Synergistic effects of TNF-α and melphalan in an isolated limb perfusion model of rat sarcoma: a histopathological, immunohistochemical and electron microscopical study

Abstract

Summary Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-a) 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-a 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-cx 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-oa (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-a 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-ax 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-a with melphalan results from potentiation of the TNF-oa-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.Keywords: tumour necrosis factor alpha; melphalan; isolated limb perfusion; sarcoma; platelets Isolated limb perfusion (ILP) with TNF-a and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities (Lienard et al., 1992. ILP involves isolation of the diseased limb, its connection to a heart-lung machine and the administration of a triple drug regimen at 39-40°C (mild hyperthermia) based on the reported synergism of TNF-a with melphalan and interferon-y (IFN-y) (Eggermont et al., 1993). The rationale of ILP is to improve the response rate by increasing the drug concentration while avoiding systemic toxicity. Morphological and immunohistochemical analysis of biopsies from patients after ILP suggested that the tumour mic...

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

“…Moreover, histopathologic observations (Nooijen et al, 1996a) closely resemble observations in patients (Renard et al, 1994;Nooijen et al, 1996b). TNF alone is not active (Posner et al, 1994) and melphalan is only marginally active in soft tissue sarcoma (Kremetz et al, 1977;Klaase et al, 1989), whereas the combination results in very high response rates in this rat model just as in melanoma patients (Liénard et al, 1992;Fraker et al, 1996) or soft tissue sarcoma patients .…”

“…Therefore, a tumour model with a highly aggressive non-immunogenic soft tissue sarcoma in BN rats was developed in our laboratory to address these questions (Manusama et al, 1996). Response after ILP with melphalan and TNF in this model correspond well to what is observed in sarcoma patients in terms of synergy between TNF and melphalan, response rate, and histopathological observations (Manusama et al, 1996;Nooijen et al, 1996a). This rat model could therefore serve as a credible model to study mechanisms and determine ways to optimize ILP efficacy for the clinical setting.…”

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-α-based isolated hepatic perfusion

“…These data indicate strongly that in vivo indirect mechanisms mediated by TNF-a in combination with melphalan determine antitumour effects in IHP. Our data support the notion that this indirect mechanism is the selective destructive effect of TNF-a on the tumour-associated vessels, thereby increasing vascular permeability (Nooijen et al, 1996;Ruegg et al, 1998). To investigate this hypothesis, the melphalan uptake in liver and tumour tissue was measured after IHP with or without TNF-a.…”

“…In our preclinical ILP model, we observed drastic alterations in tumour microvasculature integrity (Nooijen et al, 1996). Rüegg et al (1998) demonstrated elegantly that TNF-a in combination with IFN-g induced functional downregulation of avb3, resulting in detachment of the endothelial cells of the tumour vasculature.…”

In Vitro Cytotoxicity of Recombinant Human-TNF-.ALPHA. and Actinomycin D on Canine Normal and Tumor Cells.

“…TNF has been known to show marked anticancer effects on certain transplantable tumors [4]. The potential mechanisms of TNF are the vascular changes in tumor tissues [22,30], the modulation of host immunity against tumors [13,24] and the direct cytotoxicity on tumor cells by inducing apoptosis [5,29].…”