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Journal of Clinical Oncology, Vol 25, No 24 (August 20), 2007: pp. 3726-3731 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.11.4710 Association of Methylenetetrahydrofolate Reductase Gene Polymorphisms and Sex-Specific Survival in Patients With Metastatic Colon Cancer
From the Division of Medical Oncology and Departments of Preventive Medicine and Pathology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, CA Address reprint requests to Heinz-Josef Lenz, MD, FACP, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Sharon A. Carpenter Laboratory, 1441 Eastlake Ave, Ste 3456, Los Angeles, CA 90033; e-mail: lenz{at}usc.edu
Purpose Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating intracellular folate levels, which affects DNA synthesis and methylation. Two MTHFR gene polymorphisms, C677T and A1298C, are linked to altered enzyme activity. Several studies have shown these two polymorphisms to be associated with response to fluorouracil (FU) -based treatment in advanced colon cancer patients, but data are inconsistent and contradictory. Meanwhile, epidemiologic studies demonstrated that these MTHFR polymorphisms were associated with cancer risk in a sex-specific manner. We tested the hypothesis of whether these two polymorphisms are associated with sex-specific clinical outcome in metastatic colon cancer patients treated with FU-based chemotherapy. Patients and Methods This study included 318 patients (177 men and 141 women) with metastatic colon cancer treated between 1992 and 2003 at the University of Southern California/Norris Comprehensive Cancer Center or Los Angeles County/University of Southern California Medical Center. Peripheral blood samples were collected from each patient, and genomic DNA was extracted from WBCs. Two MTHFR gene polymorphisms (C677T and A1298C) were tested by fluorogenic 5'-nuclease assay. Results The A1298C polymorphism showed statistically significant differences in overall survival (OS) in female, but not male, patients with metastatic colon cancer (log-rank test, P = .038). Among females, OS was greater for patients with the A/A genotype (n = 67; median OS, 18.4 months) compared with patients with the A/C genotype (n = 50; median OS, 13.9 months) or C/C genotype (n = 10; median OS, 15.6 months). Conclusion Although preliminary, these data support the role of the A1298C polymorphism in MTHFR as prognostic marker in female patients with metastatic colon cancer. Further studies are needed to confirm these findings.
Colorectal cancer is the second most common cause of cancer-related death in the United States. In 2007, an estimated 153,000 colon cancer patients will be diagnosed, and 52,000 deaths will occur.1 Fluorouracil (FU) has been used as a cornerstone for the treatment of metastatic colorectal cancer for more than 40 years. With the addition of other chemotherapeutic drugs such as irinotecan and oxaliplatin, the median survival time of patients with advanced colorectal cancer has almost doubled from 12 months to 21 months.2-4 However, advanced colorectal cancer is still a deadly disease, with a 5-year survival rate of less than 10%.1,5 Molecular markers are needed for clinical practice to help identify patients who will likely benefit from chemotherapy and to reduce potential toxicity. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme that plays a key role in folate metabolism. This enzyme irreversibly converts 5,10-methylenetetrahydrofolate (5,10-MTHF) to 5-methyltetrahydrofolate, which is needed for remethylation of homocysteine to methionine and is required for the synthesis of S-adenosylmethionine. In addition, the substrate for MTHFR, 5,10-MTHF, is also required for conversion of deoxyuridine monophosphate to deoxythymidine monophosphate by thymidylate synthase, which then affects DNA synthesis.6 The T and C alleles of MTHFR polymorphisms, C677T and A1298C, respectively, have been associated with decreased enzyme activity of MTHFR and increased plasma levels of homocysteine in in vitro studies.7,8 Several clinical studies have associated these two MTHFR polymorphisms and clinical outcome of FU-based chemotherapy in colorectal cancer, but the data are inconsistent and controversial.9-11 Interestingly, recent epidemiologic studies revealed that these two MTHFR polymorphisms are associated with sex-specific risk difference in colorectal cancer, lung cancer, and other disease (schizophrenia and ischemic stroke).12-15 We tested the hypothesis of whether MTHFR C677T and A1298C polymorphisms could be associated with the sex-specific overall survival (OS) in patients with metastatic colon cancer treated with FU-based chemotherapy.
