Indications for Testing

• Patients anticipated to have prolonged administration of 5-FU; personal or family history of 5-FU toxicity or a therapeutic failure

Laboratory Testing

• 5-FU mutation testing
  • DPYD or TYMS mutation detected – predictive of an increased sensitivity to 5-FU that may lead to increased risk for toxicity
    • Alternative chemotherapeutic agents, therapeutic drug monitoring, altered 5-FU doses, or increased surveillance for adverse drug reactions may be indicated
  • MTHFR mutation detected – associated with decreased MTHFR activity and increased rate of TS reactivity
    • Unknown impact on 5-FU efficacy and toxicity

5-fluorouracil (5-FU) is a fluoropyrimidine drug used in the treatment of colorectal cancer and other solid tumors. Pharmacogenetic variations in genes such as DPYD, TYMS, and MTHFR may contribute to risk of toxicity and/or altered therapeutic benefits.

Epidemiology

• Prevalence
  • Heterozygosity and homozygosity for DPYD alleles with impaired function occur in 3-5% and 0.1% of the general population, respectively
  • Heterozygosity for 1494 del TTAAAG (6-bp deletion) in the 3’ untranslated region of the TYMS gene occurs in ~50% of Caucasians; 7% are homozygous
    • This deletion genotype is in strong linkage disequilibrium with the 5’ tandem repeat genotype known commonly as 3R/3R
  • Allele frequencies in the U.S. for MTHFR c.677C>T and c.1298A>C are 39% and 17%, respectively

Genetics

• At least 17 DPYD mutations identified in patients with severe 5-FU toxicity
• Five common mutations are associated with reduced enzymatic function and grade III-IV toxicity
  • *2A (IVS+14G>A) – most common and clinically significant variant
  • *9A (c.85T>C)
  • c.1590T>C
  • *13 (c.1679T>G)
  • c.2846A>T
• Polymorphisms in the TYMS gene encoding thymidylate synthase (TS) include the 6-bp deletion in the 3’ untranslated region, which contributes to decreased gene expression and reduced mRNA stability
  • Mutation is associated with increased sensitivity to 5-FU
• c.677C>T and c.1298A>T mutations in the MTHFR gene are associated with decreased MTHFR enzyme activity and increased TS enzyme reactivity; the impact of this variant on 5-FU efficacy is not clear

Pathophysiology

• Dihydropyrimidine dehydrogenase (DPD) enzyme – encoded by DPYD gene
  • Responsible for the degradation and inactivation of >80% of 5-FU
  • Initial and rate-limiting enzyme in the pathways that catabolize pyrimidines
  • Reduced DPD activity can lead to the accumulation of active 5-FU metabolite, fluorodeoxyuridine monophosphate (5-FdUMP), which leads to 5-FU sensitivity
  • DPYD mutations are associated with decreased DPD activity, leading to production of proportionately higher amounts of 5-FdUMP and an increased risk for dose-related 5-FU sensitivity
• Thymidylate synthase (TS) – encoded by TYMS gene
  • Primary target for 5-FU
  • Catalyses methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) essential for DNA replication
  • 5-FdUMP – active metabolite of 5-FU prevents DNA synthesis by forming stable complexes with TS, with folate as a cofactor, blocking the production of dTMP in cancer cells
    • TYMS gene mutations result in reduced expression of TS and may be associated with higher clinical responsiveness to 5-FU therapy and an increased risk of toxicity
• Methylenetetrahydrofolate reductase (MTHFR)
  • Involved in metabolism of folate; forms the reduced folate cofactor needed for TS inhibition
  • Mutations in MTHFR gene lead to reduced MTHFR enzyme activity, which increases intracellular folate metabolites and may increase TS reactivity

Clinical Presentation

• Grade III-IV toxicity (occurs in up to 15% of treated patients)
  • Mucositis
  • Neutropenia
  • Nausea
  • Diarrhea
  • Neurological symptoms

Tests generally appear in the order most useful for common clinical situations

General

• Hiratsuka M, Sasaki T, Mizugaki M. Genetic testing for pharmacogenetics and its clinical application in drug therapy. 2006; 363 (1-2) :177-186.PubMed
• Lee A, Ezzeldin H, Fourie J, Diasio R. Dihydropyrimidine dehydrogenase deficiency: impact of pharmacogenetics on 5-fluorouracil therapy. 2004; 2 (8) :527-532.PubMed
• Lee SY, McLeod HL. Pharmacogenetic tests in cancer chemotherapy: what physicians should know for clinical application. J Pathol. 2011; 223 (1) :15-27.PubMed
• Lentz F, Tran A, Rey E, Pons G, Treluyer JM. Pharmacogenomics of fluorouracil, irinotecan, and oxaliplatin in hepatic metastases of colorectal cancer: clinical implications. 2005; 5 (1) :21-33.PubMed
• Maring JG, Groen HJ, Wachters FM, Uges DR, de Vries EG. Genetic factors influencing pyrimidine-antagonist chemotherapy. 2005; 5 (4) :226-243.PubMed
• Omura K. Clinical implications of dihydropyrimidine dehydrogenase (DPD) activity in 5-FU-based chemotherapy: mutations in the DPD gene, and DPD inhibitory fluoropyrimidines. 2003; 8 (3) :132-138.PubMed
• Russo A, Corsale S, Cammareri P, Agnese V, Cascio S, Di Fede G, Macaluso M, Bazan V. Pharmacogenomics in colorectal carcinomas: future perspectives in personalized therapy. 2005; 204 (3) :742-749.PubMed
• Saif MW, Choma A, Salamone SJ, Chu E. Pharmacokinetically guided dose adjustment of 5-fluorouracil: a rational approach to improving therapeutic outcomes. 2009; 101 (22) :1543-1552.PubMed
• van Kuilenburg AB, De Abreu RA, van Gennip AH. Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogenase deficiency. 2003; 40 (Pt 1) :41-45.PubMed
• Yen JL, McLeod HL. Should DPD analysis be required prior to prescribing fluoropyrimidines?. 2007; 43 (6) :1011-1016.PubMed

ARUP Institute for Clinical and Experimental Pathology®

• Beumer JH, Boisdron-Celle M, Clarke W, Courtney JB, Egorin MJ, Gamelin E, Harney RL, Hammett-Stabler C, Lepp S, Li Y, Lundell GD, McMillin G, Milano G, Salamone SJ. Multicenter evaluation of a novel nanoparticle immunoassay for 5-fluorouracil on the Olympus AU400 analyzer. Ther Drug Monit. 2009; 31 (6) :688-694.PubMed

Colorectal Cancer
Pharmacogenetics