Thymidylate synthase gene expression in solid tumors predicts for response to pemetrexed in vitro

Abstract

13058 Background: Pemetrexed (P) is a novel antifolate which targets thymidilate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The aim of the present study was to identify gene expression thresholds for these enzymes in human tumor specimens in order to separate P-sensitive from P-resistant patients. Methods: Soft-agar cloning assays were performed on freshly biopsied tumor cells exposed one hour to clinically achievable concentrations of P. In parallel, RNA was isolated, transcribed to cDNA and subsequently used for multiplex real-time PCR. Gene expression data were normalized against beta-actin transcripts followed by correlation against cloning assay results. Iterative calculations (fourfold analysis) were done for each enzyme separately to find the best cutoff for prediction of sensitivity to P. Results: Sensitive and resistant tumor samples were statistically significant different in gene expression of TS, DHFR, and GARFT (p < 0.003). 81% of all tumors with a TS copy number < 144 (related to 104 copies β-actin) were sensitive to P in vitro. (specificity = 0.69; chi2 = 14.14). Statistical tests demonstrated that gene expression of TS, DHFR, and GARFT are dependent variables and that TS transcription is the leading variable. The combination of TS, DHFR, and GARFT expression data was not superior to TS alone. Conclusions: TS expression is the most meaningful predictor for sensitivity (≤ 144 copies) or resistance (> 144 copies) to Pemetrexed in fresh tumor tissue. This observation forms a rationale for clinical trials using TS expression as predictor for clinical response. No significant financial relationships to disclose.