Alterations of Circulating Bone Marrow–Derived VEGFR-2(+) Progenitor Cells in Isolated Limb Perfusion With or Without rhTNF-α

BACKGROUND: Circulating endothelial progenitor cells (cEPCs) as recruited to the angiogenic vascular system of malignant tumors have been proposed as a biomarker in malignancies. The effect of antitumor chemotherapy on cEPCs is not fully understood. We examined the level of cEPCs, vascular endothelial growth factor (VEGF), and angiopoietin-2 in the blood of sarcoma and melanoma patients before and after isolated limb perfusion (ILP) with or without recombinant human tumor necrosis factor-α (rhTNF-α). METHODS: Twenty-two patients, 11 each with soft tissue sarcoma or recurrent melanoma of the limb, were recruited. ILP was performed with rhTNF-α/melphalan (TNF) or melphalan only (no TNF). Fifteen healthy volunteers served as control subjects. Blood was sampled before and up to 6 weeks after ILP. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and annexin V-negative cells were characterized as cEPCs by triple staining for CD133(+), CD34, and VEGFR-2(+). RESULTS: Before treatment, cEPC numbers were significantly increased in sarcoma (0.179 ± 0.190 %) and melanoma patients (0.110 ± 0.073 %) versus healthy controls (0.025 ± 0.018 %; P < 0.01), but did not differ significantly between sarcoma and melanoma patients. cEPC decreased significantly after ILP in patients with no TNF compared to pretreatment values (P < 0.05) and were significantly lower at 4 h, 48 h, and 1 week compared to ILP with TNF (P < 0.05). Values 6 weeks after ILP were significantly lower than before ILP in both investigated groups (P < 0.01). CONCLUSIONS: ILP with TNF results in activation of bone marrow–derived EPCs compared to ILP without TNF. Alteration of cEPCs and angiopoietin-2 by rhTNF-α might account for the cytotoxicity and hemorrhagic effects on tumor vessels during limb perfusion procedures. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1245/s10434-012-2637-3) contains supplementary material, which is available to authorized users.