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Journal of Clinical Oncology, Vol 23, No 7 (March 1), 2005: pp. 1365-1369 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.06.219 Thymidylate Synthase and Methylenetetrahydrofolate Reductase Gene Polymorphism in Normal Tissue As Predictors of Fluorouracil SensitivityFrom the Danish Colorectal Cancer Group South, Vejle Hospital, Vejle, Denmark Address reprint requests to Anders Jakobsen, MD, Department of Oncology, Vejle Hospital, 7100 Vejle, Denmark; e-mail: aja{at}vs.vejleamt.dk
PURPOSE: To analyze thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism with respect to fluorouracil (FU) sensitivity. PATIENTS AND METHODS: The study included a retrospective analysis of 88 patients with metastatic colorectal cancer and a prospective trial with 51 patients also with measurable metastases. All patients were treated with FU and leucovorin. The analysis of gene polymorphism was performed on normal intestinal tissue and lymphocytes. RESULTS: The response rate was significantly higher in patients with TS 3R/3R or MTHFR 677 TT gene polymorphism compared with the other groups. The difference of response rate translated to a difference in time to progression. Similar results were observed in the retrospective analysis and the prospective confirmatory trial. CONCLUSION: The analysis of gene polymorphism allows delineation of a group of patients (30%) with a response rate to a single drug of approximately 50%. This information should be used in the design of tailored treatment.
Despite the development of new drugs seen in recent years fluorouracil (FU) remains a cornerstone in the treatment of colorectal cancer with antineoplastic agents. The drug has been in clinical use for almost 50 years, but there is still a lack of reliable methods that can be applied for selection of patients who will have the best chance to benefit from the treatment. Different doses and schedules have been advised, but the response rate is only approximately 25%, even if supplemented by leucovorin, which improves the effect of FU.1 This means that most of the patients undergo chemotherapy without any measurable shrinkage of their tumor, and they will only experience toxicity. Therefore, there is an obvious need to identify new markers that can be used as predictors of effect and, consequently, serve as a rational basis for treatment selection. A number of recent articles have focused on thymidylate synthase (TS) gene polymorphisms. The promoter enhancer region of the TS gene is subjected to polymorphism with double or triple repeats of a 28-base pair sequence in the 5'-untranslated region.2 The different genotypes (2R/2R, 2R/3R, and 3R/3R) occur at different frequencies in different races. In the white population, heterozygosity occurs in approximately 50% of the population, and each of the two homozygous genotypes are found in approximately 25% of the population.2 The polymorphism may affect the translational efficiency of the gene3 and predict the response to FU.4 However, contradictory results have also been published.5 Methylenetetrahydrofolate reductase (MTHFR) may also be an important predictive factor of the effect of FU. The enzyme catalyzes an irreversible conversion of 5-10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The former is essential to the DNA synthesis by acting as a cofactor in the conversion of deoxyuridine monophosphate to dexoythymidine monophosphate by TS. It also stabilizes the binding of 5-fluorodeoxyuridine monophosphate to TS, resulting in prolonged inhibition of the enzyme. The MTHFR 677 CT gene polymorphism leads to an alanine to valine conversion in the protein.6 This change results in a considerable lower activity of the enzyme in heterozygotes and especially MTHFR 677 TT homozygotes compared with MTHFR 677 CC wild-type homozygotes. Consequently, an accumulation of 5-10-methylenetetrahydrofolate is observed, and increased sensitivity to FU would be expected. The MTHFR 1298 AC gene polymorphism also causes decreased enzymatic activity but to a lesser external than the MTHFR 677 CT gene polymorphism. In vitro studies have identified the MTHFR gene polymorphism as an important predictive parameter to FU treatment, but the clinical data are sparse.7 Most studies have been based on analysis of tumor tissue. For obvious reasons, it would be a major advantage to analyze readily accessible normal tissue. It would also be important to know whether the findings for tumor tissue are similar to normal tissue findings. The objective of the present study was to analyze the TS gene polymorphism in normal tissue from patients with advanced colorectal cancer together with the MTHFR 677 CT and MTHFR 1298 AC gene polymorphisms in a retrospective analysis and confirm the results in a prospective study.
