Fluorouracil Therapy and DPYD Genotype

Introduction

Fluorouracil, or 5-flourouracil (5-FU), is a chemotherapy agent that belongs to the drug class of fluoropyrimidines. When given as an intravenous (IV) solution, 5-FU is used in the palliative management of carcinoma of many solid tumors including (but not limited to) colon, rectum, breast, esophagus, cholangiocarcinoma (bile duct cancers), stomach, and pancreas. When prescribed as a cream or solution for topical use, fluorouracil (brand names Carac, Efudex, Fluoroplex, Tolak) is used to treat multiple actinic or solar keratoses of the face and scalp. Capecitabine (brand name Xeloda or CAPE) is the oral pill form of 5-FU chemotherapy, which is used interchangeably with 5-FU IV chemotherapy. Although it is the same drug, the oral pill version has certain side effects that are more pronounced (for example, diarrhea or skin related side effects – ‘hand-foot’ syndrome). Given the common usage of 5-FU for a variety of malignancies and potentially fatal overdoses, an antidote has been developed—uridine triacetate—which may be useful for pharmacogenetic-related overdoses, as well.

The DPYD gene encodes dihydropyrimidine dehydrogenase (DPD), an enzyme that catalyzes the rate-limiting step in fluorouracil metabolism. Genetic variations in the DPYD gene can lead to enzymes with reduced or absent activity. Individuals who have at least one copy of a non-functional DPYD variant [for example, DPYD*2A (c.1905+1G>A) or DPYD*13 (c.1679T>G)] will not be able to metabolize fluorouracil at normal rates. Consequently, they are at risk of potentially life-threatening fluorouracil toxicity, such as bone marrow suppression, diarrhea, and neurotoxicity. The prevalence of DPD partial deficiency varies in different populations but is approximately 35%. Complete absence of DPD function, which is often fatal with exposure to 5-FU chemotherapy, occurs in <1% (~0.2%) of the general population.

The FDA-approved drug label for fluorouracil states that no dose of fluorouracil has been proven safe in individuals with absent DPD activity (Table 1). Fluorouracil is contraindicated in individuals who are known to have complete DPD deficiency, or when complete deficiency is suspected because of early-onset or unusually severe fluorouracil toxicity (1).

The Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG) have published dosing recommendations for fluoropyrimidines (fluorouracil and capecitabine) based on DPYD genotype (Tables 2 and 3). Both recommendations include dose reductions for intermediate metabolizers (with reduced enzyme activity) and avoiding fluorouracil or capecitabine and choosing an alternative agent for poor metabolizers (with absent enzyme activity).

Drug Class: Fluoropyrimidines

Fluoropyrimidines are a class of antimetabolite drugs that are widely used in the treatment of cancer. There are 3 types of fluoropyrimidines in clinical use: capecitabine (oral - pill) and 5-fluorouracil (5-FU – IV), which are licensed for use in the US, and tegafur, which is not available in the US. Capecitabine and tegafur are both active precursors of fluorouracil.

Fluoropyrimidines are thought to exert their chemotherapeutic effects in multiple ways, through several active metabolites. The main mechanism of action is thought to be the inhibition of thymidylate synthase, which plays an important part in the folate-homocysteine cycle, and purine and pyrimidine synthesis pathways. Also, active metabolites can be incorporated into RNA and DNA, ultimately leading to cell death (7). Based on their mechanism of action, fluoropyrimidines can cause fetal harm when administered to a pregnant woman (1).

Approximately 10–40% of individuals develop severe and potentially life-threatening toxicity early during treatment with fluoropyrimidines (8). This toxicity typically leads to an interruption or discontinuation of potentially effective anticancer therapy, and may require an emergency room visit or hospitalization in severe instances (9).

The interindividual variation in the occurrence and severity of adverse events in individuals receiving fluoropyrimidines can be partly explained by clinical factors, such as age and gender. However, much of the variability in adverse events remains unexplained (10).

Of the genetic factors thought to contribute to fluoropyrimidine intolerance, the DPYD gene has been the most studied. This gene encodes the primary enzyme involved in breaking down fluoropyrimidines to inactive metabolites. Individuals who have a deficiency of the DPD enzyme have a significantly increased risk of suffering from severe fluoropyrimidine toxicity, and the stratification of individuals on the basis of the DPYD genotype may help to prevent such adverse events (11, 12, 13, 14, 15, 16).

Drug: Fluorouracil

Fluorouracil is a form of chemotherapy that is given as an IV solution, and is used to manage many cancers, including carcinoma of the colon, rectum, breast, stomach, and pancreas. Fluorouracil may also be used topically as a cream or a solution, for the treatment of multiple actinic or solar keratoses of the face and anterior scalp.

Fluorouracil is structurally similar to pyrimidines, and the enzyme that catalyzes the rate-limiting step in the breakdown of pyrimidines (DPD) also catalyzes the rate-limiting step in 5-FU catabolism. The DPD enzyme catalyzes the conversion of fluorouracil to the non-cytotoxic dihydrofluorouracil (DHFU) (17).

The FDA states that fluorouracil therapy should be discontinued promptly whenever one of the following signs of toxicity appears:

• Cardiotoxicity including angina, myocardial infarction/ischemia, arrhythmia, and heart failure
• Hyperammonemic encephalopathy
• Neurologic toxicity, including acute cerebellar syndrome
• Mucositis, stomatitis or esophagopharyngitis, which may lead to mucosal sloughing or ulceration
• Myelosuppression, which may include neutropenia, thrombocytopenia, and anemia
• Diarrhea (grade 3 or 4), frequent bowel movements, or watery stools
• Palmar-plantar erythrodysesthesia (hand-foot syndrome), grade 2 or 3

The FDA label also indicates that uridine triacetate should be administered within 96 hours following the end of fluorouracil infusion for management of fluorouracil overdose. (1) Uridine triacetate (brand name Vistogard) was approved December 11, 2015 (18). Exogenous uridine competes with 5-FU for incorporation into RNA, thus diluting the toxic effects of high 5-FU levels. Uridine triacetate is 4–6-fold higher in bioavailability than equimolar doses of uridine (19).

Uridine triacetate is meant for overdose treatment of adults or children; however, can be considered in situations of individuals with pharmacogenetic deficiency, which is technically an overdose (20, 21). This compound can also be used in the context of capecitabine overdose (19). The high cost of a single course of uridine triacetate therapy has been cited as a potential barrier to therapy. Nevertheless, 94% of clinical trial participants treated with uridine triacetate survived the overdose event, a notable improvement over the historic mortality rate of 84% (19).

Symptomatic DPD deficiency is a rare autosomal recessive disorder with a wide range of symptoms, ranging from no symptoms or signs, to severe neurological problems. In affected individuals, the absent or greatly decreased DPD activity results in uracil and thymine accumulating in the blood, urine, and cerebrospinal fluid. Neurological symptoms typically manifest in early childhood and include seizures, small head size, and delayed cognitive and motor development (22).

Symptomatic DPD deficiency is typically caused by homozygous inactivation of DPYD; whereas individuals who are heterozygotes tend to be asymptomatic. However, all individuals with less than 70% DPD activity are considered at risk for the development of severe drug toxicity when treated with fluoropyrimidines (23). Signs of fluorouracil toxicity include severe diarrhea, severe mucositis, neutropenia, neurotoxicity, and hand-foot syndrome (redness, swelling, and blisters on the palms of the hands an soles of the feet) (1).

Gene: DPYD

The DPYD gene encodes the enzyme DPD, which catalyzes the first and rate-limiting step in the breakdown of the pyrimidine nucleotides thymine and uracil. The DPD enzyme also catalyzes the rate-limiting step in the breakdown of fluoropyrimidines.

Many DPYD variants have been described, although only a few have been demonstrated to influence DPD enzyme activity. When no variant is detected (formerly known as the *1 allele), it is associated with normal enzyme activity. Individuals who have 2 copies of normal activity DPYD alleles are known as “normal metabolizers” and have fully functional DPD enzyme activity (Table 4). The DPYD alleles c.1601G>A (*4, rs1801158), c.1627G>A (*5, rs1801159), c.2194G>A (*6, rs1801160), and c.85T>C (*9A, rs1801265) are also considered to have normal activity (24). Historically, variant haplotypes in DPYD have been identified by their star (*) allele names. However, the Pharmacogene Variation Database (PharmVar) now identifies these alleles by their dbSNP “rs” allele identifier or cDNA change based on the NM_000110.3 transcript, DPYD mRNA variant 1. All 3 of these identifiers are provided in the Nomenclature for Selected DPYD alleles table below.

The non-functional DPYD variants that have been associated with absent DPD activity and an increased risk of toxicity with fluoropyrimidines include c.1905+1G>A (*2A, rs3918290) and c.1679T>G (*13, rs55886062) (26). Variants with decreased function include rs67376798 (c.2846A>T) and HapB3, which also are associated with an increased risk of fluoropyrimidine toxicity. The most well studied variant is DPYD c.1905+1G>A (*2A, rs3918290), in which a single nucleotide substitution at the invariant splice donor site of intron 14 leads to translation skipping exon 14, resulting in the production of a truncated protein with virtually no enzyme activity.

Individuals who have combinations of one normal function and one decreased function or no function DPYD alleles are known as “intermediate metabolizers”. Individuals with 2 decreased function alleles are also categorized as intermediate metabolizers. They have partial DPD deficiency and are at increased risk of capecitabine toxicity. And individuals who have a combination of non-functional DPYD alleles, or decreased function DPYD alleles, or both, are known as “poor metabolizers”. They have complete DPD deficiency and are at an even higher risk of capecitabine toxicity.

Activity scores may be used to distinguish between the various DPYD alleles and their functionality (Table 5). The use of activity scores may result in differentiated individualized dosing advice for fluoropyrimidines, which is beneficial for reducing toxic side effects while maintaining efficacy (15).

Overall, the prevalence of individuals who are heterozygous for non-functional DPYD alleles (partially DPD deficient) and at risk of severe drug reactions is estimated to be as high as 5–8%, but this varies in different populations (8, 23, 27, 28, 29, 30, 31, 32). In Caucasians, approximately 3–5% of have partial DPD deficiency and 0.2% have complete DPD deficiency (29). Recent studies suggest that ~8% of Caucasians have at least one of the 4 well characterized altered-function alleles (33).

In African-Americans, the prevalence of decreased DPD enzyme activity is 8% (32). It is notable that despite being well studied, DPYD c.1905+1G>A (*2A, rs3918290) is incredibly rare in individuals of African ancestry (34). One study reported that the normal function c.85T>C (*9A, rs1801265) allele was present in 49% of African-American samples (35). The rs115232898 (c.557A>G) variant allele with reduced function was detected in 2.6% of African-heritage Brazilians (36).

Studies of Egyptian and Tunisian populations suggest the allelic frequencies for DPYD variants in these 2 countries are similar to Caucasian variant allele frequencies (37, 38). The frequency of the poor-metabolizer rs6376798 (c.2846A>T) allele in Mestizo and native Mexican populations is rare, but not significantly different than in MXL (Mexican Ancestry from Los Angeles USA) or CEU (Utah Residents (CEPH) with Northern and Western European Ancestry) populations in the 1000 Genomes Project (39).

Asian populations have slightly different allele frequencies as compared with African and European populations. The frequency of the c.85T>C (*9A, rs1801265) normal function variant was slightly lower in Han Chinese, Korean and Japanese populations, particularly compared with Africans, though the frequency of the c.2657G>A (*9B, rs1801267) normal function variant and c.295_298delTCAT (*7, rs72549309), c.703C>T (*8, rs1801266), and c.2983G>T (*10, rs1801268) no function alleles were similar across these groups (35). The c.1905+1G>A (*2A/*2B, rs3918290) and c.1679T>G (*13, rs55886062) no function alleles were not detected in a study of Hmong and East Asian descent individuals, underscoring the rarity of these alleles (40). An analysis of multiple genotyping studies in South Asian populations found that the rs2297595 (c.496A>G) allele was prevalent in south Asia (41).

Most individuals in the U.S. are not screened for DPD deficiency before starting fluorouracil therapy (42) and the FDA-approved label does not specifically recommend DPD testing (1). In contrast, the European Medicines Agency recommends testing for DPD deficiency before initiating treatment with any fluorouracil related substance via infusion or injection (43).

Gene: TYMS

Emerging studies and reports suggest genetic variation at another locus may also affect 5-FU efficacy and toxicity—TYMS. This gene encodes thymidylate synthase (TS), which catalyzes the methylation of deoxyuridylate to deoxythymidylate. This reaction is a rate-limiting step in production of an essential DNA synthesis precursor. The TS protein expression correlates positively with sensitivity to 5-FU, and the TS enzyme is one of the targets of 5-FU (44). While this functional link to 5-FU metabolism and tumor response has been demonstrated in multiple studies, the impact of specific genetic variants in TYMS is less clear (44, 45, 46, 47). Some TYMS alleles have been reported in a handful of studies as being associated with increased toxicity and anti-tumor cell response with fluoropyrimidines.

