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Chapter 470Statistical analysisBased on a hypothesized 1.5 fold improvement in the RR from an anticipated 33% in the non-supplemented arm to 50% in the vitamin supplemented arm, a sample size of 82 patients (41 per study arm) was required. If the true RR difference between the study regimens would be %u00b315%, there would be an approximate 90% probability of selecting the true superior arm. The unpaired t-test was used to compare RR and the stratified log rank test was used to compare survival rates between treatment groups. An intention to treat analysis was used for TTP and OS. OS and TTP were calculated using Kaplan-Meier estimates. The correlation between homocysteine levels in both treatment groups was determined with a 2-tailed t-test. P values < 0.05 were considered statistically significant.ResultsHigh folate status increases sensitivity to cisplatinUnder normal (high folate) cell culture conditions CaCo2 cells were more sensitive to cisplatin than WiDr cells (IC50 1.2 %u00b1 0.2 %u00b5M for CaCo2 vs. 6.4 %u00b1 0.5 %u00b5M for WiDr, means %u00b1 SEM, p<0.001). However, when cultured under low folate (LF) conditions these cell lines were 2-5 fold less sensitive to cisplatin (IC50 for CaCo2-LF sublines CaCo2-LF/LV, 5.6 %u00b1 0.5 %u00b5M; p<0.01 and CaCo2-LF/FA 3.4 %u00b1 0.3 %u00b5M; p<0.02), and for WiDr/LF 10.1 %u00b1 0.1 %u00b5M; p<0.02) as shown in figure 1. Addition of gemcitabine to cisplatin resulted in a slight increase in cisplatin sensitivity, but the difference in IC50 between high and low folate containing medium remained the same.In order to investigate the mechanism behind the observed difference in cisplatin sensitivity of tumor cells cultured in medium containing different folate concentrations, tumor cells were exposed to 20 %u00b5M cisplatin for 24 hr. WiDr cells accumulated more platinum than WiDr-LF cells (50 %u00b1 0.5 vs 28 %u00b1 3 pmol/106 cells, respectively; p<0.0001), while CaCo2 cells (96 %u00b1 16 pmol/106 cells) accumulated more platinum than CaCo2-LF/LV (58 %u00b1 8 pmol/106 cells; p<0.05) or CaCo2-LF/FA (44 %u00b1 6 pmol/106 cells; p<0.02). Co-incubation with gemcitabine resulted in a slight increase in cisplatin accumulation, especially under LF conditions (not shown). Overall, these data demonstrate that high folate conditions can increase sensitivity of adenocarcinoma cells to cisplatin, and that this is associated with higher intratumoral platinum accumulation, providing a rationale for supplementation of patients with folates.