BMC Cancer 2009, 9:421 http://www.biomedcentral.com/1471-2407/9/421 Table 4: Value distribution of VEGF in various response groups of NSCLC patients during first-line chemotherapy Biomarkers (unit) Remission median (range) VEGF BV1 (pg/mL) 364.0 (136.6-868.0) VEGF BV2 (pg/mL) 144.0 (34.6-588.4) VEGF BV3 (pg/mL) 119.0 (26.0-571.0) VEGF BV1-2 (Dec%) 41.6 (2.2-86.8) VEGF BV1-3 (Dec%) 63.0 (17.2-93.1) VEGF BV2-3 (Dec%) 12.7 (-62.4-87.1) No Change median (range) 180.5 (70.6-546.0) 237.8 (52.1-431.0) 269.7 (51.0-526.0) -6.6 (-90.6-64.5) -3.2 (-142.2-53.0) -21.2 (-33.6-33.8) Progression median (range) 286.4 (72.6-522.6) 325.2 (158.4-612.1) 402.8 (174.6-811.0) -18.5 (-118.2-2.7) -41.0 (-140.5- -17.8) -15.3 (-66.3-5.3) BV1, BV2, and BV3, baseline VEGF levels before therapy cycle 1 to 3, respectively; BV1-2, BV1-3 and BV2-3, kinetics of baseline VEGF levels from cycles 1 to 2, 1 to 3 and 2 to 3, respectively; Dec%, decrease in percent (negative numbers meaning increase of the VEGF levels). P values by Wilcoxon test indicate differences between patients with progressive disease (P) and those having remission and stable disease (R+NC; evaluation 1) as well as between patients with remission (R) and those having progressive and stable disease (P+NC; evaluation 2). P R+NC vs P R vs NC+P 0.589 0.001 0.001 0.0001 0.0001 0.0268 0.083 0.016 0.001 0.0001 0.0001 0.0052 When the VEGF levels at BV1, BV2 and BV3 were com- pared between R+NC versus P (evaluation 1) and R versus NC+P (evaluation 2), it was observed that pre-treatment VEGF levels (BV1) could not predict insufficient response to therapy and disease progression (Table 4). However, baseline VEGF levels before cycle 2 (BV2) and cycle 3 (BV3) were significantly able to predict insufficient response to therapy and disease progression. Addition- ally, VEGF levels showed stronger decrease from cycle 1 to 2 (BV1-2), from cycle 1 to 3 (BV1-3), and from cycle 2 to 3 (BV2-3) in patients with remission than with no change or progression (Table 4 and Figure 2). The fraction of patients with lower VEGF levels at cycle 2 (BV2) compared with at cycle 1 (BV1) in the various groups were 87.5% (R), 41.7% (NC), and 14.3% (P) and at cycle 3 (BV3) were 100% (R), 50% (NC) and 7.1% (P), respectively. VEGF levels during chemotherapy for predicting insufficient response to therapy and disease progression To test the potential of VEGF levels as a biomarker for the prediction of insufficient therapy response, we plotted receiver-operating characteristics (ROC) curves for the val- ues available before first (BV1), second (BV2) and third (BV3) therapy cycle (Figure 3). We did not consider plasma VEGF before cycle 1 (BV1) for predicting insuffi- cient therapy response because of very low area under the ROC curve (AUC = 0.339; standard error = 0.09; 95% CI = 0.157-0.520). The cut-off value of plasma VEGF levels before therapy cycle 2 (BV2) was determined as 232.1 pg/ mL using the ROC analysis (area under the curve = 0.724; standard error = 0.087; 95% CI = 0.553-0.893) According to this cut-off value, we could predict insufficient therapy response with a sensitivity and specificity of 76.9% (95% CI = 55.9-90.2) and 75.0% (95% CI = 47.4-91.7), respec- tively (Table 5). Interestingly, we observed similar sensi- tivity and specificity for plasma VEGF levels available before therapy cycle 3 (BV3; area under the ROC curve = 0.817; standard error = 0.070; 95% CI = 0.679-0.956). For the specific prediction of insufficient response to therapy, we observed very low sensitivities of 3.8% and 11.5% at 100% specificity for the VEGF levels available before sec- ond and third cycles of chemotherapy, respectively We also tested the relevance of VEGF levels available before first (BV1), second (BV2), and third (BV3) therapy cycle in predicting disease progression (Figure 4). Because of low value of plasma VEGF before cycle 1 (BV1) under the ROC curve, we did not consider it for predicting dis- ease progression (AUC = 0.552; standard error = 0.08; 95% CI = 0.376-0.729). However, plasma VEGF at BV2 and BV3 most efficiently identified patients with disease progression with area under the ROC curve being 0.805 (standard error = 0.066; 95% CI = 0.675-0.935) and 0.895 (standard error = 0.05; 95% CI = 0.797-0.993), respec- tively (Figure 4). At a cut-off of 312.6 pg/mL, determined from ROC curve for VEGF at BV2, we observed a sensitiv- ity of 71.4% (95% CI = 42.0-90.4) and specificity of 71.4% (95% CI = 51.1-86.0) for predicting disease pro- gression (Table 5). Further, at 355.4 pg/mL cut-off value for VEGF at BV3, a sensitivity of 85.7% (95% CI = 56.2- 97.5) and specificity of 89.3% (95% CI = 70.6-97.2) was achieved (Table 5). For the specific prediction of disease progression, we observed very low sensitivities of 7.1% and 21.4% at 100% specificity for the VEGF levels availa- ble before second and third cycles of chemotherapy, respectively. Circulating plasma VEGF levels as a tumor marker: calculating specificity and sensitivity using receiver- operating characteristics (ROC) curve The median plasma VEGF level (25-75th quartile) was 265.7 pg/mL (155.7-408.0 pg/mL) in patients with NSCLC (n = 134) and 74.5 pg/mL (40.3-132.0 pg/mL) in controls (n = 100). This difference in VEGF levels in case versus controls was highly significant (p < 0.0001; Table 1). Page 6 of 12 (page number not for citation purposes)