The contribution of deleterious DPYD gene sequence variants to fluoropyrimidine toxicity in British cancer patients

Purpose

The fluoropyrimidines have been extensively used for almost five decade worldwide for the treatment of solid cancers. However, severe toxicity is a major clinical problem and has been reported in association with deleterious sequence variants in dihydropyrimidine dehydrogenase (DPD) coding-gene (DPYD), causing DPD deficiency. Genetic DPD deficiency has previously been considered to be insignificant in the British population. The study aim was to assess the contribution of deleterious DPYD sequence variants to fluoropyrimidine toxicity amongst British cancer patients.

Methods

Sequencing of the coding region of DPYD was undertaken in 47 patients (27 female, mean age 61 years), mainly with GI malignancy, experiencing grade 3 or 4 toxicity on fluoropyrimidines according to CTCAE criteria.

Results

Myelotoxicity (37.5%) and diarrhoea (37.5%) were the most frequent toxicities followed by mucositis (19.6%), hand–foot syndrome (3.6%) and neurotoxicity (1.8%). 4 of 47 (8.5%) patients carried the 1905+1G>A splice site variant. All 4 cases were female and 3 of 4 suffered severe diarrhoea. A further five cases carried other sequence variants (2846A>T n = 4, 1679T>G n = 1). In total, 9 (19%) patients carried deficiency associated DPYD sequence variants.

Conclusions

Contrary to previous estimates for a UK population, genetic DPD deficiency accounts for around 19% of cases of severe fluoropyrimidine toxicity. The influence of DPD deficiency is such that toxicity can be avoided by prior testing and appropriate 5-FU dose/regimen alteration.     

Near Miss or Standard of Care? DPYD Screening for Cancer Patients Receiving Fluorouracil

5-fluorouracil (5-FU) and its pro-drug capecitabine are widely used anticancer agents. Most 5-FU catabolism is dependent on dihydropyrimidine dehydrogenase (DPD) encoded by the DPYD gene, and DPYD variants that reduce DPD function increase 5-FU toxicity. Most DPD deficient patients are heterozygous and can be treated with reduced 5-FU dosing. We describe a patient with a genotype associated with near complete absence of DPD function, and severe and likely fatal toxicity with 5-FU treatment. The patient was treated effectively with alternative systemic therapy. Routine pretreatment DPYD genotyping is recommended by the European Medicines Agency, and guidelines for use of 5-FU in DPD deficient patients are available. However, outside the province of Quebec, routine pretreatment screening for DPD deficiency remains unavailable in Canada. It is likely our patient would have died from 5-FU toxicity under the current standard of care, but instead provides an example of the potential benefit of DPYD screening on patient outcomes.     

Prospective DPYD genotyping to reduce the risk of fluoropyrimidine-induced severe toxicity: Ready for prime time

5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.     

The role of pharmacogenetics in capecitabine efficacy and toxicity

Capecitabine is an oral prodrug of 5-fluorouracil (5-FU) and approved for treatment of various malignancies. Hereditary genetic variants may affect a drug’s pharmacokinetics or pharmacodynamics and account for differences in treatment response and adverse events among patients. In this review we present the current knowledge on genetic variants, commonly single-nucleotide polymorphisms (SNPs), tested in cohorts of cancer patients and possibly useful for prediction of capecitabine efficacy or toxicity. Capecitabine is activated to 5-FU by CES, CDA and TYMP, of which SNPs in CDA and CES2 were found to be associated with efficacy and toxicity. In addition, variants in genes of the 5-FU metabolic pathway, including TYMS, MTHFR and DPYD also influenced capecitabine efficacy and toxicity. In particular, well-known SNPs in TYMS and DPYD as well as putative DPYD SNPs had an association with clinical outcome as well as adverse events. Inconsistent findings may be attributable to factors related to ethnic differences, sample size, study design, study endpoints, dosing schedule and the use of multiple agents. Of the SNPs described in this review, dose reduction of fluoropyrimidines based on the presence of DPYD variants ^*2A (rs3918290), ^*13 (rs55886062), −2846A>T (rs67376798) and −1236G>A/HapB3 (rs56038477) has already been recommended. Other variants merit further validation to establish their definite role in explanation of interindividual differences in the outcome of capecitabine-based therapy.     

Cost-effectiveness of DPYD Genotyping Prior to Fluoropyrimidine-based Adjuvant Chemotherapy for Colon Cancer

BackgroundAdjuvant fluoropyrimidine-based chemotherapy substantially reduces recurrence and mortality after resection of stage 3 colon cancer. While standard doses of 5-fluorouracil and capecitabine are safe for most patients, the risk of severe toxicity is increased for the approximately 6% of patients with dihydropyimidine dehydrogenase (DPD) deficiency caused by pathogenic DPYD gene variants. Pre-treatment screening for pathogenic DPYD gene variants reduces severe toxicity but has not been widely adopted in the United States.MethodsWe conducted a cost-effectiveness analysis of DPYD genotyping prior to fluoropyrimidine-based adjuvant chemotherapy for stage 3 colon cancer, covering the c.1129-5923C>G (HapB3), c.1679T>G (*13), c.1905+1G>A (*2A), and c.2846A>T gene variants. We used a Markov model with a 5-year horizon, taking a United States healthcare perspective. Simulated patients with pathogenic DPYD gene variants received reduced-dose fluoropyrimidine chemotherapy. The primary outcome was the incremental cost-effectiveness ratio (ICER) for DPYD genotyping.ResultsCompared with no screening for DPD deficiency, DPYD genotyping increased per-patient costs by $78 and improved survival by 0.0038 quality-adjusted life years (QALYs), leading to an ICER of $20,506/QALY. In 1-way sensitivity analyses, The ICER exceeded $50,000 per QALY when the cost of the DPYD genotyping assay was greater than $286. In probabilistic sensitivity analysis using a willingness-to-pay threshold of $50,000/QALY DPYD genotyping was preferred to no screening in 96.2% of iterations.ConclusionAmong patients receiving adjuvant chemotherapy for stage 3 colon cancer, screening for DPD deficiency with DPYD genotyping is a cost-effective strategy for preventing infrequent but severe and sometimes fatal toxicities of fluoropyrimidine chemotherapy.     

Capecitabine‐based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency

Fluoropyrimidines are frequently used anti‐cancer drugs. It is known that patients with reduced activity of dihydropyrimidine dehydrogenase (DPD), the key metabolic enzyme in fluoropyrimidine inactivation, are at increased risk of developing severe fluoropyrimidine‐related toxicity. Upfront screening for DPD deficiency and dose reduction in patients with partial DPD deficiency is recommended and improves patient safety. For patients with complete DPD deficiency, fluoropyrimidine‐treatment has generally been discouraged. During routine pretreatment screening, we identified a 59‐year‐old patient with a sigmoid adenocarcinoma who proved to have a complete DPD deficiency. Genetic analyses showed that this complete absence of DPD activity was likely to be caused by a novel DPYD genotype, consisting of a combination of amplification of exons 17 and 18 of DPYD and heterozygosity for DPYD*2A. Despite absence of DPD activity, the patient was treated with capecitabine‐based chemotherapy, but capecitabine dose was drastically reduced to 150 mg once every 5 days (0.8% of original dose). Pharmacokinetic analyses showed that the area under the concentration‐time curve (AUC) and half‐life of 5‐fluorouracil were respectively tenfold and fourfold higher than control values of patients receiving capecitabine 850 mg/m². When extrapolating from the dosing schedule of once every 5 days to twice daily, the AUC of 5‐fluorouracil was comparable to controls. Treatment was tolerated well for eight cycles by the patient without occurrence of capecitabine‐related toxicity. This case report demonstrates that a more comprehensive genotyping and phenotyping approach, combined with pharmacokinetically‐guided dose administration, enables save fluoropyrimidine‐treatment with adequate drug exposure in completely DPD deficient patients.     