The retrospective analysis included 88 patients with measurable metastatic lesions who previously entered a randomized trial comparing three different doses of bolus FU. The clinical results of this trial are reported elsewhere.8 The randomized trial included 150 patients from Denmark. Twenty patients were not assessable for response, and suitable pathologic specimens were only accessible in 88 patients. The prospective trial included 51 patients with metastatic disease. Further inclusion criteria were measurable lesions, WHO performance status  2, and informed consent. The patients were treated with FU 500 mg/m2 administered as bolus injection (intravenous push < 3 minutes) and leucovorin 60 mg/m2 administered intravenously for 30 minutes after the FU injection. The treatment was administered for 2 consecutive days every second week and continued until progression. The response was assessed every second month according to Response Evaluation Criteria in Solid Tumors Group criteria.
Gene Polymorphisms Polymerase chain reaction analysis of TS gene polymorphism was performed as previously described by Iacopetta et al.9 MTHFR 677 CT gene polymorphism was detected by restriction fragment length polymorphism and performed according to Frosst et al.6 MTHFR 1298 AC gene polymorphism was performed by restriction fragment length polymorphism as described by Skibola et al.10 In the retrospective study, the MTHFR 677 CT analysis was unsuccessful in nine patients, and the MTHFR 1298 AC analysis was unsuccessful in eight patients, leaving 79 and 80 patients available for comparison, respectively. In the prospective study, TS analysis was unsuccessful in three patients, and MTHFR 677 CT analysis was unsuccessful in two patients, leaving 48 patients available for comparison. The analyses were performed without any knowledge of the clinical results.
Statistics
Some patient and treatment characteristics are listed in Table 1. The patients in the retrospective study are comparable with the patients in the prospective study. The response rate did not differ significantly according to dose level of FU in the retrospective study, and the gene polymorphism was almost equally distributed in the three treatment groups. It also seems that 10% of patients in the retrospective study and 30% of patients in the prospective study were offered second-line treatment. The overall response rates in the retrospective and prospective studies were 33% and 31%, respectively.
Table 2 compares the TS gene polymorphism with response rate. The table shows that the frequency of homozygosity (2R/2R and 3R/3R) was identical in 24% of patients and that heterozygosity occurred in 54% of the patients. The response rates appear different. The rate is almost doubled (52%) in the 3R/3R group compared with the 2R/2R and 2R/3R groups (24% and 28%, respectively). The difference is not statistically significant, but if the 3R/3R group is compared with the two other groups taken together (2R/2R and 2R/3R), a significant difference appears (P = .03).
The MTHFR gene polymorphism is compared with response rate in Table 3. The MTHFR 677 CC and MTHFR 677 CT polymorphisms occurred with almost the same frequency (40% and 48%, respectively), but the TT polymorphism was only observed in 11% of the patients. The response rate for 677 TT was 66% compared with 33% and 21% for 677 CC and 677 CT, respectively. The difference is significant (P = .04) if 677 TT is compared with the two other groups taken together. Table 3 also lists the distribution of MTHFR 1298 AC polymorphism. It seems that only three responding patients were MTHFR 1298 CC homozygous. The figures do not indicate any obvious correlation with response. We did not find any intrapatient correlation between the 677 and 1298 MTHFR polymorphisms.
There was no correlation between TS and MTHFR homozygosity. Only three patients had both a 3R/3R TS gene polymorphism and a 677 TT MTHFR gene polymorphism. All three patients responded to treatment. Furthermore, there was no overlap between the TS 3R/3R and MTHFR 1298 AC polymorphism. However, 27 patients (30%) had either the TS 3R/3R or MTHFR 677 TT gene polymorphism. These patients had a response rate of 52% compared with 25% in all other patients (Table 4). This difference is highly significant (P = .012).
The time to progression according to TS and MTHFR gene polymorphisms is shown in Figure 1. The course seems to be different in at least the first 8 months, and the difference is, in fact, statistically significant (P = .03). Later on in the course, there is only a minor difference. The median time to progression for the group of patients with the TS 3R/3R or MTHFR 677 TT polymorphism is 7 months compared with 4 months in the group with other genomic characteristics.
Table 5 lists the distribution of TS and MTHFR gene polymorphisms in the prospective study. The frequency of TS and MTHFR homozygosity was almost the same as that in the retrospective study. It also seems that the response rate was 42% in the homozygous group versus 24% in the heterozygous group. Time to progression is shown in Figure 2. The Kaplan-Meier plot indicates a difference, which is marginally significant (P = .05), with the same division into the two groups. The progression-free interval for the TS 3R/3R or MTHFR 677 TT gene polymorphism group compared with the other groups was 8 v 4 months, respectively.
