Sample collection and genotyping
For each subject, a 5 ml peripheral blood sample was collected in EDTA anticoagulant tubes. The sample was centrifuged, and then the plasma was replaced with an equal amount of physiological solution (0.9% sodium chloride) and stored at -80°C. Genomic DNA was extracted from whole blood using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), in accordance with the manufacturer’s instructions. For the evaluation of selected SNPs in angiogenesis-related genes, DNA samples of 34 HCC patients were genotyped using the TaqMan allelic discrimination method (StepOne Plus™ Real-Time PCR System, Applied Biosystems, Monza, Italy) using commercial (VEGF-A rs2010963, VEGF-C rs4604006, HIF-1Α rs12434438, ANGP2 rs55633437, NOS3 rs2070744) genotyping assays (ThermoFisher Scientific, Waltham, MA, USA). DMET Plus assay (ThermoFisher Scientific, Waltham, MA, USA.) was performed as previously described.19-21