VWF release and platelet aggregation in human melanoma after perfusion with TNF alpha

Twenty-nine stage IIIA/B melanoma patients treated by isolated limb perfusion (ILP) with a high dose of recombinant human tumour necrosis factor alpha (rHuTNF alpha), interferon gamma (IFN gamma), and melphalan were histologically documented with emphasis on therapy-induced changes of the tumour vasculature. Sequential biopsies were taken at various intervals before and after the treatment to compare the morphological change. In order to visualize microvascular changes, immunostaining was performed for von Willebrand factor (VWF), type IV collagen, alpha-smooth muscle actin, endothelial antigen PAL-E, tissue factor, CD41 (thrombocyte marker), and fibrin. In biopsies prior to perfusion, necrosis, haemorrhage, and fibrin thrombi were not found. Within 3 h following triple combination therapy, a change in the distribution of VWF staining occurred, from a discrete endothelial pattern in the untreated lesions to a fuzzy perivascular and subepidermal pattern in the treated lesions. Within 24 h, this was accompanied by intravascular thrombocyte aggregation and erythrostasis, in the absence of tissue factor and fibrin deposits. These findings indicate that the thrombocyte aggregation observed is not caused by local procoagulant activity, but is rather the result of the therapy-associated vascular damage or haemostasis. Although it is difficult to derive the dynamics of this process from static images, we assume that TNF alpha induced endothelial cell damage, leading to VWF release. Release VWF may play a role in the adhesion between thrombocytes and the damaged endothelium or the denuded subendothelium. As a consequence, the blood flow is impaired, leading to congestion and oedema, compatible with an early stage of haemorrhagic infarction.