Gene polymorphism may be an important predictive factor in the treatment of cancer with chemotherapy. The TS gene polymorphism has attracted considerable attention in recent years. Several studies have indicated that the number of repeats is predictive of the effect of FU. In a study including 50 patients, Pullarkat et al4 found a higher response rate in 2R/2R patients compared with 2R/3R or 3R/3R patients. The results also suggested a difference in median survival. Another study11 indicated that patients with 2R/3R polymorphism had higher TS expression and a worse prognosis compared with patients with 2R/2R and 3R/3R polymorphisms. Iacopetta et al9 analyzed 240 patients with Dukes’ stage C disease and found that adjuvant FU-based treatment had no effect in 3R/3R patients compared with 2R/2R and 3R/2R patients. The 3R/3R patients may also have a lower probability of downstaging when treated for rectal cancer with preoperative chemoradiotherapy.12 In contrast with these results, the present study indicates that patients with triple homozygosity in the TS gene polymorphism responded better to FU treatment, with a higher response rate and longer time to progression. One reason for the contradictory results may be that the present study used normal tissue, which is different from the most previous investigations applying tumor tissue. The analysis of tumor tissue is, in fact, based on malignant cells with a varying admixture of normal cells, unless laser microdissection is used. The classification of a certain gene polymorphism may be hampered in a mixture of normal and malignant cells. This has been clearly illustrated by loss of heterozygosity as one example,13 which, however, probably does not explain the reverse effect of the 3R/3R TS gene polymorphism found in the present study. The use of normal tissue eliminates the problem of loss of heterozygosity, and the results presented here indicate that the gene polymorphism in normal tissue can predict the FU sensitivity of the tumor. TS gene polymorphism probably does not correlate with mRNA expression but, instead, correlates with TS protein expression,3,14 and there is some evidence that high TS protein expression may signify high sensitivity to FU in the adjuvant situation.15,16 However, it should be noted that results of adjuvant chemotherapy cannot be transferred to metastatic disease without reservation. Several studies indicate that TS, dihydropyrimidine dehydrogenase, and probably also TP gene expression are predictive markers for FU sensitivity. The present study cannot rule out that different expressions of these genes in the tumor may have contributed to the results that indicate a considerable predictive value of gene polymorphism in normal tissue. It should also be noted that FU may act as two different drugs according to mode of administration.17 Bolus FU may exert its major effect on RNA, whereas continuous infusion may have a preferential effect on TS. The predictive value of TS gene polymorphism may vary with mode of administration. In the present study, FU was administered as an intravenous injection (< 3 minutes). In a previous randomized trial, we have shown that the response rate is doubled if the injection time is reduced from 15 minutes to less than 3 minutes.18 Therefore, it is important to realize that the results presented here only apply when FU is administered as an intravenous push. In most previous trials, there is no information on injection time, and it cannot be excluded that this parameter can contribute to an explanation of the contradictory results. There are few data on the possible importance of MTHFR gene polymorphism. In vitro studies7 have indicated that the MTHFR 677 CT polymorphism is important to the effect of FU and methotrexate, and a small clinical study19 with 43 colorectal cancer patients indicated a different frequency of the T allele in responders and nonresponders. However, there was no significant difference in the frequency of objective response among CC, CT, and TT individuals. A recent in vitro study has suggested a potential role of TS and MTHFR 1298 AC polymorphisms on FU sensitivity, but there are no clinical data.20 The present study is the first to show a significant effect of the MTHFR 677 CT polymorphism in a clinical trial of reasonable size. The effect is in agreement with the fact that MTHFR 677 TT homozygotes have an enzymatic activity of 30% compared with MTHFR CC homozygotes, resulting in an accumulation of 5-10-methylenetetrahydrofolate, which stabilizes the ternary complex. The 1298 AT polymorphism, to a lesser extent, influences the enzymatic activity, and our results do not suggest an effect on the FU sensitivity. The present study is the first to relate TS and MTHFR gene polymorphisms in the same patient population to the effect of FU. Both TS and MTHFR polymorphism variants seem to have a considerable effect; in support of this fact, it was possible to allocate approximately 30% of the patients to a group with a response rate of approximately 50% and significantly longer time to progression. The response rates observed in both the retrospective analysis and the prospective trial were probably higher than that generally reported in the literature. It should be noted that the response rates were investigator defined and, consequently, should be taken with caution. However, this limitation does not explain either the difference in response rate or the difference in time to progression according to gene polymorphism. In conclusion, the present study indicates a major importance of a combination of TS and MTHFR gene polymorphisms to FU sensitivity. The results allow the delineation of approximately 30% of the patients into groups with an approximately 50% chance of effect. This information should be used in the design of tailored treatment.
