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Journal of Clinical Oncology, Vol 24, No 10 (April 1), 2006: pp. 1603-1611 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.5253 Tumor Thymidylate Synthase 1494del6 Genotype As a Prognostic Factor in Colorectal Cancer Patients Receiving Fluorouracil-Based Adjuvant Treatment
From the Laboratory of Translational Research and Departments of Medical Oncology and Cancer Epidemiology, Institut Català d'Oncologia-Institut d’Investigació de Bellvitge (IDIBELL); Department of Pathology and Department of Surgery, Hospital Universitario de Bellvitge-IDIBELL; Department of Molecular Oncology, Institut de Recerca Oncològica-IDIBELL, L'Hospitalet de Llobregat; Department of Pathology, Hospital Vall d'Hebron; and Laboratori de Bioestadística I Epidemiologia, Facultat de Medicina, Universitat Autónoma de Barcelona, Barcelona, Spain Address reprint requests to Alberto Villanueva, PhD, Laboratory of Translational Research, Institut Català d'Oncologia-IDIBELL, Hospital Duran i Reynals, L'Hospitalet de Llobregat, 08907 Barcelona, Spain; e-mail: avillanueva{at}iconcologia.net
PURPOSE: The purpose of this study was to analyze the value of germline and tumor thymidylate synthase (TS) genotyping as a prognostic marker in a series of colorectal cancer patients receiving adjuvant fluorouracil (FU) -based treatment. PATIENTS AND METHODS: One hundred twenty-nine colorectal cancer patients homogeneously treated with FU plus levamisole or leucovorin in the adjuvant setting were included. TS enhancer region, 3R G > C single nucleotide polymorphism (SNP), and TS 1494del6 polymorphisms were assessed in both fresh-frozen normal mucosa and tumor. Mutational analyses of TS and allelic imbalances were studied in all primary tumors and in 18 additional metachronic metastases. TS protein immunostaining was assessed in an expanded series of 214 tumors. Multivariate Cox models were adjusted for stage, differentiation, and location. RESULTS: Tumor genotyping (frequency of allelic loss, 26%) showed that the 3R/3R genotype was associated with a better outcome (hazard ratio [HR] = 0.38; 95% CI, 0.16 to 0.93; P = .020 for the recessive model). 3R G > C SNP genotyping did not add prognostic information. Tumor TS 1494del6 allele (frequency of allelic loss, 36%) was protective (for each allele with the deletion, based on an additive model, HR = 0.42; 95% CI, 0.22 to 0.82; P = .0034). Both polymorphisms were in strong linkage disequilibrium (D' = 0.71, P < .001), and the 3R/–6 base pair (bp) haplotype showed a significant overall survival benefit compared with the most prevalent haplotype 2R/+6bp (HR = 0.42; 95% CI, 0.20 to 0.85; P = .017). No TS point mutation was detected in primary tumors or metastases. TS protein immunostaining was not associated with survival or any of the genotypes analyzed. CONCLUSION: Tumor TS 1494del6 genotype may be a prognostic factor in FU-based adjuvant treatment of colorectal cancer patients.
Fluorouracil (FU), a thymidylate synthase (TS) –inhibitor drug, has remained the mainstay of chemotherapeutic regimens for colorectal cancers in both metastatic and adjuvant settings.1,2 Chemotherapy is indicated in all stage III (Dukes' C) patients, but some stage II (Dukes' B2) patients who may benefit from its use will not receive it.3 Therefore, it is important to identify additional factors that may help in prognostic assessment. After recurrence, the majority of patients are usually re-treated with FU in combination with other drugs, mainly oxaliplatin or irinotecan.4-6 Recently, oxaliplatin in combination with FU has been shown to be more effective than FU alone in the adjuvant setting.7 High TS protein or mRNA levels in tumors have been associated with a worse response to FU,8,9 although results have not been consistent.10 Resistance to FU treatment has been associated in vitro with the presence of point mutations in the TS gene.11,12 Recently, TS amplification has been identified as a mechanism of resistance to FU in hepatic metastases of colorectal cancer patients.13 Three polymorphisms in the TS untranslated regions (UTRs) have been identified. TS enhancer region (TSER) polymorphism is a unique tandem repeat immediately upstream of the ATG codon initiation site14 containing two (2R) or three (3R) 28–base pair (bp) repeats that can influence TS transcription or translation.15-19 A novel functional G > C single nucleotide polymorphism (SNP) present in the second repeat of 3R alleles has been identified.20 In vitro, the efficiency of translation of the 3R(C) allele is equivalent to that of the 2R allele.21 TS 1494del6, a 6-bp deletion at nucleotide 1494 in the 3'-UTR, has been associated in vitro with decreased mRNA stability and lower intratumoral TS expression20,22 and has been found in strong linkage disequilibrium with TSER.23-25 The 3R/3R genotype has been identified as a predictor of poor clinical outcome to FU-based chemotherapy in colorectal cancer in both the adjuvant and metastatic setting.14,18,26,27 However, others have reported the 3R/3R28 or 2R/3R genotypes29 as good prognostic factors in metastatic carcinoma. Moreover, combined analysis of TSER and the SNP might provide a more effective prediction of clinical outcome to FU-based chemotherapy.21,30 Because allelic losses at chromosome 18 are highly prevalent, tumor genotyping rather than germline analyses may be more informative.31-33 To evaluate the clinical usefulness of genotyping TS as a prognostic marker of response to adjuvant FU treatment, we analyzed a consecutive series of colorectal cancer patients homogeneously treated with FU. Polymorphisms were analyzed in normal and tumor tissues. Additionally, mutational analyses and TS protein immunostaining were performed.
Patients The Bellvitge Colorectal Cancer Study was designed to prospectively study novel molecular etiologic and prognostic factors in colorectal cancer. Between January 1996 and June 2000, a total of 214 consecutive patients were identified with colorectal cancer undergoing elective radical surgery, which, in 16 patients, included resection of unique liver synchronous metastases. In 129 (60%) of 214 patients, paired fresh-frozen colorectal tumor and nonadjacent areas of normal colonic mucosa tissues were simultaneously collected, and these patients constitute the cohort used for TS genotyping (Table 1). No significant differences were observed between genotyped patients and patients without frozen tissue available, although genotyped patients were more often female and had less advanced disease stage (Dukes’ B). In the first 2 years of the study, FU bolus injection plus levamisole for 1 year was the standard treatment. From 1998 and onward, FU bolus injection plus leucovorin for 6 months was administered.
Prospective clinical follow-up was available for all patients, with a median follow-up time of 58 months. Overall survival (OS) and disease-free survival (DFS) were calculated from the time of surgery until death, recurrence, or last follow-up visit. Thirty patients experienced relapse during follow-up. Three- and 5-year cause-specific OS rates were 78% and 71%, respectively. All patients gave written consent to participate, and the ethics committee of the hospital cleared the study protocol.
Samples
TS Genetic Analyses
TS 1494del6 genotype. TS 6-bp deletion polymorphism at nucleotide 1494 (TTAAAG) was amplified using primers 5'-CAAATCTGAGGGAGCTGAGT-3' and 5'-CAGATAAGTGGCAGTACAGA-3'.22 PCR conditions are available upon request. Two alleles (–6bp and +6bp) were identified after 8% PAGE and ethidium bromide staining (Fig 1C). Loss of heterozygosity analysis. Loss of heterozygosity at the TS locus was assessed analyzing heterozygous patients for any of the three intragenic polymorphisms analyzed (TSER, 3R G > C SNP, and TS 1494del6). Loss of heterozygosity was defined by loss of intensity evident to naked eye inspection in any of the alleles analyzed after analyses of paired normal and tumor tissue (Fig 1). Tumor genotype was derived for all three polymorphisms. TS mutational analysis. The complete coding region of human TS, comprising exons 1 to 7, was analyzed by PCR–single-strand conformation polymorphism in seven independent reactions using intronic primers (primers and PCR conditions are available upon request). PCR products were loaded onto a 6% polyacrylamide nondenaturing sequencing gel. Electrophoresis was carried out at room temperature under 7 W for 10 to 12 hours. Gels were silver stained and vacuum dried at 85°C. Shifted single-strand conformation polymorphism bands were excised from the gel, reamplified, and sequenced.34
Immunohistochemistry
Tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was quenched with 6% hydrogen peroxide and methanol for 10 minutes. Antigen retrieval was achieved with heating in a pressure cooker using 10 mmol/L of sodium-citrate buffer (pH 6.0). To block nonspecific binding, normal goat serum diluted in phosphate-buffered saline was used for 30 minutes. After washing with phosphate-buffered saline, sections were incubated with antibody (1:1,500 dilution) for 1 hour at room temperature, followed by 15-minute incubations with a biotinylated link antibody (containing antirabbit and antimouse immunoglobulins) and peroxidase-labeled streptavidin (DAKO K0675; DAKO Diagnostics, Barcelona, Spain). Finally, slides were incubated with diaminobenzidine chromogen solution and counterstained with hematoxylin. Positive and negative controls were included in every staining. Percentage of tumor cells expressing TS (< 50% or  50%) and staining intensity (–, undetectable; +, traces; ++, definite staining of light to moderate intensity; and +++, intense staining) were evaluated. Two pathologists (M.C. and F.V.) blindly examined all slides. Agreement between the two observers was greater than 90%. In case of disagreement, consensus was reached.
