Methylenetetrahydrofolate reductase (MTHFR) gene 677C4T and 1298A4C polymorphisms are associated with differential apoptosis of leukemic B cells in vitro and disease progression in chronic lymphocytic leukemia H Nu ¨ ckel 1,2 , UH Frey 2 , J Du ¨rig 1 , U Du ¨ hrsen 1 and W Siffert 2 1 Department of Hematology, Medical Faculty, University of Essen, Germany; and 2 Institute of Pharmacology, Medical Faculty, University of Essen, Germany Methylenetetrahydrofolate reductase (MTHFR) regulates the meta- bolism of folate and methionine, essential components of DNA synthesis and methylation. We investigated whether the two genetic MTHFR polymorphisms (677C4T and 1298A4C) are associated with an increased risk for chronic lymphocytic leukemia (CLL) or may predict disease progression. Moreover, we measured potential genotype effects on apoptosis of B-CLL cells. Allele frequencies and genotype distributions for both polymorphisms were not significantly different in 111 patients vs 92 healthy controls. While progression-free survival (PFS) was not significantly different in individuals with CLL including all stages, in patients with Binet stage A PFS was significantly longer in patients displaying the MTHFR 677CC (P ¼ 0.043) and the MTHFR 1298A/C or CC genotypes (P ¼ 0.019). In a multi- variate analysis, MTHFR haplotype (677CC plus 1298CC or A/C) was the best independent prognostic factor for PFS compared with other known prognostic factors. Spontaneous apoptosis of B-CLL cells in vitro was significantly increased in the favorable risk group with MTHFR 677CC and MTHFR 1298AC, which may constitute the cellular basis of the observed associations. While MTHFR polymorphisms do not affect the risk for B-CLL, they may be independent prognostic markers that influence the PFS in patients with early-stage B-CLL. Leukemia (2004) 18, 1816–1823. doi:10.1038/sj.leu.2403484 Published online 16 September 2004 Keywords: chronic lymphocytic leukemia; cancer; MTHFR; genetic polymorphism; apoptosis; folic acid Introduction B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical course. Staging systems devised by Rai 1 and Binet 2 are useful for predicting survival and treatment requirements in patients with CLL. However, these staging systems are of limited prognostic value in early stages of the disease (Binet A or Rai stages 0–II), which applies for most of the patients at first diagnosis. Therefore, a number of studies aimed at identifying novel prognostic markers, which may help define patient subgroups with favorable vs poor clinical outcome in early CLL. 3 The presence or absence of somatic mutations in the immunoglobulin heavy-chain variable region (IgVH) of B-CLL cells has been described as one of the most powerful prognostic factors, as B-CLL cases with mutated IgVH genes exhibit a favorable clinical course while B-CLL patients with unmutated Ig VH genes are characterized by a poor outcome in terms of reduced survival and responsiveness to chemotherapy. 4–8 Recently, Rosenwald et al 9 demonstrated that expression analysis of a single gene, the protein tyrosine kinase ZAP-70, could correctly predict the IgVH mutation status in 93% of patients. We could confirm previous studies by showing that ZAP-70 expression is a novel prognostic marker in B-CLL. Furthermore, our results and those of others suggest that a combination of prognostic factors such as ZAP-70 plus CD38 expression status may help optimize currently available prognostic instruments for the risk stratification of CLL patients in clinical trials. 10 A refined risk stratification of CLL patients, especially for those patients in stage A according to Binet, may in future guide more specific, tailored treatment decisions for an individual patient. Common single-nucleotide polymorphisms (SNPs) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (677C4T and 1298A4C) have been associated with the risk for adult and childhood acute lymphocytic leukemia. 11–13 The enzyme MTHFR catalyzes the reduction of 5,10- methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the carbon donor for de novo methionine synthesis and DNA methylation. Approximately 10% of Caucasian populations are homozygous for a common C4T substitution at nucleotide 677 of the MTHFR gene. This mutation converts an alanine to a valine at codon 225 and is associated with reduced enzyme activity. The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidy- lates for DNA synthesis and repair makes the MTHFR an attractive candidate for a cancer predisposing gene. 14–16 Recently, a second common polymorphism in the MTHFR gene has been reported that results in a glutamate to alanine (A4C) change at position 1298, which also leads to diminished enzyme activity. Frequencies of up to 33% have been reported previously for the 1298C allele. 17,18 The association of MTHFR variants with an increased or decreased risk for different neoplasia was reported in several studies. Homozygous 677T allele carriers may have a reduced incidence of colorectal cancer that can be further modified by dietary habits and life style. 19,20 In contrast, an increased frequency of 677T homozygotes was observed in cervical intraepithelial neoplasia, 21,22 esophageal, 23 endometrial, 24 sto- mach, 25 lung cancer 26,27 as well as breast carcinoma. 28,29 Here, we report a single-center retrospective study of 111 CLL patients designed to evaluate the potential of the MTHFR 677C4T and 1298A4C gene polymorphisms as a prognostic factor for the natural course of B-CLL in patients with Binet stage A. In addition, we report the results of a multivariate analysis of the genotype data with other important prognostic factors in B-CLL. Finally, we investigated which cellular phenotype might contribute to the observed association. Materials and methods Patients Between August 2001 and October 2003, 111 samples of Caucasian patients with CLL were enrolled in this retrospective Received 17 March 2004; accepted 15 July 2004; Published online 16 September 2004 Correspondence: Dr H Nu ¨ ckel, Department of Hematology, Institute of Pharmacology, University Hospital, Hufelandstrasse 55, D-45122 Essen, Germany; Fax: þþ 49-201-7235968; E-mail: holger.nueckel@uni-essen.de Leukemia (2004) 18, 1816–1823 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu