The Role of Dihydropyrimidine Dehydrogenase and Thymidylate Synthase Polymorphisms in Fluoropyrimidine-Based Cancer Chemotherapy in an Iranian Population


PDF - Export to EndNote - PubMed Central XML format - PubMed Central XML format - PubMed Central XML format
PMID: 32695278 (PubMed) - PMCID: PMC7368113 - View online: PubReader
Volume 12, Issue 3, July-September , Page 157 to 164
Monday, December 9, 2019 :Received , Monday, April 20, 2020 :Accepted



  • - Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

  • - Department of Hematology and Oncology, Hazrat Rasool-e Akram Hospital, Iran University of Medical Sciences, Tehran, Iran
    - Cancer Pharmacogenetics Research Group (CPGRG), Iran University of Medical Sciences, Tehran, Iran

  • - Cancer Pharmacogenetics Research Group (CPGRG), Iran University of Medical Sciences, Tehran, Iran
    - Department of Medical Genetic, Medical Biotechnology Ins., National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

  • - Department of Medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

  • - Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
  • Corresponding author Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran, Tel: +98 21 44787301; E-mail: Iranhealth@hotmail.com

Abstract:

Background: The fluoropyrimidine drug 5-Fluorouracil (5-FU) and the prodrug capecitabine have been extensively used for treatment of many types of cancer including colorectal, gastric, head and neck. Approximately, 10 to 25% of patients suffer from severe fluoropyrimidine-induced toxicity. This may lead to dose reduction and treatment discontinuation. Pharmacogenetics research could be useful for the identification of predictive markers in chemotherapy treatment. The aim of the study was to investigate the role of five genetic polymorphisms within two genes (DPYD, TYMS) in toxicity and efficacy of fluoropyrimidine-based chemotherapy.

Methods: Total genomic DNA was extracted from 83 cancer patients treated with fluoropyrimidine-based chemotherapy. In this study, three polymorphisms were genotyped in dihydropyrimidine dehydrogenase gene c.1905+1 G>A (DPYD*2A;  rs3918290), c.1679 T>G (I560S; DPYD*13; rs55886062), and c.2846A>T (D949V; rs67376798) and two polymorphisms, besides the Variable Number of Tandem Repeat (VNTR) polymorphism and 6-bp insertion/deletion polymorphism in thymidylate synthase gene. The analysis of polymorphisms for rs3918290, rs55886062, rs67376798 and 6-bp insertion/deletion in TYMS was done by Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCR-RFLP) TYMS VNTR analysis. 5-FU-related toxicities such as anemia, febrile neutropenia, neurotoxicity, vomiting, nausea, and mucositis were evaluated according to NCI-CTC criteria version 4.0. T-test and chi-square were used and p-values less than 0.05 were considered statistically significant.

Results: DPYD gene polymorphisms were not observed in this study. The frequency of the TYMS +6 bp allele was 40.35% and the -6 bp allele was 59.65% in this study. The frequency of VNTR 2R allele was 48.75% and 3R allele was 51.15%. Toxicity grade II diarrhea, mucositis, nausea, vomiting, and neurotoxicity was 2.2, 24.1, 15.7, 6, and 51.8%, respectively. Thymidylate synthase ins/del polymorphisms were associated with increased grade III neurotoxicity (p=0.02). Furthermore, anemia grade III was significantly associated with 2R/2R genotype (0.009).

Conclusion: Thymidylate synthase gene polymorphisms may play a key role in fluoropyrimidne -based chemotherapy. Although rare DPYD polymorphisms were not observed in our study, according to large population studies, DPYD gene polymorphisms could be used as a predictive biomarker for patient treatments.


 

 


Introduction :

The fluoropyrimidine drug 5-Fluorouracil (5-FU) and the prodrug capecitabine are used for treatment of 
a variety of solid cancers including gastrointestinal tract and breast 1.

5-FU/leucovorin combined with oxaliplatin (FOLFOX) is currently a standard chemotherapy regimen in treating colorectal and gastric cancers and has been shown clearly to improve response to treatment 2,3. However, the development of gastrointestinal, hematological and neurological toxicities is a major clinical problem following FOLFOX treatment 4. The clinical utility of DPYD polymorphisms has been demonstrated in a panel of markers to predict clinically actionable 5-FU toxicity. Other polymorphisms have been suggested as markers in TYMS, Enolase Superfamily Member 1 (ENOSF1) 5, MethyleneTetraHydroFolate Reductase (MTHFR) 6, ATP-binding cassette sub-family B member 1 (ABCB1) 7, and Cytidine Deaminase (CDA) 8.

Approximately, 85% of administered 5-FU dose is degraded via dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) 9. Functional Single-Nucleotide Polymorphisms (SNPs) in the DPYD gene alter DPD activity which may lead to the development of severe 5-FU related toxicities. The clinically most relevant variant is DPYD*2A (c.1905+1G>A, previously named IVS14+1G>A or DPD*2A), or rs3918290 10-13; DPYD*13 (c.1679T>G (rs55886062) and c.2846A>T (rs67376798) also result in low DPD activity and/or 5-fluorouracil toxicity 12-17.

A Variable Number of 28 bp Tandem Repeat (VNTR) within the promoter enhancer region (TSER) of TYMS (Thymidylate Synthase gene) is usually presented as a double-tandem repeat (2R) or a triple-tandem repeat (3R) 18-20. Second polymorphism is a 6-bp insertion/deletion in the 3ʹ-untranslated region (3ʹ-UTR) of TYMS 21. These two polymorphisms have been associated with altered TYMS expression, toxicity and an improved clinical response 13,22,23.

The aim of this study was to determine whether the five genetic polymorphisms within two genes (DPYD, TYMS) are associated with severe toxicity in patients with cancer receiving fluoropyrimidine-based chemotherapy.

 


Materials and Methods :

Patients and study design: Eighty three cancer patients received 5-FU-based chemotherapy at Hazrat-e Rasool hospital and Masoud clinic, Tehran, Iran between February 2014 and June 2016.  The cancer types among the patients were distributed as follows: 28 colon (33.7%), 37 rectum and rectosigmoid (44.5%), and 18 stomach (21.6%). Patients were questioned about nausea and vomiting, mucositis, diarrhea, hand-foot syndrome, and neutropenia at every cycle. Eligible patients were treated with FOLFOX4 (Oxaliplatin 85 mg/m2 (2 hr infusion on day 1), leucovorin (100 mg/m2 as 2 hr infusion on day 1), 5- fluorouracil bolus (400 mg/m2) and 22 hr infusion (600 mg/m2) on days 1 and 2 every 2 weeks for 6 months (12 cycles). Toxicity was evaluated according to Common Terminology Criteria for Adverse Events (CTCAE), version 4 24. Neurotoxicity was evaluated according to the oxaliplatin-specific scale. The study was approved by the ethics committees (NIGEB) (940101-IV-503). All patients signed a written informed consent before entering the study.

Genotyping: Total genomic DNA was extracted from 200 µl whole blood using the GeneAll® kit (South Korea) and stored at -20°C until genotyping. The TYMS VNTR polymorphism was amplified by polymerase chain reaction with Taq DNA Polymerase 2×Master Mix RED (Amplicon, Denmark). Other polymorphisms were performed using a PCR–RFLP technique. RFLP analysis was performed with fast digest enzymes (Thermo Fisher Scientific, the USA). After restriction enzyme analysis, PCR fragments were visualized in a 2.5-3% agarose gel. The information of studied genetic variants is shown in table 1.

Statistical analysis: Statistical analysis was done by SPSS software version 21. The quantitative analysis was performed by descriptive variables. T-test was used for comparison of different groups and chi-square was used for qualitative analysis. p-value less than 0.05 was considered statistically significant.

 


Results :

Genotyping: Eighty three cancer patients (58 males, 25 females, and mean age of 57.17 years old, range of 23-86 years) were evaluated in this study. Three variants in DPYD gene were not polymorphic in this study (Table 2, Figure 1). The distribution of the rs45445694 genotype was 27.7% for 2R/2R, 30.1% for 2R/3R and 42% for 3R/3R. The distribution of the rs16430 was 38.5% for del6/del6, 42.1% for del6/ins6 and 19% for ins6/ ins6 (Table 3).

Toxicity: Toxicity data included hematological toxicities (anemia and neutropenia) in 50 patients and nonhematologic toxicities (nausea, vomiting, diarrhea, mucositis, neurotoxicity) in 83 patients. In this study, 20% (17/83) developed non-hematological grade III-IV toxicity and 29% (13/44) developed hematological grade III-IV toxicity. Anemia (18%) was the most common severe hematological toxicity, whereas mucositis (3%) and neurotoxicity (2%) were the most frequent non-hematological severe toxicities. Also, 68% of patients experienced neurotoxicity as the most severe toxicity of any grade developed during the treatment.  Thymidylate synthase ins/del polymorphisms were significantly associated with increased grade III neurotoxicity (p=0.02). Anemia grade III was significantly associated with 2R/2R genotype (0.009) (Table 4).

 


Discussion :

Colorectal cancer is the second most common cancer in women and the third most common cancer in men worldwide 31. Colorectal cancer incidence is increasing in Iran, China and South Korea 32,33. Fluorouracil in combination with leucovorin, and oxaliplatin (FOLFOX) or with irinotecan (FOLFIRI) is the backbone of chemotherapy treatment for colorectal and gastric cancers 28,34,35. Gene polymorphisms are associated with cancer susceptibility and response to treatment in different types of cancer 36.

The aims of this study were to investigate the association of DPYD rare risk variants and TYMS polymorphisms and toxicity that may help predict the response to fluorouracil-based chemotherapy. These polymorphisms were evaluated in 83 colorectal and gastric cancer patients treated with FOLFOX regimen.

Dihydropyrimidine Dehydrogenase (DPD) is responsible for conversion of 5-FU to 5-fluorodihydro-uracil (5-FUH2) and plays a key role in the catabolism of 5-FU. Deficiency in DPD activity leads to severe toxicity which may lead to treatment discontinuation or even death. Three DPYD variants of rs3918290, rs55886062 and rs67376798 were associated with decreased DPD enzyme activity and a high risk of severe 5-FU toxicity 5,37-39. The development of severe toxicity results in dose reduction, discontinuation of treatment or even death in cancer patients 40-43.

Regarding the low minor allele frequency of the DPYD rare risk variants, three DPYD variants of rs3918290, rs55886062 and rs67376798 were not identified in the study group. This finding is consistent with some small studies 26. Similar to our study, rs3918290 polymorphism was not found in Korean, Taiwanese, Turkish, Caucasian, and African populations 44-47. In some large studies, heterozygous patients for the rs3918290 experienced severe toxicity 23,48. Some reported toxicity in just 46% of patients with rs3918290 polymorphism 13. For the rs67376798 variant, previous studies reported that 75% of patients with a heterozygous genotype experienced severe toxicity in the first two cycles of therapy 23. Other studies showed 60% or 62% of heterozygous patients experiencing toxicity within the first two cycles 13,48. The single patient with a heterozygous rs55886062 genotype experienced late toxicity after cycle 2. Loganayagam et al reported, in the largest study to date, the incidence of grade ≥3 5FU-toxicities in rs3918290, rs55886062, and rs67376798 carriers as 88.0, 50.0, and 81.5%, respectively, whereas incidence of grade≥3 overall toxicities was 88.0, 75.0, and 88.9%, respectively. Carrier of rs3918290, rs55886062 and rs37376798 had at least one grade IV toxicity of  64.0, 25.0 and 66.7%, respectively 10.

The Clinical Pharmacogenetics Implementation Consortium (CPIC) recommended reduction of fluoropyrimidine dosage in patients who are heterozygous for rs3918290, rs55886062 and rs67376798 variants which may prevent severe and possibly life-threatening toxicities. The use of 5-fluorouracil or capecitabine is not recommended in patients who are homozygous for rs3918290, rs55886062 and rs67376798 16.

Thymidylate synthase is a main intracellular target of the active 5-FU metabolite, FdUMP, which forms a ternary complex with TS and 5, 10-MTHF. rs3474303 as a variable number of tandem repeats (VNTR) is present in the 5ʹ-untranslated region (5ʹ-UTR) of the TS gene (TYMS). The three 28-bp repeat (3R) is associated with 3-4-fold translational efficiency, compared to two 28-bp repeat. In our study, the two 28-bp repeat and the 6-bp deletion allele frequencies of the TYMS gene were 48.75 and 59.65%, respectively. Significant associations were found between anemia grade III and 2R/2R TYMS genotype (0.009). In our study, association between polymorphism rs3474303 with toxicity is the same as other studies 13,20,29,49-51. 2R/3R or 3R/3R genotypes were significantly associated with a lower risk of toxicity. Although some studies have shown association between rs3474303 52 and toxicity, this association is likely to be small and clinical test would not be useful 5.

The 6-bp deletion in the 3'-UTR region of the TYMS gene affects TYMS mRNA expression 21. In our study, the del/del genotype in rs16430 was significantly associated with increased grade III neurotoxicity (p=0.02). Other studies have shown del/del genotype in rs16430 associated with diarrhea, neutropenia and mucositis (p=0.0123) 23. However, certain other studies have failed to show an association between a homozygous del/del genotype and severe toxicity 53,54.

TS mRNA level predicts fluoropyrimidine and raltitrexed sensitivity in gastric cancer 55. A meta-analysis has shown that response rates for fluoropyrimidine-based chemotherapy were significantly lower in gastric cancer patients with high TS expression 56. In Meulendijks et al’s study, TYMS VNTR 3 R/3 R genotype was formally associated with Objective Response Rate (ORR) 57.

Homozygous genotype rs1801159A/A was associated with response to fluorouracil-based adjuvant chemotherapy in gastric cancer patients 58. Another study revealed that gene polymorphisms of DPYD could be considered as a biomarker for prediction of gastric cancer patients survival treated with 5-fluorouracil-based adjuvant chemotherapy 59.

 


Conclusion :

In conclusion, an association was found between VNTR and TYMS 1494 ins/del polymorphism in TYMS gene and anemia and neurotoxicity, respectively. Due to the relatively limited sample size, DPYD rare risk variants of rs3918290, rs55886062 and rs67376798 were not identified in our study. Further investigations on larger sample size are needed to demonstrate the role of rare risk variants of DPYD gene. In addition, to achieve better biomarkers for FOLFOX chemotherapy regimen, additional variation in genes involved in metabolism of other drugs like oxaliplatin should be investigated.

 


Acknowledgement :

This work is supported in part by a grant BS-1394-01-05 from the Institute for Research in Fundamental Sciences (IPM), Tehran, Iran.The authors acknowledge the assistance of Kioomars Saliminejad in primer design and technical assistance of Shamila Darvishalipoor.

 


Conflict of Interest :

 The authors declare that they have no competing interests.

 



<p>Figure 1. Polymorphism analysis of dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS) polymorphism. A) rs67376798; PCR product digested with BclI; lanes 1,3,5,7 and 9:113+23 <em>bp</em> (Wild type); lanes 2, 4, 6 and 8:136 <em>bp</em> (Undigested control); marker 100 <em>bp</em> (SinaClon, Tehran, Iran). B) rs55886062; PCR product digested with Bsp119I; lanes 1-10: 469+265 <em>bp</em> (Wild type); lanes 11:724 <em>bp</em> (Undigested control) marker 100 <em>bp</em> (Sina-Clon, Tehran, Iran). C) rs 3918290; product digested with Hpy CH4; lanes 1,3,5,7,9,11: 190+54 <em>bp</em> (Wild type); lanes 2,4,6,8,10,12: 244 <em>bp</em> (Undigested control); marker 50 <em>bp</em> (SinaClon, Tehran, Iran). D) rs45445694; lanes 3,9: 276 <em>bp</em> (2 repeat of 28 <em>bp</em> VNTR,2R); lanes 1,4,10: 304 <em>bp</em> (3 repeat of 28 <em>bp</em> VNTR,3R); lanes 2,5,6,7,8: 276+ 304 (2R3R); marker 50 <em>bp</em> (SinaClon, Tehran, Iran). E) TYMS 1494-del TTAAAG; lanes 1,2,10: 60+63 <em>bp</em> (6 <em>bp</em> deletion, del/del); lanes 7,11,12: 123 <em>bp</em> (6 <em>bp</em> insertion,ins/ins) lanes 3-6,8,9: 60+63+123 (ins/del); marker 50 <em>bp</em> (SinaClon, Tehran, Iran).</p>

Figure 1. Polymorphism analysis of dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS) polymorphism. A) rs67376798; PCR product digested with BclI; lanes 1,3,5,7 and 9:113+23 bp (Wild type); lanes 2, 4, 6 and 8:136 bp (Undigested control); marker 100 bp (SinaClon, Tehran, Iran). B) rs55886062; PCR product digested with Bsp119I; lanes 1-10: 469+265 bp (Wild type); lanes 11:724 bp (Undigested control) marker 100 bp (Sina-Clon, Tehran, Iran). C) rs 3918290; product digested with Hpy CH4; lanes 1,3,5,7,9,11: 190+54 bp (Wild type); lanes 2,4,6,8,10,12: 244 bp (Undigested control); marker 50 bp (SinaClon, Tehran, Iran). D) rs45445694; lanes 3,9: 276 bp (2 repeat of 28 bp VNTR,2R); lanes 1,4,10: 304 bp (3 repeat of 28 bp VNTR,3R); lanes 2,5,6,7,8: 276+ 304 (2R3R); marker 50 bp (SinaClon, Tehran, Iran). E) TYMS 1494-del TTAAAG; lanes 1,2,10: 60+63 bp (6 bp deletion, del/del); lanes 7,11,12: 123 bp (6 bp insertion,ins/ins) lanes 3-6,8,9: 60+63+123 (ins/del); marker 50 bp (SinaClon, Tehran, Iran).





<p>Table 1. Characteristics of studied DPYD and TYMS polymorphisms</p>
<p>a: Underlined capital letter labeled nucleotides show mismatch to complementary DNA.</p>
<p>b: F=forward; R=reverse; N=normal; M=mutant.</p>

Table 1. Characteristics of studied DPYD and TYMS polymorphisms

a: Underlined capital letter labeled nucleotides show mismatch to complementary DNA.

b: F=forward; R=reverse; N=normal; M=mutant.





<p>Table 2. Genotype frequencies of DPYD rs3918290, rs67376798, rs55886062 polymorphism</p>

Table 2. Genotype frequencies of DPYD rs3918290, rs67376798, rs55886062 polymorphism





<p>Table 3. Genotype frequencies of TYMS rs45445694 and rs16430 polymorphisms</p>

Table 3. Genotype frequencies of TYMS rs45445694 and rs16430 polymorphisms





<p>Table 4. Association between TYMS polymorphisms, rs16430 and rs3474303, and toxicity after FOLFOX chemotherapy</p>
<br />
a) Toxicity was evaluated according to Common Terminology Criteria for Adverse Events (CTCAE). Neurotoxicity was evaluated according to the oxaliplatin specific scale.
<br />
b) 50 patients were evaluable for anemia.
<br />
c) Chi-square was performed for analyzing association between TYMS polymorphisms, rs16430 and rs3474303, and toxicity. p-values less than 0.05 are statistically significant.

Table 4. Association between TYMS polymorphisms, rs16430 and rs3474303, and toxicity after FOLFOX chemotherapy


a) Toxicity was evaluated according to Common Terminology Criteria for Adverse Events (CTCAE). Neurotoxicity was evaluated according to the oxaliplatin specific scale.
b) 50 patients were evaluable for anemia.
c) Chi-square was performed for analyzing association between TYMS polymorphisms, rs16430 and rs3474303, and toxicity. p-values less than 0.05 are statistically significant.





References :
  1. de Gramont Ad, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 2000;18(16):2938-2947.   [PubMed]
  2. Sargent D, Sobrero A, Grothey A, O'Connell MJ, Buyse M, Andre T, et al. Evidence for cure by adjuvant therapy in colon cancer: observations based on individual patient data from 20,898 patients on 18 randomized trials. J Clin Oncol 2009;27(6):872-877.   [PubMed]
  3. Bokemeyer C, Bondarenko I, Hartmann J, De Braud F, Schuch G, Zubel A, et al. Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Ann Oncol 2011;22(7):1535-1546.   [PubMed]
  4. Bohanes P, Rankin CJ, Blanke CD, Winder T, Ulrich CM, Smalley SR, et al. Pharmacogenetic analysis of INT 0144 trial: association of polymorphisms with survival and toxicity in rectal cancer patients treated with 5-FU and radiation. Clin Cancer Res 2015;21(7):1583-1590.
  5. 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.   [PubMed]
  6. Jiang H, Shen Y. Methylene tetrahydrofolate reductase (MTHFR) gene rs1801133 C> T polymorphisms and response to 5-FU based chemotherapy in patients with colorectal cancer: a meta-analysis. Pteridines 2019;30(1):126-132.
  7. Chen J, Ying X, Zhang L, Xiang X, Xiong J. Influence of TS and ABCB1 gene polymorphisms on survival outcomes of 5‑FU-based chemotherapy in a Chinese population of advanced gastric cancer patients. Wiener klinische Wochenschrift 2017;129(11-12):420-426.   [PubMed]
  8. Liu D, Li X, Li X, Zhang M, Zhang J, Hou D, et al. CDA and MTHFR polymorphisms are associated with clinical outcomes in gastroenteric cancer patients treated with capecitabine-based chemotherapy. Cancer Chemother Pharmacol 2019;83(5):939-949.   [PubMed]
  9. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3(5):330-338.   [PubMed]
  10. 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). pii: dju298.
  11. Boisdron-Celle M, Remaud G, Traore S, Poirier A, Gamelin L, Morel A, et al. 5-Fluorouracil-related severe toxicity: a comparison of different methods for the pretherapeutic detection of dihydropyrimidine dehydrogenase deficiency. Cancer Lett 2007;249(2):271-282.   [PubMed]
  12. Morel A, Boisdron-Celle M, Fey L, Soulie P, Craipeau MC, Traore S, et al. Clinical relevance of different dihydropyrimidine dehydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther 2006;5(11):2895-2904.   [PubMed]
  13. Schwab M, Zanger UM, Marx C, Schaeffeler E, Klein K, Dippon Jr, et al. Role of genetic and nongenetic factors for fluorouracil treatment-related severe toxicity: a prospective clinical trial by the German 5-FU Toxicity Study Group. J Clin Oncol 2008;26(13):2131-2138.   [PubMed]
  14. Offer SM, Lee AM, Mattison LK, Fossum C, Wegner NJ, Diasio RB. A DPYD variant (Y186C) in individuals of African ancestry is associated with reduced DPD enzyme activity. Clin Pharmacol Ther 2013;94(1):158-166.   [PubMed]
  15. Johnson MR, Wang K, Diasio RB. Profound dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype. Clin Cancer Res 2002;8(3):768-774.   [PubMed]
  16. Caudle KE, Thorn CF, Klein TE, Swen JJ, McLeod HL, Diasio RB, et al. Clinical Pharmacogenetics Implementation Consortium guidelines for dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing. Clin Pharmacol Ther 2013;94(6):640-645.   [PubMed]
  17. Iachetta F, Bonelli C, Romagnani A, Zamponi R, Tofani L, Farnetti E, et al. The clinical relevance of multiple DPYD polymorphisms on patients candidate for fluoropyrimidine based-chemotherapy. An Italian case-control study. Br J Cancer 2019;120(8):834-839.   [PubMed]
  18. Horie N, Aiba H, Oguro K, Hojo H, Takeishi K. 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 1995;20(3):191-197.   [PubMed]
  19. Kawakami K, Salonga D, Park JM, Danenberg KD, Uetake H, Brabender J, 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 2001;7(12):4096-4101.   [PubMed]
  20. Pullarkat S, Stoehlmacher J, Ghaderi V, Xiong Y, Ingles S, Sherrod A, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 2001;1(1):65-70.   [PubMed]
  21. Mandola MV, Stoehlmacher J, Zhang W, Groshen S, Yu MC, Iqbal S, et al. A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 2004;14(5):319-327.   [PubMed]
  22. Meulendijks D, Jacobs BA, Aliev A, Pluim D, Van Werkhoven E, Deenen MJ, et al. Increased risk of severe fluoropyrimidine‐associated toxicity in patients carrying a G to C substitution in the first 28‐bp tandem repeat of the thymidylate synthase 2R allele. Int J Cancer 2016;138(1):245-253.   [PubMed]
  23. Loganayagam A, Hernandez MA, Corrigan A, Fairbanks L, Lewis C, Harper P, et al. Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity. Br J Cancer 2013;108(12):2505-2515.   [PubMed]
  24. Health UDo, Services H. Common terminology criteria for adverse events (CTCAE) version 4.0. National Cancer Institute. 2009(09-5410).
  25. 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 Can Res 2011;17(10):3455-3468.   [PubMed]
  26. 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.   [PubMed]
  27. Rosmarin D, Palles C, Church D, Domingo E, Jones A, Johnstone E, 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.   [PubMed]
  28. 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.   [PubMed]
  29. Lecomte T, Ferraz JM, Zinzindohoué F, Loriot MA, Tregouet DA, Landi B, et al. Thymidylate synthase gene polymorphism predicts toxicity in colorectal cancer patients receiving 5-fluorouracil-based chemotherapy. Clin Cancer Res 2004;10(17):5880-5888.   [PubMed]
  30. Gusella M, Frigo A, Bolzonella C, Marinelli R, Barile C, Bononi A, et al. Predictors of survival and toxicity in patients on adjuvant therapy with 5-fluorouracil for colorectal cancer. Br J Cancer 2009;100(10):1549-1557.   [PubMed]
  31. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015;65(2):87-108.   [PubMed]
  32. Zayeri F, Sheidaei A, Mansouri A. Clustering Asian and North African countries according to trend of colon and rectum cancer mortality rates: an application of growth mixture models. Asian Pac J Cancer Prev 2015;16(9):4115-4121.   [PubMed]
  33. Fang JY, Dong HL, Sang XJ, Xie B, Wu KS, Du PL, et al. Colorectal cancer mortality characteristics and predictions in China, 1991-2011. Asian Pac J Cancer Prev 2015;16(17):7991-7995.   [PubMed]
  34. Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med 2005;352(5):476-487.   [PubMed]
  35. Murad AM, Santiago FF, Petroianu A, Rocha PR, Rodrigues MA, Rausch M. Modified therapy with 5‐fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer. Cancer 1993;72(1):37-41.   [PubMed]
  36. Abbasian MH, Abbasi B, Ansarinejad N, Motevalizadeh Ardekani A, Samizadeh E, Gohari Moghaddam K, et al. Association of interleukin-1 gene polymorphism with risk of gastric and colorectal cancers in an Iranian population. Iran J Immunol 2018;15(4):321-328.
  37. Terrazzino S, Cargnin S, Del Re M, Danesi R, Canonico PL, Genazzani AA. DPYD IVS14+ 1G> A and 2846A> T genotyping for the prediction of severe fluoropyrimidine-related toxicity: a meta-analysis. Pharmacogenomics 2013;14(11):1255-1272.   [PubMed]
  38. 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 2015;34(3):227-234.   [PubMed]
  39. Del Re M, Cinieri S, Michelucci A, Salvadori S, Loupakis F, Schirripa M, et al. DPYD* 6 plays an important role in fluoropyrimidine toxicity in addition to DPYD* 2A and c. 2846A> T: a comprehensive analysis in 1254 patients. Pharmacogenomics J 2019;19(6):556-563.
  40. Van Kuilenburg A, Baars J, Meinsma R, Van Gennip A. Lethal 5-fluorouracil toxicity associated with a novel mutation in the dihydropyrimidine dehydrogenase gene. Ann Oncol 2003;14(2):341-342.   [PubMed]
  41. Tsalic M, Bar-Sela G, Beny A, Visel B, Haim N. Severe toxicity related to the 5-fluorouracil/leucovorin combination (the Mayo Clinic regimen): a prospective study in colorectal cancer patients. Am J Clin Oncol 2003;26(1):103-106.   [PubMed]
  42. Mercier C, Ciccolini J. Profiling dihydropyrimidine dehydrogenase deficiency in patients with cancer undergoing 5-fluorouracil/capecitabine therapy. Clin Colorectal Cancer 2006;6(4):288-296.   [PubMed]