Statistical Analysis
TSER Genotype The frequency of each genotype was similar to previous reports in white populations, and allelic distribution was in Hardy-Weinberg equilibrium (Table 2). No association was observed with sociodemographic or tumor characteristics (Table 1). Survival analysis showed a modest trend towards better OS for patients with the germline 3R allele (P = .099 in an additive model; Table 2 and Fig 3A). Tumor allelic imbalances were detected in 16 (26%) of 61 informative 2R/3R tumors (11 lost 2R allele and five lost 3R allele). Tumor genotyping confirmed the association of the 3R/3R genotype with better prognosis (HR = 0.38; 95% CI, 0.16 to 0.93; P = .020 for recessive model; Table 2 and Fig 3B). SNP genotyping of germline 3R alleles, which resulted in an extensive reclassification of patients (Table 2), did not provide additional prognostic information. The frequency of allelic imbalances for this locus was 38% [five of 13 informative 3R(G)/3R(C) tumors: three lost 3R(C) and two lost 3R(G)]. Finally, classification of alleles according to their theoretical functional status failed to provide information when both germline and somatic genotypes were considered. No association was observed with DFS.
TS 1494del6 Genotype The frequency of each genotype was similar to previous reports, and allelic distribution was in Hardy-Weinberg equilibrium (Table 3). The presence of the –6bp allele in normal mucosa was associated with more advanced stages of disease and well-differentiated tumors (Table 1). This allele was also associated with a reduced risk of death (Table 3). In fact, no deaths were observed in the 10 –6bp/–6bp patients (P = .011 in an additive model; Table 3 and Fig 4A). Allelic imbalances were observed in 17 (36.2%) of 47 heterozygous patients showing similar loss rates for each allele (+6bp allele: seven of 17 patients, 41.2%; –6bp allele: 10 of 17 patients, 58.8%; Table 3). Reclassification according to tumor genotype increased the number of homozygotes to 17 and strengthened the protective role of the –6bp/–6bp allele (for each –6bp allele, the HR was 0.42; 95% CI, 0.22 to 0.82; P = .0034 in an additive model; Table 3 and Fig 4B). No significant association was observed between the TS 1494del6 germline of tumor genotypes and any of the clinicopathologic features analyzed. Allelic imbalances per se were not associated with poor outcome (data not shown). No association was observed with DFS.
Haplotype Analysis As previously described in other populations,23-25 TSER and TS 1494del6 polymorphisms are in strong linkage disequilibrium in our population (D' = 0.71; P < .001). Haplotypes could be only estimated from germline genotypes as a result of allelic imbalances observed in tumors. Patients harboring the 3R/–6bp haplotype showed a significant OS benefit compared with patients with the most prevalent haplotype 2R/+6bp (P = .017; P for association = .077; Table 4 and Fig 5). The other haplotype harboring the –6bp allele was also associated with a reduced risk of death but was present in a minority of patients. After 3R SNP allele redistribution, the results remained essentially identical. In a Cox model that included Dukes' stage, degree of differentiation, and tumor site, only TS 1494del6 added prognostic information (P = .0091), supporting a predominant role of the –6bp allele in the association observed in the haplotype analysis.
Mutational Analyses of the TS Gene Mutational analyses of the 129 primary tumors revealed no somatic point mutations and the presence of four different polymorphisms. A nonconservative change (P > S) at amino acid 15 was detected in two patients, and three changes in intron-exon boundaries were detected. A 7-bp (TTGGATG) deletion at nt 2,839 through 2,845 and a 2-bp (CG) insertion at nt 14,123 were detected in a single patient each. In contrast, an A > G substitution at nt 15,589 was detected in 28.5% of the patients (TS Genebank D00596 sequence). The latter, which was close to a splicing regulating sequence, was further explored. In 10 paired normal colonic mucosa and colorectal tumors, reverse-transcriptase PCR of the TS gene did not depict additional bands indicative of splicing changes (data not shown). Also, no association was observed with survival. In a second step, 18 metachronic hepatic metastases resected after FU treatment were also analyzed. Again, no somatic mutations were depicted.
Immunostaining of TS Protein
We have carried out a comprehensive analysis of TS alterations in a consecutive series of colorectal cancer patients receiving FU-based adjuvant treatment. Our study shows that tumor TS 1494del6 and TSER 3R/3R genotypes and, to a lesser extent, the corresponding normal mucosa genotypes are good prognostic factors in these patients. TS 1494del6 polymorphism causes in vitro message instability and is associated with decreased intratumoral TS mRNA levels.36 In cell lines, this polymorphism did not correlate with FU sensitivity.29 Also, in the only study that prospectively assessed its prognostic value in colorectal cancer, TS 1494del6 was not associated with survival,25,37 although information regarding chemotherapy was not available. In our series of homogenously FU-treated patients, tumor –6bp/–6bp genotype is a strong prognostic factor that may be of benefit for up to 20% of patients. In our study, 3R/3R tumor allele showed a better survival, which is in line with recent observations by Jakobsen et al28 in metastatic carcinoma. This is in contrast with the majority of reports associating 3R/3R TSER genotype with poor response for colon and rectal tumors in the adjuvant setting.14,17,18,26,27 Tsuji et al38 showed, in an Asian population, that this genotype was not efficacious in predicting response in the adjuvant setting. Nonetheless, results from the latter series cannot be easily extrapolated to whites because of the distinct TSER allele distribution observed. It has been suggested that the recently described functional SNP within the 3R allele20 could account for some of the contradictory results. In our series, classification of patients taking into account the predicted functional role of the alleles has been of no help. This is in contrast with results by Kawakami et al21 in Asian patients receiving oral fluoropyrimidine adjuvant therapy. Also, in disseminated disease, Marcuello et al30 showed that the 3R(G) allele was a poor prognostic marker. Although there is no good explanation for these discrepancies, it may be that the functional predictions based on in vitro tests may not reflect the real situation in vivo. In fact, the only study assessing TS protein activity in human tumors observed an increased TS protein activity in tumors harboring the 2R/3R genotype.29 We have confirmed that both polymorphisms analyzed are in Hardy-Weinberg equilibrium and in strong linkage disequilibrium.23-25,37 This is in contrast with findings in Hungarian patients, in whom an unbalanced distribution of the haplotypes was observed.39 In our series, the 3R/-6bp haplotype, which represents 22.8% of the population analyzed, showed a better response rate. The strength of the association was higher with haplotypes harboring the TS 1494del6 allele, suggesting a prominent role of the 3'-UTR polymorphism. Short-arm chromosome 18 allelic losses are a frequent event in human colorectal tumors that can modify tumor genotype. Tumor genotyping has proved more informative than normal mucosa, as previously reported.31,40 Tumor information has consistently confirmed trends already observed in normal mucosa, further reinforcing the validity of our findings. It must be taken into account that the 30% incidence of allelic imbalances observed in our series is among the lowest reported in comparable settings.31,32 In vitro resistance to FU mainly occurs by acquisition of specific TS point mutations or gene amplification.12,41 We have shown that TS point mutations are not related in vivo to FU resistance in colorectal patients. We have not analyzed TS amplification, but it has been reported amplified in colorectal hepatic metastases.13 Because TS has recently described as an oncogene,42 it is likely that allelic imbalances observed may reflect TS amplification rather than allelic loss. If this is the case, it may have only limited clinical relevance because the presence of allelic imbalances has not associated with worse outcome. A recent meta-analysis showed that, in the adjuvant setting, protein expression analysis did not seem to predict outcome in patients treated with surgery and adjuvant FU.43 Because heterogeneity and possible publication bias were observed, authors recommended the performance of additional studies.43 Here, using a newly generated polyclonal antibody, immunohistochemical TS protein analyses did not provide any prognostic information. We speculate that semiquantitative immunohistochemistry does not provide an accurate estimate of TS protein levels. Whether this is a result of the use of distinct antibodies, differences in tissue handling, or the use of subjective parameters remains to be elucidated. We did not detect any significant correlation between TS polymorphisms and immunohistochemical TS protein levels. Kawakami et al31 have shown that genotype may influence the total amount of active protein, although significant individual variability was observed. As stated earlier, technical limitations may account for this observation. We have not measured mRNA levels, which might add information,17 although its true value has not been confirmed. In summary, we have shown that 3R/–6bp tumor haplotype provides prognostic information in FU-treated patients in the adjuvant setting. Also, TS 1494del6 may play a more prominent role in FU response than what was previously expected. The use of a homogenous patient series, which is likely to reflect the experience of a referral hospital and which shows a balanced allele distribution, makes significant bias unlikely. The consistency of findings in normal and tumor genotype strengthens our observations regarding TS 1494del6 allele, which certainly need replication in independent series. Finally, our results further reinforce the notion that the clinical usefulness of TS genotyping or protein assessment is still a matter of controversy and that caution is warranted when translating it into the clinics.