Eligible Patients A total of 318 patients with metastatic colon cancer treated at the University of Southern California/Norris Comprehensive Cancer Center (USC/NCCC) or the Los Angeles County/University of Southern California Medical Center (LAC/USCMC) between 1992 and 2003 were eligible for the present study. This population included only metastatic or recurrent colon cancer patients. All patients in this study signed informed consents and were enrolled onto protocols designed to study the molecular determinants of colon cancer. These protocols permitted blood collection (protocol 0S-99-10) and/or tissue collection (protocol 0S-00-15) before chemotherapy start. All patients were entered and followed in an institutional database. Patient information was collected through database review and retrospective chart review when additional patient information was necessary. A large number of the patients (220 of 318 patients, 69%) were initially treated at an outside institution until, because of failure to respond to prior treatment, they were referred to USC/NCCC or LAC/USCMC for future treatments. All 318 patients were enrolled in at least one chemotherapy clinical trial while being treated at the institution (USC/NCCC or LAC/USCMC). All patients were treated with FU-based chemotherapy regimens. Response to chemotherapy was not investigated as an end point for this study. This was a heavily pretreated cohort, with 20 patients (6%) treated with one line of chemotherapy, 19 patients (6%) treated with two different chemotherapy regimens, 183 patents (58%) treated with three chemotherapy regimens, and 96 patients (30%) treated with four or more chemotherapy regimens. Although the treatment regimens varied among patients, the majority of the patients were exposed to similar chemotherapies. All 318 patients received treatment with FU, 298 patients (94%) received treatment with FU/irinotecan, and 279 patients (88%) received treatment with FU/oxaliplatin.
Genotyping
Statistical Analysis The associations between MTHFR C677T and A1298C polymorphisms, sex, and other demographic and clinical characteristics were examined using contingency tables and the Fisher's exact test. The associations of MTHFR C677T and A1298C polymorphisms with OS were analyzed individually using Kaplan-Meier curves and the log-rank test. In the univariate analysis, the estimate of relative risk (RR) with 95% CIs was based on the log-rank test.16 The independent effects of two MTHFR polymorphisms on OS were examined using the Cox proportional hazards model. Interactions between sex and MTHFR polymorphisms were examined using stratified models and were tested by comparing corresponding likelihood ratio statistics between the baseline and nested Cox proportional hazards models that included the multiplicative product terms.17 Linkage disequilibrium between the two MTHFR variants was assessed using D' and r2 values, and haplotype frequencies were inferred using the program Haploview version 3.32 (www.broad.mit.edu/mpg/haploview/). All reported P values were two sided. All analyses were performed using the SAS Statistical Package Version 9.0 (SAS Institute Inc, Cary, NC) and Epilog Plus Version 1.0 (Epicenter Software, Pasadena, CA).
This population contained a total of 318 patients, 141 women (44%) and 177 men (56%). Within the two populations, males and females, there were no statistically significant variations with regard to age, race/ethnicity, location of the primary tumor, or location of first metastatic site. The median age at the time of diagnosis was 58 years, with a range of 25 to 86 years. The racial/ethnic distribution of study participants was 234 whites (73%), 43 Asians (14%), 24 Hispanics (8%), 15 blacks (5%), and two Native Americans (1%). The location of the primary tumor within the colon was as follows: 144 left-sided tumors (54%), 124 right-sided tumors (46%), and 50 tumors where the side was unknown. The location of the first metastatic site was liver in 156 patients (49%), intra-abdominal in 56 patients (18%), and other (lung, bone, stomach, spleen, pancreas, or gallbladder) in 46 patients (15%); there were 57 patients (18%) who presented with two or more metastatic sites at the onset of metastatic disease (Table 1).
