5-FU based chemotherapy is the most common first line regimen used for metastatic colorectal cancer (mCRC). Identification of predictive markers of response to chemotherapy is a challenging approach for drug selection. The present study analyzes the predictive role of 5-FU degradation rate (5-FUDR) and genetic polymorphisms (MTHFR, TSER, DPYD) on survival.
Materials and Methods
Genetic polymorphisms of MTHFR, TSER and DPYD, and the 5-FUDR of homogenous patients with mCRC were retrospectively studied. Genetic markers and the 5-FUDR were correlated with clinical outcome.
133 patients affected by mCRC, treated with fluoropyrimidine-based chemotherapy from 2009 to 2014, were evaluated. Patients were classified into three metabolic classes, according to normal distribution of 5-FUDR in more than 1000 patients, as previously published: poor-metabolizer (PM) with 5-FU-DR ≤ 0,85 ng/ml/106 cells/min (8 pts); normal metabolizer with 0,85 < 5-FU-DR < 2,2 ng/ml/106 cells/min (119 pts); ultra-rapid metabolizer (UM) with 5-FU-DR ≥ 2,2 ng/ml/106 cells/min (6 pts). PM and UM groups showed a longer PFS respect to normal metabolizer group (14.5 and 11 months respectively vs 8 months; p = 0.029). A higher G3-4 toxicity rate was observed in PM and UM, respect to normal metabolizer (50% in both PM and UM vs 18%; p = 0.019). No significant associations between genes polymorphisms and outcomes or toxicities were observed.
5-FUDR seems to be significantly involved in predicting survival of patients who underwent 5-FU based CHT for mCRC. Although our findings require confirmation in large prospective studies, they reinforce the concept that individual genetic variation may allow personalized selection of chemotherapy to optimize clinical outcomes.
Citation: Botticelli A, Borro M, Onesti CE, Strigari L, Gentile G, Cerbelli B, et al. (2016) Degradation Rate of 5-Fluorouracil in Metastatic Colorectal Cancer: A New Predictive Outcome Biomarker? PLoS ONE 11(9): e0163105. https://doi.org/10.1371/journal.pone.0163105
Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES
Received: June 16, 2016; Accepted: September 4, 2016; Published: September 22, 2016
Copyright: © 2016 Botticelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: The authors do not receive funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Colorectal cancer (CRC) is the second highest cause of cancer death in Western countries. The combinations of fluoropyrimidine with oxapliplatin or irinotecan and biological agents are the most common first line chemotherapy regimens used for mCRC. [1–4]
5-Fluorouracil is an antimetabolite of the pyrimidine analogue type, that inhibits DNA and RNA synthesis, with its active metabolites, resulting from anabolism of about 1–2% of the drug. 5-FU active metabolites form an inactive ternary complex with thymidylate synthase (TS) and 5–10-methylenetetrahydrofolate (MTHF). TS optimal inhibition requires an elevated level of MTHF, regulated by the methylenetetrahydrofolate reductase (MTHFR).  As a consequence, polymorphisms in TS enhancing region (TSER) and MTHFR gene are presumed to be determinants for 5-FU clinical response, even if their clinical utility is still controversial. [6–14]
Dihydropyrimidine dehydrogenase (DPD) polymorphically expressed enzyme, encoded by DPD gene (DPYD) play a crucial role in the pharmacology of fluoropyrimidines, as it inactivates up to 85% of 5-FU to 5,6-dihydro-5-fluorouracil. [15, 16] Genetic polymorphism in DPYD has shown to be potentially responsible for lethal toxicity after 5FU-based chemotherapy. [17, 18]
Knowledge of the clinical impact of gene polymorphisms involved in the pharmacokinetics and pharmacodynamics of fluoropyrimidines may provide opportunities for patient-tailored chemotherapy, resulting in decreased incidence of severe side effects, reduced numbers of treatment delays or discontinuations, and possibly increased survival probability.
In a previous study high-performance liquid chromatography (HPLC) was used to identify an index of DPD metabolic activity, measuring uracil/dihydrouracil (U/UH2) ratio in plasma.  In 2009 we proposed the determination of 5-FU degradation rate (5-FU-DR) by intact peripheral blood monocuclear cells (PBMC) as a useful pre-screening test to evaluate drug toxicity.  Furthermore, a genotype-phenotype correlation in 5-FU metabolism was demonstrated, through an association analysis between DPYD single nucleotide polymorphisms (SNPs) and 5-FUDR.  Finally, we analysed the effects of the individual 5-FUDR on 5-FU toxicity in a population of 433 CRC patients. We found that both the poor metabolizer (PM) subjects, defined by a 5-FUDR ≤ 5th centile, and the ultra-rapid metabolizer (UM) patients, defined by a 5-FUDR ≥ 95th centile, are at higher risk to develop G3-4 toxicity, with an OR of 3.47 and 3.34, respectively, compared to normal metabolizers (5th < 5FUDR < 95th centiles). 
In the present study the Authors aim is to evaluate the influence of genetic polymorphisms of the genes involved in 5-FU metabolism and the of 5-FUDR on progression free survival in a population of metastatic colorectal cancer patients.
Materials and Methods
From 2009 to 2014 patients with a histologically confirmed metastatic adenocarcinoma of the colon and rectum undergoing fluoropyrimidine-based chemotherapy at the Sant’Andrea Hospital of Rome, were enrolled in this retrospective study. Each patient records were de-identified and analyzed anonymously.
The inclusions criteria were: patients with measurable disease, adequate organ function and performance status grade 0, 1 or 2 as defined by the Eastern Cooperative Oncology Group; patients who undergone 5-FU and Capecitabine based chemotherapy (FOLFOX, XELOX, FOLFIRI and Capecitabine monotherapy) alone or in combination with biological agents; patients who undergone pre-treatment assay of 5-FUDR and characterization of polymorphisms of TSER, MTHFR and DPYD genes.
The exclusion criteria were: relevant diseases within 6 months (i.e.: myocardial infarction, lung fibrosis, etc); 5FU based chemotherapy in the past.
The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the institutional ethic committee.
Germinal polymorphisms were analyzed. Genomic DNA was isolated from peripheral blood using the X-tractor Gene system (Corbett Life Science, Australia). The splice-site polymorphism, IVS14+1G>A in the DPYD gene, C677T and A1298C SNPs in MTHFR gene were analyzed using the commercial kit for fluoropyrimidine response (Diatech, Jesi, Italy) according to manufacturer’s protocol. Briefly, region covering the SNP of interest was amplified by PCR, using specific primers, and then sequenced, using the Pyrosequencer PyroMark ID system (Biotage AB and Biosystems, Uppsala, Sweden). The variable number of tandem repeats (VNTR; 2R or 3R) in TSER was determined by PCR according to manufacturer’s protocol (fluoropyrimidine response—Diatech, Jesi, Italy) and visualized onto 2,2% agarose gel.
Determination of the individual 5FU degradation rate.
The test was performed using a HPLC-MS/MS instrument including an Agilent 1100 chromatographic system coupled to an API 3200 triple quadrupole (ABSCIEX, Framingham, MA, USA).  Briefly, freshly prepared peripheral blood mononuclear cells (2.5–3.5 x 106 cells) are incubate at 37°C, with shaking, with a known amount of 5-FU. Cells aliquots are drawn at time 0, 1 h and 2 h, lysed and centrifuged and the concentration of 5-FU in the supernatants is quantified by HPLC-MS/MS. The 5-FUDR is expressed as ng 5-FU/ml/106 cells/min.
Chemotherapy response, toxicity and survival
Chemotherapy cycles were administered every 2 or 3 weeks until disease progression or the development of unacceptable toxicity. We focused in patients undergoing chemotherapy consisting of FOLFOX, FOLFIRI, XELOX and Capecitabine with or without BEVACIZUMAB or CETUXIMAB.
Radiological response was assessed with RECIST Criteria. All toxicity was graded according to the National Cancer Institute Common Toxicity Criteria and toxicity assessments performed at day 1 of each cycle until the end of treatment. Patients were also analyzed according to disease control rate (complete response, partial response and stable disease) and progressive disease. Progression-free survival (PFS) was defined as the time from treatment beginning until the first documented tumour progression or death from any cause. Overall survival (OS) was defined as the time from treatment beginning to death from any cause.
Data analysis and statistics
Patients' data were shown as mean ± SD or median (range) as appropriate. Metabolic classes were determined according to the degradation rates as reported in our previous published article.  Box plots of time to progression according the metabolic classes and toxicity were used to show variability among groups. Chi-square test was calculated according to investigated groups.
Patients were also analyzed according to disease control rate (complete response, partial response and stable disease) and progressive disease.
Kaplan-Meier curves were generated according the metabolic classes. Cox multivariate analysis was calculated.
All tests were two-sided, and differences were considered significant at P < 0.05.
All statistics were calculated using R-Package (version 3.1).
133 metastatic colorectal cancer patients were evaluated in this study. Clinical characteristics and genotype frequencies for MTHFR677/1298, TSER and DPYD are reported in Table 1.
MTHFR677, MTHFR1298 and TSER resulted mutated (heterozygous or homozygous mutated) in 72.2%, 48.2% and 73% of cases, respectively, while DPYD was heterozigously mutated in only one case (0.8%).
The median 5-FUDR value in the overall population was 1.610 (0.460–2.570) ng/ml/106 cells/min.
Overall, 5-FUDR was ≤ 0.85 ng/mL/min in 8 patients (6% of the cases as poor metabolizers, PM) between 0.85 and 2.2 ng/mL/min in 119 patients (89% of the cases as normal metabolizers) and ≥ 2.2 ng/mL/min in 6 patients (5% of the cases as ultra-rapid metabolizers, UM).
Information on clinical response to treatment was available for all the 133 patients studied. The median PFS was 8 months, while the median OS was 28 months. No significant associations at the univariate and multivariate analysis were observed between OS and genetic polymorphisms or metabolic classes.
PFS of patients with normal 5-FU-DR was lower than PFS of patients with altered 5-FUDR (8 vs 11 months, p = 0.03). (Fig 2)
The boxplot of time to progression according to the metabolic classes is shown in Fig 3. The median time to progression was higher for patient with a poor than normal and ultra-metabolizer (14.5 vs. 7.5 and 10.5 months respectively).
Severe toxicities (grade 3 or 4 toxicity) were encountered in 28 patients (21%) and were found to be significantly associated (p = 0.019) with a 5-FUDR below the 5th centile (PM) or above the 95th centile (UM). In particular, the severe toxicity rate was 50% in PM and UM while it was 17.6% in the remaining patients.
In the investigated cohort, 37 patients received a reduced chemotherapy dose. 7 PM subjects received a reduced dose (88.5% of the cases); while in normal and UM groups 23.5% and 33.3% of patients received a reduced dose of 5-FU (p = 0.0004). (Table 3)
The aims of this study was to investigate the efficacy of 5-FU degradation rate and genetic polymorphisms (MTHFR, DPYD, and TSER) as prognostic and predictive parameter for progression free survival.
So far, some studies have investigated the MTHFR, DPYD and TSER genotypes as predictors of toxicity to 5-FU-based chemotherapy. [17, 18, 23–33] The most consistent evidence concerns the DPYD gene, demonstrating an association between severe toxicity and the presence of the polymorphism. [17, 18, 29–33] Interestingly, a case-cohort analysis on the patients enrolled in the phase III CAIRO2 trial showed that DPYD polymorphisms are related to grade 3–4 toxicities, with a trend toward increased overall survival.  Moreover, a recent published study, performed on 2038 patients, demonstrated that with a pharmacokinetically guided dose adjustments of 5-FU the incidence and severity of adverse events were significantly reduced, with drug related death decrease from 10% to 0% and G3-4 toxicities risk reduced from 73% to 28%. Unfortunately, the authors did not present any results about clinical outcomes, despite 50% dose reduction.  Recently, an increasing interest has been shown in identifying prognostic and predictive factor through gene polymorphisms’analysis. Actually, several studies have shown an association between response to treatment and polymorphisms in genes encoding enzymes involved in 5-fluorouracil metabolism, but none of these is considered a prognostic factor in clinical practice. [34–46]
In this study, the distribution of MTHFR677/1298, TSER and DPYD polymorphisms was similar to those described in other Caucasian populations. [47, 48] In our series only one patient presented mutation for DPYD so we could not adequately evaluate the association to efficacy and safety. The effect of genetic polymorphisms of DPYD, TSER, MTHFR and degradation rate of 5-FU on survival was studied. No significant differences in terms of PFS and OS related with MTHFR677/1298, TSER and DPYD polymorphisms were found. These results concur with several other studies that used FOLFOX or FOLFIRI [49–51], but not with tree others that used 5-FU monotherapy and reported better response for patients with the mutated MTHFR677/1298 genotypes [52–54].
Interesting associations with outcomes were found when 5-FUDR was studied. Patients with a low 5-FUDR (≤ 0.85 ng/ml/min) and high 5-FUDR (≥ 2,2 ng/ml/min) presented a significant increase in PFS at the univariate analysis, compared to patients with a normal 5-FUDR. Instead we didn’t find any associations between 5-FUDR and OS, but it could depend that patients lost at follow up reduced the sample size for the analysis of OS.
Moreover, poor metabolizing patients presented a better progression free survival, even though 7 of 8 patients received a reduced dose of 5-FU. This result, enlightens the remarkable finding that probably the pharmacogenetic of these patients allows a longer and effective persistence of 5-FU during treatment.
Surprisingly, a better outcome and higher toxicity grade (3 patients of 6; 50%) was observed also in UM group. So far a relationship between increased toxicity and/or better outcomes with fast drug metabolism was not reported in literature. The faster 5-FU consumption, expressed with a higher value of 5-FUDR, should be related to an increased DPD activity, with 5-FU inactive metabolites raise, or with an augmented activity of enzymes involved in 5-FU active metabolites production, i.e. orotate phosphoribosyltransferase (ORPT), thymidine phosphorylase (TP) and uridine phosphorilase (UP).  In this regard, literature data showed that 5-FU sensitivity is associated with OPRT gene polymorphisms and OPRT/DPD activity ratio. [56–58] This finding leads to the hypothesis that UM show better PFS and higher rate of severe toxicities, due to the increased amount of 5-FU active metabolites. (Fig 4)
The present study focused on the importance of degradation rate of 5-FU and genetic polymorphisms in predicting toxicity and survival of metastatic colorectal cancer patients who underwent 5-FU based chemotherapy. The Authors demonstrated the relevance of 5-FU degradation rate analysis on avoidance of adverse event occurrence, and its role in predicting survival.
Further prospective studies are needed in order to validate and verify these novel and relevant findings. OPRT, UP and TP gene analysis and the dosage of 5-FU metabolites are required to better understand pharmacokinetics mechanisms involved.
- Conceptualization: AB MB.
- Data curation: AB MO.
- Formal analysis: LS.
- Investigation: AB GG LL.
- Methodology: AB MB.
- Project administration: MS PM FM.
- Resources: GG LL.
- Software: LS.
- Supervision: MS PM FM.
- Validation: LS.
- Writing – original draft: AB CEO.
- Writing – review & editing: BC AR LM CS.
- 1. Giachetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, 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. 2000; 18 (1): 136–147. pmid:10623704
- 2. De Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, et al. Leucovorin and fluorouracil with or without oxaliplatin as firstline treatment in advanced colorectal cancer. J Clin Oncol. 2000; 18 (16): 2938–2947. pmid:10944126
- 3. Cassidy J, Tabernero J, Twelves C, Brunet R, Butts C, Conroy T, et al. XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer. J Clin Oncol. 2004; 22 (11): 2084–2091. pmid:15169795
- 4. Colucci G, Gebbia V, Paoletti G, Giuliani F, Caruso M, Gebbia N, et al. Phase III randomised trial of FOLFIRI versus FOLFOX4 in the treatment of advanced colorectal cancer: a multicenter study of the Gruppo Oncologico Dell’italia Meridonale. J Clin Oncol. 2005; 23 (22): 4866–4875. pmid:15939922
- 5. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003; 3 (5):330–338 pmid:12724731
- 6. Marcuello E, Altes A, Menoyo A, Rio ED, Baiget M. Methylenetetrahydrofolate reductase gene polymorphisms: genomic predictors of clinical response to fluoropyrimidine-based chemotherapy? Cancer Chemother Pharmacol. 2006; 57 (6): 835–840. pmid:16187112
- 7. Sharma R, Hoskins JM, Rivory LP, Zucknick M, London R, Liddle C, et al. Thymidylate Synthase and Methylenetetrahydrofolate Reductase Gene Polymorphisms and Toxicity to Capecitabine in Advanced Colorectal Cancer Patients. Clin Cancer Res. 2008;14 (3): 817–825 pmid:18245544
- 8. Jakobsen A, Nielsen JN, Gyldenkerne N, Lindeberg J. Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol. 2005; 23 (7): 1365–1369. pmid:15735113
- 9. Etienne-Grimaldi M-C, Francoual M, Formento JL, Milano G. Methylenetetrahydrofolate reductase (MTHFR) variants and fluorouracil-based treatments in colorectal cancer. Pharmacogenomics. 2007; 8 (11): 1561–1566. pmid:18034621
- 10. Etienne-Grimaldi MC, Bennouna J, Formento JL, Douillard JY, Francoual M, Hennebelle I, et al. Multifactorial pharmacogenetic analysis in colorectal cancer patients receiving 5-fluorouracil-based therapy together with cetuximab-irinotecan. Br J Clin Pharmacol. 2012; 73 (5): 776–785. pmid:22486600
- 11. Thomas F, Motsinger-Reif AA, Hoskins JM, Dvorak A, Roy S, Alyasiri A, et al. Methylenetetrahydrofolate reductase genetic polymorphisms and toxicity to 5-FU-based chemoradiation in rectal cancer. Br J Cancer. 2011; 105 (11): 1654–1662. pmid:22045187
- 12. Etienne MC, Formento JL, Chazal M, Francoual M, Magne N, Formento P, et al. Methylenetetrahydrofolate reductase gene polymorphisms and response to fluorouracil-based treatment in advanced colorectal cancer patients. Pharmacogenetics. 2004; 14 (12): 785–792. pmid:15608557
- 13. Ruzzo A, Graziano F, Loupakis F, Rulli E, Canestrari E, Santini D, et al. Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFOX-4 chemotherapy. J Clin Oncol. 2007; 25 (10): 1247–1254. pmid:17401013
- 14. Capitain O, Boisdron-Celle M, Poirier AL, Abadie-Lacourtoisie S, Morel A, Gamelin E. The influence of fluorouracil outcome parameters on tolerance and efficacy in patients with advanced colorectal cancer. Pharmacogenomics J. 2008; 8 (4): 256–267. pmid:17700593
- 15. Deenen MJ, Rosing H, Hillebrand J, Schellens JH, Beijnen JH. Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2013; 913–914: 30–40. pmid:23270936
- 16. Heggie GD, Sommadossi JP, Cross DS, Huster WJ, Diasio RB. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res. 1987; 47 (8): 2203–2206. pmid:3829006
- 17. Deenen MJ, Tol J, Burylo AM, Doodeman VD, de Boer A, Vincent A, et al. Relationship between single nucleotide polymorphisms and haplotypes in DPYD and toxicity and efficacy of capecitabine in advanced colorectal cancer. Clin Cancer Res. 2011; 17 (10): 3455–3468. pmid:21498394
- 18. Deenen MJ, Meulendijks D, Cats A, Sechterberger MK, Severens JL, Boot H, et al. Upfront genotyping of DPYD*2A to individualize fluoropyrimidine therapy: a safety and cost analysis. J Clin Oncol. 2016; 34(3): 227–234. pmid:26573078
- 19. Ciccolini J, Mercier C, Blachon MF, Favre R, Durand A, Lacarelle B. A simple and rapid high-performance liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD) activity. J Clin Pharm Ther. 2004; 29 (4): 307–315. pmid:15271097
- 20. Lostia AM, Lionetto L, Ialongo C, Gentile G, Viterbo A, Malaguti P, et al. A liquid chromatography-tandem mass spectrometry method for the determination of 5-Fluorouracil degradation rate by intact peripheral blood mononuclear cells. Ther Drug Monit. 2009; 31(4): 482–488. pmid:19571774
- 21. Gentile G, Botticelli A, Lionetto L, Mazzuca F, Simmaco M, Marchetti P, et al. Genotype-phenotype correlations in 5-fluorouracil metabolism: a candidate DYPD haplotype to improve toxicity prediction. Pharmacogenomics J. 2015 Jul 28.
- 22. Mazzuca F, Borro M, Botticelli A, Mazzotti E, Marchetti L, Gentile G, et al. Pre-treatment evaluation of 5-fluorouracil degradation rate: association of poor and ultra-rapid metabolism with severe toxicity in a colorectal cancer patients cohort. Oncotarget. 2016 Mar 8.
- 23. Joerger M, Huitema AD, Boot H, Cats A, Doodeman VD, Smits PH, et al. Germline TYMS genotype is highly predictive in patients with metastatic gastrointestinal malignancies receiving capecitabine-based chemotherapy. Cancer Chemother Pharmacol. 2015; 75(4):763–772. pmid:25677447
- 24. Loganayagam A, Arenas Hernandez M, Corrigan A, Fairbanks L, Lewis CM, Harper P, et al. Pharmacogenetic variants in the DYPD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity. Br J Cancer. 2013; 108 (12): 2505–2515. pmid:23736036
- 25. Chua W, Goldstein D, Lee CK, Dhillon H, Michael M, Mitchell P, et al. Molecular markers of response and toxicity to FOLFOX chemotherapy in metastatic colorectal cancer. Br J Cancer. 2009; 101 (6): 998–1004. pmid:19672255
- 26. Sharma R, Hoskins JM, Rivory LP, Zucknick M, London R, Liddle C, et al. Thymidylate Synthase and Methylenetetrahydrofolate Reductase Gene Polymorphisms and Toxicity to Capecitabine in Advanced Colorectal Cancer Patients. Clin Cancer Res- 2008; 14 (3): 817–825. pmid:18245544
- 27. Rosmarin D, Palles C, Church D, Domingo E, Jones A, Jonhstone et al. Genetic markers of toxicity from capecitabine and other fluorouracil-based regimens: investigation in the QUASAR2 study, systematic review, and meta-analysis. J Clin Oncol. 2014; 32 (10): 1031–1039. pmid:24590654
- 28. Afzal S, Gusella M, Vainer B, Vogel UB, Andersen JT, Broedbaek K, et al. Combinations of polymorphisms in genes involved in the 5-Fluorouracil metabolism pathway are associated with gastrointestinal toxicity in chemotherapy-treated colorectal cancer patients. Clin Cancer Res. 2011;17(11):3822–3829. pmid:21471424
- 29. Leung HW, Chan AL. Association and prediction of severe 5-fluorouracil toxicity with dihydropyrimidine dehydrogenase gene polymorphisms: A meta-analysis. Biomed Rep. 2015; 3(6): 879–883. pmid:26623034
- 30. Meulendijks D, Henricks LM, Sonke GS, Deneen MJ, Froehlich TK, Amstutz U, et al. Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data. Lancet Oncol. 2015;16(16):1639–1650. pmid:26603945
- 31. Toffoli G, Giodini L, Buonadonna A, Berretta M, De Paoli A, Scalone S, et al. Clinical validity of a DPYD-based pharmacogenetic test to predict severe toxicity to fluoropyrimidines. Int J Cancer. 2015;137(12):2971–80. pmid:26099996
- 32. Lee AM, Shi Q, Pavey E, Alberts SR, Sargent DJ, Sinicrope FA, et al. DPYD variants as predictors of 5-fluorouracil toxicity in adjuvant colon cancer treatment (NCCTG N0147). J Natl Cancer Inst. 2014;106(12).