The rs45445694 variant is the basis of the TYMS “2R” allele, which has been associated with clinical response and severe toxicity events, either in homozygosity or heterozygosity (20, 48, 49, 50). This 2R variant occurs in the 5’UTR and is duplication of a 28 base pair (bp) repeat. This same locus can have variable tandem repeats between 0 and 9 copies, and studies suggest that the increased copy number of the repeat is associated with increased TYMS expression and TS protein levels (51).

One additional variant in TYMS has been found in association with adverse reactions to fluoropyrimidine therapy: a 3'UTR 9bp-indel (rs11280056) (49, 51). There are conflicting reports as to whether this is a 6- or 9-bp indel. One variant (rs2853542) within the TYMS enhancer region in the context of the 28bp tandem repeat triplication, called 3RG or 3RC based on the specific nucleotide present, has also been reported in association with neurotoxicity during 5-FU treatment (52). The presence of the C nucleotide at rs2853542 has been associated with decreased expression of TYMS mRNA (53).

PharmGKB has described TYMS as a ‘Very Important Pharmacogene’, though the level of evidence for TYMS and capecitabine/5-fluorouracil interaction is limited (PharmGKB “level 3”) (51). The CPIC also views this interaction as having limited evidence and thus provides no prescribing recommendations for these pharmacogenetic variants (54).

Linking Gene Variation with Treatment Response

Standard doses of fluorouracil increase the risk of severe toxicity in individuals who are carriers of specific DPYD variant alleles, and no dose of fluorouracil is considered safe among individuals with absent DPD activity (1, 55). Multiple studies have found that preemptive DPYD screening for individuals with cancer can significantly improve individual safety (56, 57, 58, 59, 60, 61). Of note, at least one case report indicated that the cost of administering uridine triacetate and palliative care following an adverse, overdose reaction to 5-FU was roughly $180,000 USD (20).

Genetic Testing

The NIH Genetic Testing Registry, GTR, displays genetic tests that are available for the DPYD gene and the fluorouracil drug response. The DPYD c.1905+1G>A (*2A, rs3918290) variant is the most commonly tested; however, newly discovered and rare variants may also lead to enzyme inactivation and toxicity to fluoropyrimidine-based chemotherapy (36, 62, 63, 64, 65).

Biochemical genetic tests may also be used, which assess the activity of the DPD enzyme. These tests include biochemical assays such as analyte testing (for example, measuring the amount of thymine and uracil in the urine or blood) or an enzyme assay (for example, directly measuring the activity of DPD using RNA extracted from blood cells and measuring the DPD mRNA copy number) (66, 67, 68).

The GTR provides a list of biochemical tests that assess the levels of thymine and uracil analytes, and the activity of the enzyme DPD.

Therapeutic Recommendations based on Genotype

This section contains excerpted¹ information on gene-based dosing recommendations. Neither this section nor other parts of this review contain the complete recommendations from the sources.

Nomenclature for Selected Alleles

Acknowledgments

The author would like to thank Ayman Madi, MD, MCRP, Consultant Medical Oncologist, The Clatterbridge Cancer Centre NHS Foundation Trust, Liverpool, UK; Masahiro Hiratsuka, PhD, Associate Professor, Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences Tohoku University, Sendai, Japan; Linda M. Henricks, PharmD, Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands; Pashtoon Kasi, MD, MS, Clinical Assistant Professor of Internal Medicine - Hematology, Oncology and Blood and Marrow Transplantation, Department of Internal Medicine, University of Iowa Health Care, Iowa City, IA, USA; and Mandy van Rhenen, PharmD, Royal Dutch Pharmacists Association, Drug Information Centre KNMP, The Hague, the Netherlands for reviewing this summary.

2016 Edition

The author would like to thank George P. Patrinos, Associate Professor of Pharmacogenomics and Pharmaceutical Biotechnology, University of Patras, Department of Pharmacy, Patras, Greece; Mohamed Nagy, Clinical Pharmacist, Head of the Personalised Medication Management Unit, Department of pharmaceutical Services, Children's Cancer Hospital, Cairo, Egypt; and Victoria M. Pratt, PhD, FACMG, Director, Pharmacogenomics Laboratory, Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA for reviewing this summary.

Version History

To view the 2016 version of this summary (created 3 November 2016), please click here.

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