OS differences in this population were not associated with sex differences, racial/ethnic distribution, location of primary tumor, or location of first metastatic lesion (data not shown). The MTHFR polymorphisms were not associated with the age, sex, racial/ethnic distribution, location of primary tumor, or location of first metastatic lesion (data not shown).
MTHFR C677T and A1298C Polymorphisms
The MTHFR C677T and MTHFR A1298C polymorphisms were analyzed with regard to OS. When the population was not separated by sex, the genotypes were not associated with OS. However, when the patient population was separated by sex, the MTHFR A1298C polymorphism was found to be significantly associated with OS (log-rank test, P = .038) in females (Table 3; Fig 1). Among females, OS was greater for patients with the A/A genotype (n = 67; median OS, 18.4 months) compared with patients with the A/C genotype (n = 50; median OS, 13.9 months) or C/C genotype (n = 10; median OS, 15.6 months). Female patients with the C/C genotype (RR = 1.67; 95% CI, 0.78 to 3.6) and those with the A/C genotype (RR = 1.53; 95% CI, 1.01 to 2.3) had a greater risk of dying than noncarriers of the A allele. However, there were no statistically significant differences between A1298C polymorphism and OS in male patients (Table 3; Fig 2). We also did not find any OS difference between the MTHFR 677C wild-type allele and MTHFR 677T mutant allele.
Haplotype Analysis The MTHFR C677T and MTHFR A1298C polymorphisms were in strong linkage disequilibrium, with a D' value of 0.962 (95% CI, 0.82 to 1.0) but with a low r2 value of 0.171. Haplotypes were constructed from those two polymorphisms. The three common haplotypes were found and account for more than 99% of all haplotypes. Female patients, but not male patients, carrying the C-C haplotype had a higher risk of dying compared with patients with the most prevalent haplotype, C-A (P = .014 for females; P = .90 for males; Table 4). There were no significant differences in OS comparing the haplotype T-A with the haplotype C-A regardless of sex, which indicated that the MTHFR A1298C polymorphism, not C677T, was associated with OS in females.
To our knowledge, our data from this study demonstrated for the first time that MTHFR A1298C polymorphism is associated with OS in female metastatic colorectal cancer patients but not in male patients. The female colorectal cancer patients with MTHFR mutant 1298CC genotype survived a median of 15.6 months compared with 13.9 months for patients with heterozygous A/C genotype and 18.4 months for patients with homozygous A/A genotype (log-rank test, P = .038). However, this survival discrepancy associated with the MTHFR A1298C polymorphism was not found in the male population (log-rank test, P = .89). Folate metabolism is critical for DNA synthesis and methylation. MTHFR is a key enzyme that plays a major role in the metabolism of folate. MTHFR irreversibly converts 5,10-MTHF to 5,10-methyltetrahydrofolate, which is required for homocysteine remethylation to methionine. Methionine is then activated to S-adenosylmethionine, which is linked to the methylation of DNA. In addition, the substrate for MTHFR, 5,10-MTHF, is also required for conversion of deoxyuridine monophosphate to deoxythymidine monophosphate by thymidylate synthase, which then affects DNA synthesis. Recent in vitro data have demonstrated that antisense-mediated inhibition of MTHFR results in reduced cancer cell survival and decreased tumor growth in human colon and lung xenografts. These data suggest that MTHFR may be a potential target for antitumor therapy.18 In vitro studies have shown two functional polymorphisms of MTHFR, C677T and A1298C. The mutant alleles are associated with decreased MTHFR enzyme activity, although the 1298C allele is associated to a lesser extent than the 677T allele. Several clinical studies have correlated these two MTHFR polymorphisms with clinical outcome in colorectal cancer patients treated with FU-based chemotherapy, but the results are inconsistent and contradictory. Etienne et al10 analyzed these two polymorphisms in 98 advanced colorectal cancer patients with unresectable liver metastases. They found that, for MTHFR C677T polymorphism, the response rate was significantly increased in patients with 677 mutant T allele compared with patients with wild-type 677C allele. They also found that the MTHFR A1298C polymorphism was associated with OS, with patients with the 1298 mutant C allele having a poorer prognosis compared with patients with the 1298 wild-type A allele.10 Wisotzkey et al19 determined MTHFR C677T genotype in 51 stage III colon cancer patients and found, in a small subgroup of patients, that those patients with homozygous wild-type C/C genotype survived longer compared with patients with C/T and T/T genotypes. Meanwhile, Marcuello et al9 tested these two polymorphisms in 94 patients diagnosed with