INTRODUCTION:Expression of the DLL4 (a notch pathway ligand) by tumor-associated endothelium is a postulated marker of vascular maturity and functionality. As vascular functionality is an important parameter defining chemotherapy and oxygen intra-tumoral distribution, we investigated the role of DLL4 expression in tumour vasculature in the efficacy of radio-chemotherapy for HNSCC patients. MATERIALS AND METHODS:Sixty-five biopsy specimens from HNSCC patients with inoperable disease were immunohistochemically examined using anti-CD31 (pan-endothelial cell marker) and anti-DLL4 antibodies and the vascular density (VD) was recorded. Patients were treated with platinum based hypofractionated accelerated conformal radiotherapy. The median follow-up period was 24 months (4-80 months). RESULTS:Using the 33rd and 66th percentiles cases were grouped in three categories of low, medium and high CD31+ or DLL4+ VD. The percentage of vessels expressing DLL4 (DLL4-ratio) ranged from 17 to 100 % (mean 71 %), showing substantial variation among cases. In accordance with previous published studies, a biphasic pattern of association of CD31+ VD with poor outcome was noted. Cases with a medium VD had a significantly better local relapse free survival (LRFS) compared to cases of high VD (p = 0.0005, HR 0.15) and of low VD (p = 0.02, HR 0.28). High DLL4/CD31 ratio defined improved LRFS in both these subgroups of poor prognosis. CONCLUSIONS:The expression of DLL4 is associated with reduced radio-resistance, presumably by reducing hypoxia and improving chemotherapy accessibility. Using the combination of CD31 and DLL4 staining, a classification is suggested so that HNSCCs are categorized in sub-groups to be targeted by different anti-angiogenic and hypoxia targeting agents.