Intratumoural expression of TNF-R1 and EMAP-II in relation to response of patients treated with TNF-based isolated limb perfusion

Tumour necrosis factor-alpha (TNF) has been used in the clinic for more than 10 years in an isolated limb perfusion (ILP). However, intra-tumoural expression of TNF receptor-1 (TNF-R1) and TNF-R1 upregulating factors are unknown. We determined the expression of TNF-R1, proEMAP and endothelial monocyte-activating polypeptide-II (EMAP-II) before and after ILP and evaluated this against clinical response. Tumour biopsies were taken before and after ILP of patients (n = 27) with advanced sarcoma or metastatic melanoma. Biopsies were randomly analysed by western blotting for proEMAP/EMAP-II and TNF-R1 expression. Appropriate melanoma biopsies were stained for EMAP-II, TNF-R1, CD31 and CD68. For melanomas we found that an up-regulation of EMAP-II, in contrast to proEMAP or TNF-R1, directly after ILP significantly correlated with a complete tumour response. No correlation was found for sarcoma patients. In a comparative analysis we found that the overall proEMAP and EMAP-II expression was higher in melanoma as compared to sarcoma cases and measurements in cell lines revealed high proEMAP expression by melanoma cells. We report high EMAP-II expression by endothelial cells and association with macrophages. In addition, macrophages are recruited to vessel-remnants after ILP. An upregulation of EMAP-II directly after ILP of melanoma patients correlates with and might predict a complete response to TNF-based ILP. The association of macrophages with EMAP-II expression and vascular damage suggests a role for EMAP-II in regulating the TNF-based anti-tumour effects observed with an ILP. Analysis of EMAP-II expression in melanoma biopsies should be implemented in the ILP procedure.