metastatic colorectal cancer and receiving FU-containing chemotherapy as first-line treatment. Their results do not show any correlation between these two MTHFR genotypes and clinical outcome (response and OS) of chemotherapy. Interestingly, recent epidemiologic studies have shown that the MTHFR C677T and A1298C polymorphisms are actually associated with cancer risk in a sex-specific manner. Curtin et al12 studied these two polymorphisms in an incident case-control colon cancer risk study; they found that the 677CC/1298CC genotype was associated with a statistically significant lower risk among women but not men. When the polymorphism is considered individually, for MTHFR A1298C polymorphism, a significant risk reduction with the mutant 1298CC genotype was seen among women only.12 This sex-specific difference was also found in a lung cancer risk study. Shi et al13 investigated the association of these two MTHFR polymorphisms and lung cancer risk in a hospital-based case-control study of 1,051 lung cancer patients and 1,141 cancer-free controls in a non-Hispanic white population. Their results showed that the 1298CC genotype was associated with a significantly increased risk of lung cancer in women but not in men. Also, the MTHFR 677TT genotype was associated with a significantly decreased risk of lung cancer in women but not in men.13 A similar sex-specific difference with these two MTHFR polymorphisms was also found in an ischemic stroke study in Japanese patients and in a study to associate these two polymorphisms with schizophrenia risk.14,15 These findings all point out that sex may play an important role in determining association between MTHFR polymorphisms and cancer or disease risk. The underlying biologic mechanism of why sex may affect the association of MTHFR polymorphism and clinical outcome or cancer risk is not clear. One possible hypothesis is through the link with homocysteine levels. Studies have shown that there is evidence for an interaction between estrogen status and homocysteine levels. Lower levels of homocysteine are observed in pregnant and premenopausal women and postmenopausal women who are on hormonal replacement therapy when compared with age-matched men and postmenopausal women who are not on hormonal replacement therapy.20-23 Castro et al24 tested MTHFR C677T and MTHFR A1298C polymorphisms in a prospective study, where they observed a significant effect of the 1298 A to C mutation on plasma homocysteine levels; patients bearing the 1298CC genotype had significantly higher homocysteine levels than patients with the wild-type 1298AA genotypes. This is also true for the C677T mutation. Here, we postulate that female colon cancer patients' estrogen status may interact with MTHFR genotype through decreasing homocysteine levels, but this impact is diminished in male colon cancer patients. A second possible explanation is that the MTHFR genotype may affect methylation capacity. One striking finding from Sarter et al25 points out that there was a significant sex difference in methylation pattern of the MTHFR gene in a Singapore Chinese cohort study. They compared relative levels of MTHFR gene methylation between 134 males and 257 females. Their data suggest that sex is a strong predictor of methylation levels in the MTHFR gene, with males showing higher relative methylation levels.25 Our study did not find an association with MTHFR C677T polymorphism and OS in female patients, male patients, or both sexes combined, although in vitro studies have demonstrated that MTHFR C677T mutant allele leads to more severe decreasing enzyme activities than MTHFR A1298C mutant allele. Also, our study, along with other studies, found that the MTHFR A1298C mutant C allele, which is associated with decreasing enzyme activities, has the worst survival in female metastatic colorectal cancer patients. The possible explanation that MTHFR A1298C polymorphism associated with sex-specific survival other than C677T may depend on this polymorphism could be more affected by hormone status and/or methylation status than C677T. Although A1298C polymorphism changes the enzyme activities to a lesser extent than C677T. The exact mechanism for this discrepancy between in vitro functional study and clinical observation is worth further investigation. Our findings present some limitations. Because of the nature of our retrospective study, we cannot correlate these two MTHFR polymorphisms with clinical response to FU-based chemotherapy. Further well-designed prospective studies are needed to confirm our data and address this tumor response issue. In summary, these data suggest a role for the MTHFR gene as a prognostic marker in metastatic colon cancer in a sex-specific manner. To our knowledge, this is the first study that shows a relationship between MTHFR A1298C polymorphism and sex-specific OS. Future studies are needed to validate our preliminary findings.