  43. Graham JS, Saunders J, Naylor G, Crearie C, Campbell W, Abdullah T, et al. Prospective DPYD testing and dose adjustment in colorectal cancer patients prior to fluoropyrimidine-based chemotherapy: Experience in a regional cancer center. J Clin Oncol (American Society of Clinical Oncology) 2020;38(4):93.
  44. Cho HJ, Park YS, Kang WK, Kim JW, Lee SY. Thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) polymorphisms in the Korean population for prediction of 5-fluorouracil-associated toxicity. Ther Drug Monit 2007;29(2):190-196.   [PubMed]
  45. Hsiao HH, Yang MY, Chang JG, Liu YC, Liu TC, Chang CS, et al. Dihydropyrimidine dehydrogenase pharmacogenetics in the Taiwanese population. Cancer Chemother Pharmacol 2004;53(5):445-451.   [PubMed]
  46. Süzen H, Yüce N, Güvenç G, Duydu Y, Erke T. TYMS and DPYD polymorphisms in a Turkish population. Eur J Clin Pharmacol 2005;61(12):881.   [PubMed]
  47. Ahluwalia R, Freimuth R, McLeod HL, Marsh S. Use of pyrosequencing to detect clinically relevant polymorphisms in dihydropyrimidine dehydrogenase. Clin Chem 2003;49(10):1661-1664.   [PubMed]
  48. 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.   [PubMed]
  49. Campbell JM, Bateman E, Peters MD, Bowen JM, Keefe DM, Stephenson MD. Fluoropyrimidine and platinum toxicity pharmacogenetics: an umbrella review of systematic reviews and meta-analyses. Pharmacogenomics 2016;17(4):435-451.   [PubMed]
  50. Gallegos-Arreola MP, Zúñiga-González GM, Sánchez-López JY, Naranjo-Cruz AY, Peralta-Leal V, Figuera LE, et al. TYMS 2R3R polymorphism and DPYD [IVS] 14+ 1G> A mutation genes in Mexican colorectal cancer patients. Acta Biochim Pol 2018;65(2):227-234.   [PubMed]
  51. Castro-Rojas CA, Esparza-Mota AR, Hernandez-Cabrera F, Romero-Diaz VJ, Gonzalez-Guerrero JF, Maldonado-Garza H, et al. Thymidylate synthase gene variants as predictors of clinical response and toxicity to fluoropyrimidine-based chemotherapy for colorectal cancer. Drug Metabolism and Personalized Therapy 2017;32(4):209-218.
  52. Martinez-Balibrea E, Abad A, Martínez-Cardús A, Ginés A, Valladares M, Navarro M, et al. UGT1A and TYMS genetic variants predict toxicity and response of colorectal cancer patients treated with first-line irinotecan and fluorouracil combination therapy. Br J Cancer 2010;103(4):581-589.   [PubMed]
  53. 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.   [PubMed]
  54. Braun MS, Richman SD, Thompson L, Daly CL, Meade AM, Adlard JW, et al. Association of molecular markers with toxicity outcomes in a randomized trial of chemotherapy for advanced colorectal cancer: the FOCUS trial. J Clin Oncol 2009;27(33):5519-5528.   [PubMed]
  55. Shen J, Wang H, Wei J, Yu L, Xie L, Qian X, et al. Thymidylate synthase mRNA levels in plasma and tumor as potential predictive biomarkers for raltitrexed sensitivity in gastric cancer. Int J Cancer 2012;131(6):E938-E945.
  56. Gao Y, Cui J, Xi H, Cai A, Shen W, Li J, et al. Association of thymidylate synthase expression and clinical outcomes of gastric cancer patients treated with fluoropyrimidine-based chemotherapy: a meta-analysis. Oncotargets and Therapy 2016;9:1339-1350.   [PubMed]
  57. Meulendijks D, Rozeman E, Cats A, Sikorska K, Joerger M, Deenen M, et al. Pharmacogenetic variants associated with outcome in patients with advanced gastric cancer treated with fluoropyrimidine and platinum-based triplet combinations: a pooled analysis of three prospective studies. Pharmacogenomics J 2017;17(5):441-451
  58. Zhang XP, Bai ZB, Chen BA, Feng JF, Feng Y, Jiang Z, et al. Polymorphisms of dihydropyrimidine dehydrogenase gene and clinical outcomes of gastric cancer patients treated with fluorouracil-based adjuvant chemotherapy in Chinese population. Chinese Med J 2012;125(5):741-746.   [PubMed]
  59. Grau JJ, Caballero M, Monzó M, Muñoz‐García C, Domingo‐Domenech J, Navarro A, et al. Dihydropyrimidine dehydrogenases and cytidine‐deaminase gene polymorphisms as outcome predictors in resected gastric cancer patients treated with fluoropyrimidine adjuvant chemotherapy. J Surg Oncol 2008;98(2):130-134.   [PubMed]