The capacity of thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms to predict outcome after adjuvant cyclophosphamide, methotrexate and 5-fluorouracil in patients with early breast cancer

Thymidylate synthase (1YMS) catalyses the conversion of dUMP to TMP and is the target of 5-fluorouracil (5FU). Expression of 1YMS modulates sensitivity to 5FU and polymorphisms in the TYMS gene have been associated with expression and response to 5FU. Methylenetetrahydrofolate reductase (MTHFR) catalyses the reductive methylation of 5,10-methylenetetrahydrofolate, a critical component of the complex formed when 5FU binds and inhibits 1YMS. A polymorphism of MTHFR leads to a thermolabile enzyme with reduced activity, and may alter sensitivity to 5FU. The majority of studies examining the relationship between TYMS and MTHFR and response to 5FU have been carried out in Gl cancers with conflicting results. The aim of this study was to examine the relationship between TYMS and MTHFR polymorph isms, and 1YMS protein expression and outcome, in women with early breast cancer who received adjuvant cyclophosphamide, methotrexate and 5FU (CMF). DNA was extracted from archival tumour tissue blocks, using laser capture microdissection where appropriate, from eighty-six women who received CMF, and from blood samples from 190 women who received doxorubicin and cyclophosphamide (AC). TYMS polymorphisms were analysed using PCR and restriction fragment length polymorphism techniques. PCR products were analysed by high performance capillary polyacrylamide gel electrophoresis with fluorescence detection. The MTHFR polymorphism was analysed by pyrosequencing. 1YMS protein expression was measured in a subset of CMF patients using immunohistochemistry. For patients receiving CMF, carrying a TYMS 3R allele was associated with a significantly increased risk of relapse and death (DFS HR=5.1 [95% er 1.2-21.9], p=D.O'l ; OS HR=4.3 [95% Cl 1.0-18.5], p=0.03). The TSdel6 polymorphism appeared to have some association with DFS and OS, with the 6 bp insertion (+6) homozygotes having the best outcome (p=0.08 and p=0.07, respectively). Patients who were homozygous for both the 2R and +6 alleles had significantly longer DFS (HR=30.1 [95% Cl 0.4-2400], p=O.Ol) and OS (HR=30 [95% er 0.2-3600], p=0.02) compared to all others, and none relapsed or died during the follow up period. These findings were not observed in the AC patients, indicating the differences seen in the CMF patients were treatment related. 1YMS protein expression was not associated with outcome after CMF, nor did it correlate with TS VNTR genotype, suggesting that expression was not the mechanism underlying the association between genotype and outcome. No association was observed between MTHFR C677T genotype and outcome in either the CMF or AC treated patients. The TS VNTR and TSdel6 polymorph isms yielded a significant association with outcome, and the 2R2R-+6+6 diplotype selected a group of patients who had not relapsed or died during the follow up period. The TS VNTR-deI6 diplotype may select breast cancer patients who would benefit from adjuvant CMF chemotherapy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo