BMC Cancer 2009, 9:421 http://www.biomedcentral.com/1471-2407/9/421 lected before second (BV2) and third (BV3) cycles of chemotherapy from 42 patients receiving platinum-based chemotherapy. Within one hour, samples were centri- fuged (2500 g, 10 min), and the plasma was removed, aliquoted and stored at -80°C until analysis. The VEGF was assayed by commercially available sandwich enzyme- linked immunosorbent assay kits (Calbiochem, Darm- stadt, Germany) according to the manufacturer's instruc- tions. The limit of sensitivity of the VEGF assay was 9.0 pg/ mL. The coefficient of variation was less than 5.0%. The study was approved by the institute's ethics committee. Informed written consent was obtained from all the sub- jects. In the present study, we compared plasma VEGF levels in patients with progressive disease (P) with those having remission and stable disease (R+NC) to predict disease progression (evaluation 1). Also, plasma VEGF levels in patients with remission (R) were compared with those having progressive and stable disease (P+NC) in an effort to predict insufficient response to therapy (evaluation 2). Statistical analysis To test the association of therapy at time of staging inves- tigations after 3rd cycle of chemotherapy with overall sur- vival of the patients, Kaplan-Meier curves and log-rank analyses were established for various response groups. Concerning the biochemical variables, the baseline values of all markers before the first, second, and third cycles (BV1, BV2, and BV3) and the percentage changes (BV1-2 and BV1-3) were considered for statistical analyses. In a first step, all biomarkers were evaluated on their power to univariately discriminate between (a) patients with pro- gression (P) and non-progression (R + NC; evaluation 1) as well as between (b) patients with remission (R) and non-remission (P + NC; evaluation 2) by Wilcoxon test. To identify the diagnostic biomarker for insufficient ther- apeutic efficacy and progression, receiver operating char- acteristic curves and corresponding areas under the curve (AUC) was calculated. In addition, sensitivity and specifi- Table 1: Characteristics of the subjects Characteristics Age (years), median (range)‡ Sex# Male Female Smoking status† Never smokers Smokers VEGF levels (pg/mL) * Median (25-75% quartile) ‡p = 0.642;# p = 0.009;† p = 0.026; * p < 0.0001 city (with 95% confidence interval) was calculated at defined cut-off VEGF levels for detecting insufficient ther- apeutic efficacy and progression. Demographic information between cases and controls was compared using unpaired t-test for continuous varia- bles and Chi-square test for categorical variables. The comparison of the different patient groups and the circu- lating VEGF variables was performed using unpaired t- test. All statistical comparisons for VEGF were performed with logarithmically transformed data as VEGF-values were not distributed normally. Median and interquartile ranges for VEGF is given because of their skewed distribu- tions. p value < 0.05 was considered statistically signifi- cant. All statistical analyses were performed using SPSS software program for Windows (SPSS 9.1; STATA Corpo- ration, Texas, 77845, USA). Results Subject characteristics The general characteristics of the patients and controls are given in table 1. The median age was 56.5 years (range: 33-85) in non-small cell lung cancer (NSCLC) patients and 56.5 years (range: 35-86) in controls. One hundred and twenty one (90.3%) patients were male, while sev- enty eight (78.0%) controls were male. Twenty six (19.4%) patients and 33 (33.0%) controls were never smokers. The demographic characteristics of NSCLC patients and controls were significantly different for sex and smoking status. The characteristics of patients with NSCLC, like performance status, histology, stage, tumor size, nodal status, presence of metastasis, smoking and tobacco habits are given in table 2. Briefly, there were 103 patients with squamous cell carcinoma, and 31 with ade- nocarcinoma. According to TNM classification, 87 patients had stage III disease and 47 patients had stage IV disease. Correlation of patient's characteristics with survival At staging investigations after third cycle of chemother- apy, 16 (38.1%) of the 42 patients had partial remission, Cases (n = 134) 56.5 (33-85) 121 (90.3%) 13 (9.7%) 26 (19.4%) 108 (80.6%) 265.7 (155.7-408.0) Controls (n = 100) 56.5 (35-86) 78 (78.0%) 22 (22.0%) 33 (33.0%) 67 (67.0%) 74.5 (40.3-132.0) Page 3 of 12 (page number not for citation purposes)