The authors indicated no potential conflicts of interest.
We thank Lawrence A. Loeb (University of Washington, Seattle, WA) for kindly providing the plasmid containing the human TS cDNA.
Supported by Grant Nos. SAF2002-02265 and AGL2004-07579-04 from the Spanish Ministry of Science and Technology and Grant No. FIS03-0114 from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III. The group belongs to and gets financial support from the Spanish Networks Red de Centros de Investigación en Epidemiología y Salud Pública (C03/09) and Red Temática de Investigación Cooperativa de Centros de Cáncer (C03/10) from Instituto de Salud Carlos III. A.V. is an investigator from the Ramon y Cajal program. M.M.I. is a recipient of an IDIBELL predoctoral fellowship, and F.V. is a recipient of a Becas de Formación del Fondo de Investigaciones Sanitarias grant from the Instituto de Salud Carlos III. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Danenberg PV: Thymidylate synthetase: A target enzyme in cancer chemotherapy. Biochim Biophys Acta 473:73-92, 1977[Medline] 2. Longley DB, Harkin DP, Johnston PG: 5-Fluorouracil: Mechanisms of action and clinical strategies. Nat Rev Cancer 3:330-338, 2003[CrossRef][Medline] 3. Allegra CJ, Parr AL, Wold LE, et al: Investigation of the prognostic and predictive value of thymidylate synthase, p53, and Ki-67 in patients with locally advanced colon cancer. J Clin Oncol 20:1735-1743, 2002 4. Giacchetti S, Perpoint B, Zidani R, et al: Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol 18:136-147, 2000 5. de Gramont A, Figer A, Seymour M, et al: Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 18:2938-2947, 2000 6. 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 7. Andre T, Boni C, Mounedji-Boudiaf L, et al: Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 350:2343-2351, 2004 8. Johnston PG, Lenz HJ, Leichman CG, et al: Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res 55:1407-1412, 1995 9. Lenz HJ, Hayashi K, Salonga D, et al: p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: An analysis of response and survival. Clin Cancer Res 4:1243-1250, 1998[Abstract] 10. 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 11. Kitchens ME, Forsthoefel AM, Barbour KW, et al: Mechanisms of acquired resistance to thymidylate synthase inhibitors: The role of enzyme stability. Mol Pharmacol 56:1063-1070, 1999 12. Landis DM, Loeb LA: Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases. J Biol Chem 273:25809-25817, 1998 13. Wang TL, Diaz LA Jr, Romans K, et al: Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients. Proc Natl Acad Sci U S A 101:3089-3094, 2004 14. 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] 15. Kaneda S, Takeishi K, Ayusawa D, et al: Role in translation of a triple tandemly repeated sequence in the 5'-untranslated region of human thymidylate synthase mRNA. Nucleic Acids Res 15:1259-1270, 1987 16. Horie N, Aiba H, Oguro K, et al: Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 20:191-197, 1995[Medline] 17. 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 18. 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] 19. Kawakami K, Omura K, Kanehira E, et al: Polymorphic tandem repeats in the thymidylate synthase gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Res 19:3249-3252, 1999[Medline] 20. Mandola MV, Stoehlmacher J, Muller-Weeks S, et al: A novel single nucleotide polymorphism within the 5' tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity. Cancer Res 63:2898-2904, 2003 21. Kawakami K, Watanabe G: Identification and functional analysis of single nucleotide polymorphism in the tandem repeat sequence of thymidylate synthase gene. Cancer Res 63:6004-6007, 2003 22. Ulrich CM, Bigler J, Velicer CM, et al: Searching expressed sequence tag databases: Discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 9:1381-1385, 2000 23. Lecomte T, Ferraz JM, Zinzindohoue F, et al: Thymidylate synthase gene polymorphism predicts toxicity in colorectal cancer patients receiving 5-fluorouracil-based chemotherapy. Clin Cancer Res 10:5880-5888, 2004 24. Graziano F, Kawakami K, Watanabe G, et al: Association of thymidylate synthase polymorphisms with gastric cancer susceptibility. Int J Cancer 112:1010-1014, 2004[CrossRef][Medline] 25. Ulrich CM, Bigler J, Bostick R, et al: Thymidylate synthase promoter polymorphism, interaction with folate intake, and risk of colorectal adenomas. Cancer Res 62:3361-3364, 2002 26. 