SYNERGISTIC EFFECTS OF TNF-ALPHA AND MELPHALAN IN AN ISOLATED LIMB PERFUSION MODEL OF RAT SARCOMA - A HISTOPATHOLOGICAL, IMMUNOHISTOCHEMICAL AND ELECTRON-MICROSCOPIC STUDY

Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-a lpha) and melphalan has shown impressive results in patients with irre sectable soft tissue sarcomas and stage III melanoma of the extremitie s. The mechanisms of the reported in vivo synergistic anti-tumour effe cts of TNF-alpha adn melphalan are not precisely understood. We have d eveloped and ILP model in the rat using a non-immunogenic sarcoma in w hich similar in vivo synergy is observed. The aim of this present stud y was to analyse the morphological substrate for this synergistic resp onse of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumors from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tu mour 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 alternations of permeability and integri ty of the microvasculature. Using light and electron-microscopy, we ob served that haemorrhage preceded generalised platelet aggregation. The refore, we suggest that the observed platelet aggregation was the resu lt of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, prefusion with the combination of TNF-alpha and melphalan (n = 24) showed more extens ive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other grou ps of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more pro minent 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-i nduced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells.