344 • Clinical Colorectal Cancer January 2006 Introduction The enzyme methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes the conversion of 5,10- methylenetetrahydrofolate (methyleneTHF) to 5- methyltetrahydrofolate (methylTHF), which is the major circulating form of folates. The 5-methylTHF is needed for the remethylation of homocysteine to methionine, which is linked to S-adenosylmethionine (SAM). S-adenosylmethionine, in turn, is required in several DNA methylation reactions. Abnormal DNA methylation can cause decreases or increases in the activity of the genes affected. A common polymorphism in the MTHFR gene is C677T, ie, a conversion of cytosine to thymine at nucleotide 677. This mutation results in the amino acid substitution of alanine to valine in the enzyme at codon 222, which is an enzymatically important region. 1 Individuals with CT and TT genotypes might have decreased enzyme activity 2 resulting in low cellular 5-methylTHF concentrations, 3 increased plasma homocysteine levels, and hypomethylation of DNA. 4,5 Previous studies have found a significantly decreased risk of Elisabeth Odin, 1 Yvonne Wettergren, 1 Göran Carlsson, 1 Peter V. Danenberg, 2 Angelo Termini, 1 Roger Willén, 3 Bengt Gustavsson 1 Abstract Submitted: Oct 6, 2005; Revised: Nov 11, 2005; Accepted: Jan 13, 2006 1 Department of General Surgery, Sahlgrenska University Hospital/Östra, Göteborg, Sweden 2 Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles 3 Department of Pathology and Cytology, Uppsala University Hospital, Uppsala, Sweden Contribution Original Background: The aim of the study was to investigate the influence of methylenetetrahydrofolate reductase (MTHFR) gene expression levels and MTHFR polymorphism C677T on the outcome of patients with colorectal cancer (CRC). Furthermore, we wanted to evaluate the interaction between MTHFR and thymidylate synthase (TS) and folylpolyglutamate synthase (FPGS) and to investigate the impact of folate concentration on patients with CRC with different MTHFR genotypes. Patients and Methods: The frequency of MTHFR polymorphism C677T was determined (n = 147), and gene expression levels of MTHFR, TS, and FPGS were quantified with real-time polymerase chain reaction (n = 157). Reduced folates in tissue were measured with a binding assay (n = 40). Results: We observed a significantly lower concentration of tetrahydrofolate (THF) in patients with CT or TT genotypes compared with patients having the CC genotype.Twenty-six patients with Dukes A to C tumors who had not been subjected to chemotherapy relapsed. Out of these, 18 had CT or TT genotypes, and only 8 had the CC genotype (P = 0.045). Furthermore, 75 patients did not relapse, and out of these, 35 had CT or TT genotypes, and 40 had the CC genotype.The relative gene expression level of MTHFR in patients subgrouped by CC and CT or TT genotypes was significantly lower in carcinomas compared with adjacent mucosa (P < 0.0001 and P < 0.0001, respectively). A significant difference in MTHFR expression level was also observed according to MTHFR genotype in the tumor but not in adjacent mucosa.The MTHFR gene expression level in mucosa was a prognostic parameter independent of the clinicopathologic factors with regard to survival for patients with MTHFR C677T mutation. Conclusion: Our results showed that it is possible to identify patients with CRC with a higher risk for relapse. Furthermore, patients with a mutant genotype in combination with low MTHFR expression have a poor clinical outcome. Clinical Colorectal Cancer, Vol. 5, No. 5, 344-349, 2006 Key words: C677T, Folate, Folylpolyglutamate synthase, Thymidylate synthase Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1533-0028, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400. Address for correspondence: Elisabeth Odin, Surgical-Oncology Laboratory, Göteborg University, plan1 CK, Sahlgrenska University Hospital/Östra, Göteborg, S-416 85 Sweden Fax: 46-31-3435930; e-mail: elisabeth.odin@dep-surg.gu.se Expression and Clinical Significance of Methylenetetrahydrofolate Reductase in Patients with Colorectal Cancer