour necrosis factor-alpha and melphalan has emerged as a very promising option for the limb-saving management of locally advanced STS. In recent studies, complete response rates of approximately 30% and partial remission rates of 50% have been achieved, while the overall limb-salvage rate is more than 80%.

A syngeneic rat liver metastasis model, the CC531 colon carcinoma cell line in Wag rats, was used to study the homing properties and anti-tumor effects of adoptively transferred, interleukin-2 (IL-2)-activated, cultured natural killer (A-NK) cells. To identify the route of administration that gives the highest tumor infiltration, 1.5 x 10(8) A-NK cells were dyed with fluorescent rhodamine and injected via 4 different routes into rats, bearing subcapsularly induced (day 10) liver metastases. The routes chosen were: jugular vein, portal vein, hepatic artery and directly into the peritoneal cavity (i.p). The rats were sacrificed 20 hr after administration of A-NK cells. The highest (p0.05) infiltration of tumors by A-NK cells was found both at the tumor border and in the tumor center after injection via the hepatic artery: 65 +/- 7 A-NK cells/mm2 at the tumor border and 26 +/- 14 A-NK cells/mm2 in the center of the tumor (jugular vein infusion: 32 +/- 10 and 9 +/- 5 A-NK cells/mm2, respectively; portal vein infusion: 36 +/- 13 and 7 +/- 4 A-NK cells/mm2, respectively). No A-NK cells were detected in the liver after i.p. injection. Rats bearing day 5 tumors were injected with 1.5 x 10(8) A-NK cells via the hepatic artery or via the jugular vein (n = 5 and n = 6 respectively). Regional administration of A-NK cells via the hepatic artery resulted in a significant (p0.05) lower weight (35 +/- 23 mg) of tumors than did systemic administration (70 +/- 10 mg). Our results suggest that both the level of tumor infiltration by adoptively transferred A-NK cells and the therapeutic outcome depend on the route of administration.

The clinical success of the application of tumor necrosis factor-alpha (TNFalpha) in isolated limb perfusions in patients with advanced sarcomas, melanomas and other tumors has renewed the interest in this agent as an anticancer drug. At the Rotterdam Cancer Center, we have developed an interactive preclinical-clinical TNFalpha program that explores new methods to use TNFalpha in various settings. Regional organ perfusion models were developed and the effectivity of targeting of TNFalpha to the tumor by means of systemic administration of liposomes are tested. Furthermore various drugs and mechanisms that may enhance the activity of TNFalpha are under investigation. A summary of this comprehensive program is presented here.

The unique property of high dose recombinant tumor necrosis factor alpha (rTNF alpha) is to activate and selectively destroy the tumor-associated microvasculature. For the systemic application of rTNF alpha it has been shown that the maximum tolerated dose (MTD) is 10 times less than the effective dose in animals. The main toxicity corresponds to systemic inflammatory response syndrome with a decrease in vascular resistance and hypotension. We found that it is possible to administer rTNF alpha at 10 times the MTD in an isolated limb perfusion (ILP) system with heart-lung machine, for locally advanced extremity soft tissue sarcomas. One hundred forty patients received an ILP with high-dose TNF alpha. In 55 patients treated with the combination of high-dose rTNF alpha + interferon-gamma + melphalan an overall objective response rate of 87% with 36% complete responses was observed; it was 81% and 28%, respectively, in a group treated with TNF alpha and melphalan (n = 85). Angiographic and immunohistological studies showed the selective and early damage of the sarcoma-associated microvasculature preceded by the upregulation of adhesion molecules and intratumoral leak of von Willebrand factor. Tumor invasion by platelets and, in some cases, by polymorphonuclear cells, appeared within hours after the application of rTNFa long before the lysis of the tumor. Thus, ILP with high-dose TNF alpha and chemotherapy seems to act through a dual targeting: TNF hits the tumor associated vasculature, and chemotherapy attacks the tumor cells. Therefore, ILP with TNF is a new option in the management of locally advanced soft tissue sarcoma of the extremities.

Various adjuvant therapy trials in high risk melanoma are at present ongoing in the world. Interferon-alpha (IFN-alpha) and ganglioside vaccines are currently being evaluated on both sides of the Atlantic Ocean. In Europe, the two largest randomized trials are being carried out by the EORTC Melanoma Cooperative Group. In patients with a moderate risk (about 35%) of having micrometastatic disease, EORTC trial 18961 will investigate placebo versus vaccination with the ganglioside GM2 vaccine (GM2-KLH/QS-21) in 1000 patients with stage IIA melanoma (T3N0M0). This trial will be started during spring 1997. In stage IIB-IIIB (T4N0M0-TxN1-2M0) patients with a very high risk (about 80%) of having micrometastatic disease, EORTC trial 18952 (1000 patients) will investigate placebo versus adjuvant therapy with high or low intermediate doses of IFN-alpha2b (10 MU subcutaneously every day, 5 days per week for 4 weeks, followed by either 10 MU subcutaneously, three times weekly for 1 year, or 5 MU subcutaneously, three times weekly for 2 years). This review will discuss the trials and the philosophy of the programme, which allows more toxic treatments to be investigated in patients with a very high risk, and only treatments with little or no toxicity to be evaluated in patients with only a modest risk of disease. Other recently completed and ongoing trials are also discussed. From all programmes it is clear that in the case of medical efficacy, the toxicity, impact on quality of life, and costs of the treatment determine whether a treatment will be accepted and implemented.