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Heinz-Josef Lenz, Imclone Systems Inc, Novartis, Sanofi-Aventis, Bristol-Myers Squibb Co, Genentech, Merck KGaA, Pfizer Inc, Response Genetics Stock Ownership: None Honoraria: Heinz-Josef Lenz, Novartis, Merck KGaA, Sanofi-Aventis, Bristol-Myers Squibb Co, Genentech, Pfizer Inc Research Funding: Heinz-Josef Lenz, National Cancer Institute, Cancer Therapy Evaluation Program, Southwest Oncology Group Expert Testimony: None Other Remuneration: None
Conception and design: Wu Zhang, Oliver A. Press, Dongyun Yang, Heinz-Josef Lenz Financial support: Heinz-Josef Lenz Provision of study materials or patients: Anthony El-khoueiry, Syma Iqbal, Andy E. Sherrod, Heinz-Josef Lenz Collection and assembly of data: Wu Zhang, Oliver A. Press, Christopher A. Haiman, William Fazzone, Georg Lurje Data analysis and interpretation: Dong Yun Yang, Georg Lurje Manuscript writing: Wu Zhang, Oliver A. Press, Christopher A. Haiman, Dong Yun Yang, Michael A. Gordon, Heinz-Josef Lenz Final approval of manuscript: Oliver A. Press, Georg Lurje, Heinz-Josef Lenz
Supported by Grant No. 5 P30CA14089-27l from the National Institutes of Health, the San Pedro Guild Research Fund, and the Dhont Foundation. W.Z. and O.A.P. contributed equally to this work. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Jemal A, Siegel R, Ward E, et al: Cancer statistics, 2007. CA Cancer J Clin 57:43-66, 2007 2. Douillard JY: Irinotecan and high-dose fluorouracil/leucovorin for metastatic colorectal cancer. Oncology (Williston Park) 14:51-55, 2000[Medline] 3. Saltz LB, Cox JV, Blanke C, et al: Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer: Irinotecan Study Group. N Engl J Med 343:905-914, 2000 4. Kohne CH, van Cutsem E, Wils J, et al: Phase III study of weekly high-dose infusional fluorouracil plus folinic acid with or without irinotecan in patients with metastatic colorectal cancer: European Organisation for Research and Treatment of Cancer Gastrointestinal Group Study 40986. J Clin Oncol 23:4856-4865, 2005 5. Cunningham D, Humblet Y, Siena S, et al: Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 351:337-345, 2004 6. Rosenblatt DS: Methylenetetrahydrofolate reductase. Clin Invest Med 24:56-59, 2001[Medline] 7. van der Put NM, Gabreels F, Stevens EM, et al: A second common mutation in the methylenetetrahydrofolate reductase gene: An additional risk factor for neural-tube defects? Am J Hum Genet 62:1044-1051, 1998[CrossRef][Medline] 8. Ma J, Stampfer MJ, Giovannucci E, et al: Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 57:1098-1102, 1997 9. Marcuello E, Altes A, Menoyo A, et al: Methylenetetrahydrofolate reductase gene polymorphisms: Genomic predictors of clinical response to fluoropyrimidine-based chemotherapy? Cancer Chemother Pharmacol 57:835-840, 2006[CrossRef][Medline] 10. Etienne MC, Formento JL, Chazal M, et al: Methylenetetrahydrofolate reductase gene polymorphisms and response to fluorouracil-based treatment in advanced colorectal cancer patients. Pharmacogenetics 14:785-792, 2004[CrossRef][Medline] 11. 