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 27. 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] 28. Jakobsen A, Nielsen JN, Gyldenkerne N, et al: Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol 23:1365-1369, 2005 29. 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 30. Marcuello E, Altes A, Del Rio E, et al: Single nucleotide polymorphism in the 5' tandem repeat sequences of thymidylate synthase gene predicts for response to fluorouracil-based chemotherapy in advanced colorectal cancer patients. Int J Cancer 112:733-737, 2004[CrossRef][Medline] 31. Kawakami K, Ishida Y, Danenberg KD, et al: Functional polymorphism of the thymidylate synthase gene in colorectal cancer accompanied by frequent loss of heterozygosity. Jpn J Cancer Res 93:1221-1229, 2002[CrossRef][Medline] 32. 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 33. Vogelstein B, Fearon ER, Kern SE, et al: Allelotype of colorectal carcinomas. Science 244:207-211, 1989 34. Villanueva A, Garcia C, Paules AB, et al: Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells. Oncogene 17:1969-1978, 1998[CrossRef][Medline] 35. Schaid DJ, Rowland CM, Tines DE, et al: Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 70:425-434, 2002[CrossRef][Medline] 36. Mandola MV, Stoehlmacher J, Zhang W, et al: A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 14:319-327, 2004[CrossRef][Medline] 37. Chen J, Hunter DJ, Stampfer MJ, et al: Polymorphism in the thymidylate synthase promoter enhancer region modifies the risk and survival of colorectal cancer. Cancer Epidemiol Biomarkers Prev 12:958-962, 2003 38. 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 39. Adleff V, Hitre E, Koves I, et al: Heterozygote deficiency in thymidylate synthase enhancer region polymorphism genotype distribution in Hungarian colorectal cancer patients. Int J Cancer 108:852-856, 2004[CrossRef][Medline] 40. Zinzindohoue F, Ferraz JM, Laurent-Puig P: Thymidylate synthase promoter polymorphism. J Clin Oncol 19:3442, 2001 41. Tong Y, Liu-Chen X, Ercikan-Abali EA, et al: Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis: Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89. J Biol Chem 273:31209-31214, 1998 42. Rahman L, Voeller D, Rahman M, et al: Thymidylate synthase as an oncogene: A novel role for an essential DNA synthesis enzyme. Cancer Cell 5:341-351, 2004[CrossRef][Medline] 43. Popat S, Matakidou A, Houlston RS: Thymidylate synthase expression and prognosis in colorectal cancer: A systematic review and meta-analysis. J Clin Oncol 22:529-536, 2004 Submitted July 25, 2005; accepted January 31, 2006. This article has been cited by other articles:
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. Survival curves were estimated using the Kaplan-Meier method, and differences between individual curves were evaluated by multivariate analyses using Cox proportional hazards regression models. Analyses were always adjusted for Dukes' stage, differentiation degree, and tumor location. Hazard ratios (HRs) and 95% CIs were calculated. Likelihood ratio tests were used to assess the prognostic value of the genotypes and haplotypes. P = .05 was considered significant. Each polymorphism was tested to ensure that it fitted Hardy-Weinberg equilibrium. Genotypes for each SNP were analyzed as a three-group categoric variable (codominant model), and they were also grouped according to the dominant, recessive, and additive model. This latter model corresponds to the trend test calculated by assigning a linear score to each genotype (1 for homozygous common allele, 2 for heterozygous common allele, and 3 for homozygous rare allele). The best inheritance model was chosen based on Akaike Information Criteria. Haplotypes were analyzed using the haplo.stats package,