- 33. Rosmarin D, Palles C, Pagnamenta A, Kaur K, Pita G, Martin M, et al. A candidate gene study of capecitabine-related toxicity in colorectal cancer identifies new toxicity variants at DPYD and a putative role for ENOSF1 rather than TYMS. Gut. 2015;64(1):111–120. pmid:24647007
- 34. Huang K, Shen Y, Zhang F, Wang S, Wei X. Evaluation of effects of thymidylate synthase and excision repair cross-complementing 1 polymorphism on chemotherapy outcome in patients with gastrointestinal tumors using peripheral venous blood. Oncol Lett. 2016;11(5):3477–3482. pmid:27123139
- 35. Wu NC, Su SM, Lin TJ, Chin J, Hou CF, Yang JY, et al. Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and fluorouracil-based treatment in Taiwan colorectal cancer. Anticancer Drugs. 2015;26(8):888–93. pmid:26111049
- 36. Fernández-Peralta AM, Daimiel L, Nejda N, Iglesias D, Medina Arana V, González-Aguilera JJ. Association of polymorphisms MTHFR C677T and A1298C with risk of colorectal cancer, genetic and epigenetic characteristic of tumors, and response to chemotherapy. Int J Colorectal Dis. 2010;25(2):141–151. pmid:19669769
- 37. Yhim HY, Cho SH, Kim SY, Cho IS, Lee KT, Lee WS, et al. Prognostic implications of thymidylate synthase gene polymorphisms in patients with advanced small bowel adenocarcinoma treated with first-line fluoropyrimidine-based chemotherapy. Oncol Rep. 2015;34(1):155–64. pmid:25955097
- 38. Sun W, Yan C, Jia S, Hu J. Correlation analysis of peripheral DPYD gene polymorphism with 5-fluorouracil susceptibility and side effects in colon cancer patients. Int J Clin Exp Med. 2014;7(12):5857–5861. pmid:25664120
- 39. Zhao J, Li W, Zhu D, Yu Q, Zhang Z, Sun M, et al. Association of single nucleotide polymorphisms in MTHFR and ABCG2 with the different efficacy of first-line chemotherapy in metastatic colorectal cancer. Med Oncol. 2014;31(1):802. pmid:24338217
- 40. Kumamoto K, Ishibashi K, Okada N, Tajima Y, Kuwabara K, Kumagai Y, et al. Polymorphisms of GSTP1, ERCC2 and TS-3'UTR are associated with the clinical outcome of mFOLFOX6 in colorectal cancer patients. Oncol Lett. 2013;6(3):648–654. pmid:24137384
- 41. Jang MJ, Kim JW, Jeon YJ, Chong SY, Hong SP, Hwang SG, et al. Polymorphisms of folate metabolism-related genes and survival of patients with colorectal cancer in the Korean population. Gene. 2014;533(2):558–564. pmid:24100087
- 42. Sulzyc-Bielicki D, Binczak-Kuleta A, Kaczmarczyk M, Pioch W, Machoy-Mokrzynska A, Ciechanowicz A, et al. Thymidylate synthase gene polymorphism and survival of colorectal cancer patients receiving adjuvant 5-fluorouracil. Genet Test Mol Biomarkers. 2013;17(11):799–806. pmid:23968134
- 43. Teh LK, Hamzah S, Hashim H, Bannur Z, Zakaria ZA, Hasbullani Z, Shia JK, Fijeraid H, Md Nor A, Zailani M, Ramasamy P, Ngow H, Sood S, Salleh MZ. Potential of dihydropyrimidine dehydrogenase genotypes in personalizing 5-fluorouracil therapy among colorectal cancer patients. Ther Drug Monit. 2013;35(5):624–630. pmid:23942539
- 44. Wang YC, Xue HP, Wang ZH, Fang JY. An integrated analysis of the association between Ts gene polymorphisms and clinical outcome in gastric and colorectal cancer patients treated with 5-FU-based regimens. Mol Biol Rep. 2013;40(7):4637–4644. pmid:23645036
- 45. Negandhi AA, Hyde A, Dicks E, Pollett W, Younghusband BH, Parfrey P, et al. MTHFR Glu429Ala and ERCC5 His46His polymorphisms are associated with prognosis in colorectal cancer patients: analysis of two independent cohorts from Newfoundland. PLoS One. 2013;8(4):e61469. pmid:23626689
- 46. Zhu L, Wang F, Hu F, Wang Y, Li D, Dong X, et al. Association between MTHFR polymorphisms and overall survival of colorectal cancer patients in Northeast China. Med Oncol. 2013;30(1):467. pmid:23392576
- 47. Ruzzo A, Graziano F, Loupakis F, Santini D, Catalano V, Bisonni R, et al. Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFIRI chemotherapy. Pharmacogenomics J. 2008; 8(4): 278–288. pmid:17549067
- 48. Seck K, Riemer S, Kates R, Ulrich T, Lutz V, Harbeck N, et al. Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals. Clin Cancer Res. 2005; 11(16): 5886–5892. pmid:16115930
- 49. Marcuello E, Altes A, Menoyo A, Rio ED, Baiget M. Methylenetetrahydrofolate reductase gene polymorphisms: genomic predictors of clinical response to fluoropyrimidine-based chemotherapy? Cancer Chemother Pharmacol 2006; 57 (6): 835–40. pmid:16187112
- 50. Suh KW, Kim JH, Kim DY, Kim BY, Lee C, Choi S. Which gene is a dominant predictor of response during FOLFOX chemotherapy, the MTHFR or XRCC1 gene? Ann Surg Oncol. 2006; 13 (11): 1379–1385. pmid:17009149
- 51. Ruzzo A, Graziano F, Loupakis F, Rulli E, Canestrari E, Santini D, et al. Pharmacogenomic profiling in patients with advanced colorectal cancer treated with first-line FOLFOX-4 chemotherapy. J Clin Oncol. 2007; 25 (10): 1247–1254. pmid:17401013
- 52. Jakobsen A, Nielsen JN, Gyldenkerne N, Lindeberg J. Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol. 2005; 23 (7): 1365–1369. pmid:15735113
- 53. Etienne-rimaldi M-C, Francoual M, Formento JL, Milano G. Methylenetetrahydrofolate reductase (MTHFR) variants and fluorouracil-based treatments in colorectal cancer. Pharmacogenomics. 2007; 8 (11): 1561–1566. pmid:18034621
- 54. Zhang W, Press OA, Haiman CA, Yang DY, Gordon MA, Fazzone W, et al. Association of methylenetetrahydrofolate reductase gene polymorphisms and sex-specific survival in patients with metastatic colorectal cancer. J Clin Oncol. 2007; 25 (24): 3726–3731. pmid:17704422
- 55. Sakamoto E, Nagase H, Kobunai T, Oie S, Oka T, Fukushima M, et al. Orotate phosphoribosyltrasferase expression level in tumors is a potential determinant of the efficacy of 5-fluorouracil. Biochem Biophys Res Commun. 2007; 363 (1): 216–222. pmid:17854773
- 56. Tsunoda A, Nakao K, Watanabe M, Matsui M, Ooyama A, Kusano M. Associations of various gene polymorphisms with toxicity in colorectal cancer patients receiving oral uracil and tegafur plus leucovorin: a prospective study. Ann Oncol. 2011; 22 (2): 355–361. pmid:20647221
- 57. Furuse H, Hirano Y, Harada M, Hong Ming L, Aoki T, Kurita Y, et al. Significance of 5-fluorouracil-related enzyme activities in predicting sensitivity to 5-fluorouracil in bladder carcinoma. Anticancer Res. 2009; 29 (4): 1001–1008. pmid:19414338
- 58. Ochiai T, Umeki M, Miyake H, Iida T, Okumura M, Ohno K, et al. Impact of 5-fluorouracil metabolizing enzymes on chemotherapy in patients with resectable colorectal cancer. Oncol Rep. 2014; 32 (3): 887–892. pmid:24994673