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 12. Curtin K, Bigler J, Slattery ML, et al: MTHFR C677T and A1298C polymorphisms: Diet, estrogen, and risk of colon cancer. Cancer Epidemiol Biomarkers Prev 13:285-292, 2004 13. Shi Q, Zhang Z, Li G, et al: Sex differences in risk of lung cancer associated with methylene-tetrahydrofolate reductase polymorphisms. Cancer Epidemiol Biomarkers Prev 14:1477-1484, 2005 14. Wu Y, Tomon M, Sumino K: Methylenetetrahydrofolate reductase gene polymorphism and ischemic stroke: Sex difference in Japanese. Kobe J Med Sci 47:255-262, 2001[Medline] 15. Sazci A, Ergul E, Kucukali I, et al: Association of the C677T and A1298C polymorphisms of methylenetetrahydrofolate reductase gene with schizophrenia: Association is significant in men but not in women. Prog Neuropsychopharmacol Biol Psychiatry 29:1113-1123, 2005[CrossRef][Medline] 16. Berry G, Kitchin RM, Mock PA: A comparison of two simple hazard ratio estimators based on the logrank test. Stat Med 10:749-755, 1991[Medline] 17. Rothman KJ, Greenland S: Modern Epidemiology (ed 2). Philadelphia, PA, Lippincott-Raven, 1998, p 737 18. Stankova J, Shang J, Rozen R: Antisense inhibition of methylenetetrahydrofolate reductase reduces cancer cell survival in vitro and tumor growth in vivo. Clin Cancer Res 11:2047-2052, 2005 19. Wisotzkey JD, Toman J, Bell T, et al: MTHFR (C677T) polymorphisms and stage III colon cancer: Response to therapy. Mol Diagn 4:95-99, 1999[CrossRef][Medline] 20. Mijatovic V, Kenemans P, Jakobs C, et al: A randomized controlled study of the effects of 17beta-estradiol-dydrogesterone on plasma homocysteine in postmenopausal women. Obstet Gynecol 91:432-436, 1998[CrossRef][Medline] 21. Smolders RG, van der Mooren MJ, Sipkema P, et al: Estrogens, homocysteine, vasodilatation and menopause: Basic mechanisms, interactions and clinical implications. Gynecol Endocrinol 17:339-354, 2003[Medline] 22. Mijatovic V, Netelenbos C, van der Mooren MJ, et al: Randomized, double-blind, placebo-controlled study of the effects of raloxifene and conjugated equine estrogen on plasma homocysteine levels in healthy postmenopausal women. Fertil Steril 70:1085-1089, 1998[CrossRef][Medline] 23. Giri S, Thompson PD, Taxel P, et al: Oral estrogen improves serum lipids, homocysteine and fibrinolysis in elderly men. Atherosclerosis 137:359-366, 1998[CrossRef][Medline] 24. Castro R, Rivera I, Ravasco P, et al: 5,10-Methylenetetrahydrofolate reductase 677C–>T and 1298A–>C mutations are genetic determinants of elevated homocysteine. QJM 96:297-303, 2003 25. Sarter B, Long TI, Tsong WH, et al: Sex differential in methylation patterns of selected genes in Singapore Chinese. Hum Genet 117:402-403, 2005[CrossRef][Medline] Submitted February 27, 2007; accepted May 29, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