The authors indicated no potential conflicts of interest.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
1. Advanced Colorectal Cancer Meta-Analysis Project: Modulation of fluorouracil by leucovorin in patients with advanced colorectal cancer: Evidence in terms of response rate. J Clin Oncol 10:896-903, 1992[Abstract] 2. Marsh S, McKay JA, Cassidy J, et al: Polymorphism in the thymidylate synthase promoter enhancer region in colorectal cancer. Int J Oncol 19:383-386, 2001[Medline]
3. Kawakami K, Salonga D, Park JM, et al: Different lengths of a polymorphic repeat sequence in the thymidylate synthase gene affect translational efficiency but not its gene expression. Clin Cancer Res 7:4096-4101, 2001 4. Pullarkat ST, Stoehlmacher J, Ghaderi V, et al: Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 1:65-70, 2001[Medline]
5. Tsuji T, Hidaka S, Sawai T, et al: Polymorphism in the thymidylate synthase promoter enhancer region is not an efficacious marker for tumor sensitivity to 5-fluorouracil-based oral adjuvant chemotherapy in colorectal cancer. Clin Cancer Res 9:3700-3704, 2003 6. Frosst P, Blom HJ, Milos R, et al: A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 10:111-113, 1995[CrossRef][Medline]
7. Sohn KJ, Croxford R, Yates Z, et al: Methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst 96:134-144, 2004 8. Jakobsen A, Berglund A, Glimelius B, et al: Dose-effect relationship of bolus 5-fluorouracil in the treatment of advanced colorectal cancer. Acta Oncol 41:525-531, 2002[CrossRef][Medline] 9. Iacopetta B, Grieu F, Joseph D, et al: A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil. Br J Cancer 85:827-830, 2001[CrossRef][Medline]
10. Skibola CF, Smith MT, Kane E, et al: Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc Natl Acad Sci U S A 96:12810-12815, 1999
11. Etienne MC, Chazal M, Laurent-Puig P, et al: Prognostic value of tumoral thymidylate synthase and p53 in metastatic colorectal cancer patients receiving fluorouracil-based chemotherapy: Phenotypic and genotypic analyses. J Clin Oncol 20:2832-2843, 2002
12. Villafranca E, Okruzhnov Y, Dominguez MA, et al: Polymorphisms of the repeated sequences in the enhancer region of the thymidylate synthase gene promoter may predict downstaging after preoperative chemoradiation in rectal cancer. J Clin Oncol 19:1779-1786, 2001
13. Uchida K, Hayashi K, Kawakami K, et al: Loss of heterozygosity at the thymidylate synthase (TS) locus on chromosome 18 affects tumor response and survival in individuals heterozygous for a 28-bp polymorphism in the TS gene. Clin Cancer Res 10:433-439, 2004 14. Ishida Y, Kawakami K, Tanaka Y, et al: Association of thymidylate synthase gene polymorphism with its mRNA and protein expression and with prognosis in gastric cancer. Anticancer Res 22:2805-2809, 2002[Medline]
15. Kornmann M, Schwabe W, Sander S, et al: Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression levels: Predictors for survival in colorectal cancer patients receiving adjuvant 5-fluorouracil. Clin Cancer Res 9:4116-4124, 2003
16. Edler D, Glimelius B, Hallstrom M, et al: Thymidylate synthase expression in colorectal cancer: A prognostic and predictive marker of benefit from adjuvant fluorouracil-based chemotherapy. J Clin Oncol 20:1721-1728, 2002
17. Sobrero A, Aschele C, Bertino JR: Fluorouracil in colorectal cancer: A tale of two drugs—Implications for biochemical modulation. J Clin Oncol 15:368-381, 1997 18. Glimelius B, Jakobsen A, Graf W, et al: Bolus injection (2-4 min) versus short-term (10-20 min) infusion of 5-fluorouracil in patients with advanced colorectal cancer: A prospective randomised trial. Eur J Cancer 34:674-678, 1998
19. Cohen V, Panet-Raymond V, Sabbaghian N, et al: Methylenetetrahydrofolate reductase polymorphism in advanced colorectal cancer: A novel genomic predictor of clinical response to fluoropyrimidine-based chemotherapy. Clin Cancer Res 9:1611-1615, 2003 20. Etienne MC, Ilc K, Formento JL, et al: Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms: Relationships with 5-fluorouracil sensitivity. Br J Cancer 90:526-534, 2004[CrossRef][Medline] Submitted June 28, 2004; accepted November 29, 2004.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