Tumor necrosis factor alpha (TNF-alpha) is thought to have a key role in metabolic changes of muscle tissue during inflammatory diseases. It is unknown whether TNF-alpha affects muscle metabolism directly or whether these changes are mediated by secondary mediators. We studied 6 patients undergoing isolated limb perfusion with TNF-alpha for irresectable soft-tissue sarcoma or in-transit melanomas. Glucose, lactate, ammonia and amino-acid consumption or production were measured in the perfusate during 3 perfusion periods: before, after TNF-alpha and after the combined administration of TNF alpha and melphalan. Arterial glucose, lactate, ammonia and amino-acid concentrations were monitored to detect metabolic effects of TNF-alpha after it entered the systemic circulation. Glucose uptake and lactate release by the limb remained unchanged after the injection of TNF-alpha alone, as well as after the combination of TNF-alpha and melphalan. Furthermore, glutamine, alanine, phenylalanine, tyrosine and total amino-acid release into the perfusate did not increase during TNF-alpha and melphalan treatment, indicating that muscle metabolism was not changed. After the isolated limb perfusion, TNF-alpha entered the systemic circulation and induced metabolic changes resulting in a doubling of arterial lactate concentrations, decreased arterial glucose concentrations and decreased arterial amino-acid concentrations. Our study shows that regional administration of TNF-alpha alone or in combination with melphalan does not directly affect muscle glucose and protein metabolism. The data suggest that systemic metabolic changes induced by TNF-alpha are mediated through secondary, centrally produced, factors.

New developments in the field of (chemo-)immunotherapy for the management of metastatic melanoma can be devided in 1. new systemic treatments of systemic micrometastatic disease; 2. new regimens for systemic macrometastatic disease and 3. new biochemotherapeutic approaches in the field of isolated limb perfusion for the treatment of multiple intransit metastases confined to the extremities.

Sixty percent of the 6000 new cases of soft tissue sarcomas diagnosed in the United States each year occur in the extremities and are often large at the time of initial diagnosis [1]. Surgery for locally advanced extremity soft tissue sarcomas may consist of an amputation or exarticulation. Limb-sparing surgical procedures in these advanced cases, if at all possible, usually consist of extensive but still marginal resections requiring adjuvant radiation therapy to a large area. In those cases where location of the tumor dictate the sacrifice of functionally crucial muscles and or joint-spanning radiation therapy treatment may mutilate and compromise limb function considerably, some form of induction therapy prior to surgery should be considered. Preservation of the limb and limb function has become all the more important in light of evidence that amputations do not improve survival rates because the prognosis in patients with large (>5 cm) deep-seated, high-grade soft tissue sarcomas is not, or is only marginally, influenced by the extent of the procedure dealing with the primary tumor [2]. Although local recurrence rates vary according to the initial surgical procedure and improved local control has been reported to correlate with better survival [3], several recent studies have not been able to show that ‘inadequate treatment,’ that is, marginal excisions with a high risk for local recurrence, influences survival significantly [2,4-7].

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.

Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities. The mechanisms of the reported in vivo synergistic anti-tumour effects of TNF-alpha and melphalan are not precisely understood. We have developed an ILP model in the rat using a non-immunogenic sarcoma in which similar in vivo synergy is observed. The aim of this present study was to analyse the morphological substrate for this synergistic response of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumours from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tumour cells in over 80% of the cross-sections. Interstitial oedema and coagulation necrosis were observed in the remaining part of the tumour. Haemorrhage was virtually absent. TNF-alpha (n = 22) induced marked oedema, hyperaemia, vascular congestion, extravasation of erythrocytes and haemorrhagic necrosis (20-60% of the cross-sections). Oedema and haemorrhage suggested drastic alterations of permeability and integrity of the microvasculature. Using light and electron-microscopy, we observed that haemorrhage preceded generalised platelet aggregation. Therefore, we suggest that the observed platelet aggregation was the result of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, perfusion with the combination of TNF-alpha and melphalan (n = 24) showed more extensive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other groups of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more prominent at all time points after perfusion. In conclusion, our findings suggest that the enhanced anti-tumour effect after the combination of TNF-alpha with melphalan results from potentiation of the TNF-alpha-induced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells. Images Figure 2 Figure 4 Figure 5

In this study the role of MHC class I antigen expression in rat natural killer (NK) cell-mediated lysis was investigated. Various rat tumor cell lines and two Adenovirus (Ad)-transformed rat cell lines were tested for their expression levels of total MHC class I and two MHC class I alleles, RT1.A and RT1.C, by flow cytometry. Their susceptibility to NK cell-mediated lysis in relation to MHC class I expression was determined by 51Cr release assays. IFN-gamma is know to increase the expression of MHC class I. Therefore target cell with and without prior IFN-gamma treatment were examined for MHC class I expression and its effect on NK lysis. An significant inverse exponential relationship was found. To investigate the effect of virus infection on MHC class I expression and target cell lysis by NK cells, rat embryonal fibroblasts (REF) were infected with cytomegalovirus (CMV) and used as target cells for NK cell-mediated lysis. Results showed that these virus-infected cells were less susceptible to NK lysis than non-infected cells. Moreover, the non-infected cells expressed less MHC class I than the infected cells, indicating that also in this case, there was an inverse correlation between MHC class I expression and susceptibility to lysis by NK cells. Subsequently, we showed that sorted subsets of predominantly CD8-positive and CD8-negative NK cells lysed a MHC class I-positive tumor cell line at the same level. This suggests that CD8 is not likely to participate as a receptor for MHC class I in NK cell-mediated lysis in a syngeneic rat model.