Evaluation of 5-FU metabolism-relating enzyme gene expression levels using quantitative real-time RT-PCR from formalin fixed parafine embedded samples

Abstract

13034 Background: We have previously reported that tumors with high orotate phosphoribosil transferase (OPRT) and low dihydropyrimidine dehydrogenase (DPD) mRNA expression levels showed remarkable sensitivity to 5-fluorouracil (5-FU) by real-time RT-PCR using fresh frozen (FF) tumor samples. However, the use of fresh frozen samples has some limitations. The use of formalin fixed parafine embedded (FFPE) samples has great advantage to apply these technologies for clinical settings. In order to investigate the feasibility of real-time RT-PCR from FFPE samples to predict sensitivitiy to 5-fluorouracil (5-FU), we investigated the gene expression levels of 5-FU metabolism-relating enzymes by real-time RT-PCR from FFPE samples and compared the results from FF samples in gastric and colorectal cancer. Methods: FFPE samples and FF samples were obtained from 46 patients with gastric cancer and 29 patients with colorectal cancer. Gene expression levels of tymidylate synthase (TS), OPRT, thymidine phosphorylase (TP) and DPD were determined by quantitative real-time RT-PCR. In FFPE samples tumor tissue was obtained using laser captured microdissection (LCM). Tumor sensitivity to 5-FU was evaluated by in vitro ATP assay. Results: Gene expression levels of TS, TP, DPD determined from FFPE samples significantly correlated with those from FF samples. Although respective gene expression levels alone failed to show signifcianct correlation with the in vitro 5-FU sensitivity, statistically significant correlation was noted either from the samples of FF or FFPE in both gastric (FF: r = 0.660, FFPE: r = 0.780) and colorectal cancer (FF: r = 0.780, FFPE: r = 0.660), when OPRT/DPD mRNA ratio was applied for comparison with the results of 5-FU sensitivity. Thus, high OPRT/DPD ratio determined from FFPE samples resulted in high sensitivity to 5-FU. Conclusions: From these results, it is suggested that sensitivity to 5-FU is predictable by quantitative RT-PCR using FFPE samples. Measurement of 5-FU metabolism-relating enzyme gene expression level from FFPE samples appeared to be feasible for predicting 5-FU sensitivity and to have great advantage to apply the molecular predicting assay in clinical settings. [Table: see text]