Plasma samples were centrifuged for 15 minutes at 3,000 rpm within 30 minutes of the col lection time. The supernatant was collected with a pipette and placed in Eppendorf tubes before freezing at 80 C. All samples were stored frozen at the Univer sity of Ioannina Cancer Biobank Center. Collection of all of the samples therefore had been completed within a period of 2 months. RNA isolation For RNA extraction, the PAXgene blood RNA extraction kit was used according to the manufacturers instructions. RNA integrity was checked by RNA electrophoresis. Only RNAs with RNA integrity number 7 were used for reverse transcription and further processing. Purified RNA samples were then subjected to quantitative analysis, using the NanoDrop ND 1000 spectophotometer, with measurements in a range of 230 to 350 nm.

A260A280 values were about 2 for all samples, indicating highly purified Inhibitors,Modulators,Libraries RNA. Qualitative ana lysis was performed with RNA gel electrophoresis 1. 2%. For reverse transcription, the SABiosciences RT2 First Strand kit was used. In all cases 0. 5 ug RNA were reversed tran scribed. Simultaneous quantification of Inhibitors,Modulators,Libraries 84 gene tran scripts involved in angiogenesis Inhibitors,Modulators,Libraries was performedusing the angiogenesis RT2 profiler PCR Array. Relative expression was determined with the LightCycler 480 instrument and the Ct method. Vascular endothelial growth factor A, fibroblast growth factor 2 and interleukin 8 protein measurements EDTA plasma samples were analyzed using commercial ELISA kits for vascular endothelial growth factor A, fibroblast growth factor 2 and CXCL8 IL8.

Aliquots of 100 ul were used for VEGFA Inhibitors,Modulators,Libraries and FGF2 and 50 ul for IL8 analysis. All analyses and calibrations were carried out in duplicate. The calibration on each microplate used recombinant VEGFA, FGF2 and IL8 standards, respectively. Optical densities were determined using a microplate Inhibitors,Modulators,Libraries reader at 450 nm. The blank was subtracted from the duplicate readings for each standard and sample. A standard curve was created by plotting the logarithm of the mean absorbance of each standard biological activity versus the logarithm of the VEGFA, FGF2 and IL8 concen trations. VEGFA, FGF2 and IL8 concentrations are reported as pgml. The sensitivity of the assay for VEGFA is 9 pgml with a detection range of 31. 2 to 2,000 pgml. The ELISA kit for CXCL8IL8 has a sensi tivity of 1. 5 to 7. 5 pgml and the reliable standard curve ranges from 31. 2 to 2,000 pgml. The Quantikine Human FGF2 immunoassay kit has a minimum detec table dose less than 3 pgml with a detection range of 10 to 640 pgml. Patient outcome All patients were assessed for local recurrence or development of metastatic disease according to stan dard follow up protocols. Duration of follow up ranged from 25 to 37 months.