DPYD*2A mutation: the most common mutation associated with DPD deficiency

Background Dihydropyrimidine dehydrogenase (DPD) enzyme is responsible for the elimination of approximately 80% of administered dose of 5-FU. DPD deficiency has been associated with severe 5-FU toxicity. Syndrome of DPD deficiency manifests as diarrhea, stomatitis, mucositis, and neurotoxicity and in some cases death. This is a true pharmacogenetic syndrome, with symptoms being unrecognizable until exposure to the drug. Patients and methods A 75-year-old patient with metastatic pancreatic adenocarcinoma developed grade 4 thrombocytopenia, grade 3 coagulopathy, and grade 3 neurologic toxicity with a fatal outcome following administration of 5-FU. Due to pancytopenia, DPD activity could not be determined in peripheral blood mononuclear cells (PBMC) using a previously described radioassay. Therefore, screening and genotypic analysis of homozygous and heterozygous, known and unknown sequence variants, in the DPYD gene were performed using DHPLC as previously described. All DPYD sequence variants identified by DHPLC were confirmed by DNA sequencing using a dideoxynucleotide chain termination method and capillary electrophoresis on an ABI 310 Automated DNA Sequencer. Results Genotyping analysis of the DPYD gene revealed the presence of the heterozygous mutation, IVS14 + 1 G > A, DPYD*2A. Conclusion Genotypic analysis using DHPLC can be employed to screen DPD deficiency in a patient with severe neutropenia. The mutation IVS14 + 1 G > A, DPYD*2A, is the most common mutation associated with DPD deficiency. A G > A base change at the splice recognition sequence of intron 14, leads to exon skipping and results in a 165-bp deletion in the DPD mRNA. We have previously demonstrated that a homozygote DPYD*2A genotype results in complete deficiency while the heterozygous DPYD*2A genotype results in partial deficiency of DPD. This work was supported in part by NIH grant CA 62164.     

Can inhibiting dihydropyrimidine dehydrogenase limit hand-foot syndrome caused by fluoropyrimidines?

Hand-foot syndrome (HFS) is a cutaneous adverse event that occurs in some patients treated with fluoropyrimidines. Although it is not life threatening, HFS can severely disrupt the daily lives of patients. HFS appears more frequently with 5-fluorouracil (5-FU) delivered by continuous infusion or with the 5-FU oral derivative capecitabine than with bolus 5-FU therapy. HFS is a leading cause of treatment interruption, dosage reduction, or, even, therapy discontinuation for patients on a capecitabine regimen. Interestingly, addition of a dihydropyrimidine dehydrogenase (DPD) inhibitor, such as uracil, 5-chloro-2,4-dihydroxypyridine, or eniluracil, to the fluoropyrimidine treatment regimen significantly diminishes the incidence of HFS. DPD inhibitors were initially combined with fluoropyrimidines to increase the efficacy of the drugs by impairing the DPD-mediated catabolism of 5-FU. However, with the accumulating findings from clinical trials that show the benefits of DPD inhibition on decreasing the risk of HFS, consideration should be given to changing the recommendations for the treatment of cancer patients with fluoropyrimidines to include DPD inhibitor components as standard therapy.     

Population-scale predictions of DPD and TPMT phenotypes using a quantitative pharmacogene-specific ensemble classifier

Background Inter-individual differences in dihydropyrimidine dehydrogenase (DPYD encoding DPD) and thiopurine S-methyltransferase (TPMT) activity are important predictors for fluoropyrimidine and thiopurine toxicity. While several variants in these genes are known to decrease enzyme activities, many additional genetic variations with unclear functional consequences have been identified, complicating informed clinical decision-making in the respective carriers.Methods We used a novel pharmacogenetically trained ensemble classifier to analyse DPYD and TPMT genetic variability based on sequencing data from 138,842 individuals across eight populations.Results The algorithm accurately predicted in vivo consequences of DPYD and TPMT variants (accuracy 91.4% compared to 95.3% in vitro). Further analysis showed high genetic complexity of DPD deficiency, advocating for sequencing-based DPYD profiling, whereas genotyping of four variants in TPMT was sufficient to explain >95% of phenotypic TPMT variability. Lastly, we provided population-scale profiles of ethnogeographic variability in DPD and TPMT phenotypes, and revealed striking interethnic differences in frequency and genetic constitution of DPD and TPMT deficiency.Conclusion These results provide the most comprehensive data set of DPYD and TPMT variability published to date with important implications for population-adjusted genetic profiling strategies of fluoropyrimidine and thiopurine risk factors and precision public health.Subject terms: Cancer genetics, Cancer genomics, Cancer genetics     

Profiling dihydropyrimidine dehydrogenase deficiency in patients with cancer undergoing 5-fluorouracil/capecitabine therapy

Fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and capecitabine are a mainstay in the treatment of numerous solid tumors, including colorectal cancers, alone or as part of combination therapies. Cytotoxic drugs such as 5-FU and oral capecitabine display narrow therapeutic indexes combined with high interpatient pharmacokinetic variability. As a result, severe toxicities often limit or delay the administration of successive, optimal chemotherapeutic courses, leading to unfavorable clinical outcome in patients with cancer. Catabolism and deactivation of fluoropyrimidine drugs depend on a single and exclusive enzymatic step driven by dihydropyrimidine dehydrogenase (DPD). Dihydropyrimidine dehydrogenase is prone to marked circadian rhythms, drug-drug interactions, and genetic polymorphisms; influence of its erratic activity on 5-FU pharmacokinetics and toxicity profile has been extensively investigated, and it is now well known that DPD deficiency leads to severe toxicities with 5-FU or possibly capecitabine exposure. With the ever-increasing number of patients with cancer likely to be treated with fluoropyrimidines, predicting and preventing the occurrence of such toxicities is now a major issue in clinical oncology. Early determination of DPD status in patients with cancer would allow identification of those at risk and help in subsequent dose adjustment or selection of other treatment modalities. Numerous methods, either genotypic or phenotypic, have been proposed to achieve this goal. This review covers a wide range of techniques available to establish DPD status in patients with cancer.     

Clinical implications of dihydropyrimidine dehydrogenase (DPD) activity in 5-FU-based chemotherapy: mutations in the <Emphasis Type="Italic">DPD</Emphasis> gene,and DPD inhibitory fluoropyrimidines

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the degradation of pyrimidine bases. DPD is also responsible for the degradation of 5-fluorouracil (5-FU), which is the most frequently prescribed anticancer drug for the treatment of malignancies of the gastrointestinal tract. DPD could influence the antitumor effect and the adverse effects of 5-FU. High intratumoral DPD activity markedly decreases the cytotoxic effect of 5-FU. More than 80% of administered 5-FU is detoxified and excreted as F--alanine in urine. In 5-FU-based chemotherapy, escape from the degradation catalyzed by DPD is important. Recently, the dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined. To date, many DPYD variant alleles associated with a lack of DPD activity have been identified. In 5-FU-based cancer chemotherapy, severe toxicities were observed at higher rates in patients who were heterozygous for a mutant DPYD allele, compared with toxicities in patients who were homozygous for the wild DPYD allele. Furthermore, the adverse effects of 5-FU are often lethal for patients homozygous for the mutant DPYD allele. The apparently high prevalence of the DPYD mutation associated with lack of DPD activity in the normal population warrants genetic screening for the presence of these mutations in cancer patients before the administration of 5-FU. DPD inhibitory fluoropyrimidines (DIFs), including uracil plus tegafur (UFT) and tegafur plus 5-chloro-2,4-dihydroxypyridine plus potassium oxonate, in a molar ratio of 1:0.4:1 (TS-1), have recently been used in clinical settings. DIFs should provide chemotherapy that improves both quality of life and duration of survival.     

Is capecitabine safe in patients with gastrointestinal cancer and dihydropyrimidine dehydrogenase deficiency?

Patients with cancer with dihydropyrimidine dehydrogenase (DPD) deficiency are at significant risk for severe 5-fluorouracil (5-FU) toxicity, including the risk of death. Data regarding the toxicity of capecitabine, an oral fluoropyrimidine, in patients with DPD deficiency are scarce. From 2004 to 2005, 2 patients with gastrointestinal (GI) malignancies (of the pancreas and liver) experienced severe to even life-threatening toxicities during capecitabine therapy, which resulted in death for 1 patient. A DPD enzyme assay was performed as previously defined in our laboratory. Both patients were DPD deficient upon evaluation for toxicity. Capecitabine can lead to severe and sometimes life-threatening toxicities akin to toxicities caused by 5-FU in patients with DPD deficiency. In cases of unexpected severe toxicity during capecitabine treatment, DPD deficiency should be considered. We suggest that capecitabine should not be used in patients with DPD deficiency. Screening should be considered in view of the widespread use of capecitabine and 5-FU, the severe toxicity that can develop in patients with low DPD activity, and the prevalence of the mutation.     

Severe fluoropyrimidine-related toxicity: clinical implications of DPYD analysis and UH2/U ratio evaluation

The fluoropyrimidines are commonly used in chemotherapeutic cancer medicine, but many patients still experience severe adverse side effects from these drugs. We observed a severe toxicity in a 50-year-old woman treated with capecitabine and docetaxel for a metastatic breast cancer. Since dihydropyrimidine dehydrogenase (DPD) is the main candidate for pharmacogenetic studies on 5-FU toxicity, the entire coding sequence and exon-flanking intronic regions of the DPYD gene were sequenced in the patient. None of the previously described deleterious variants were detected. Also, the haplotype-based analysis failed to reveal DPYD variations associated with 5-FU toxicity. We also evaluated the UH2/U ratio in plasma as an index of 5-FU pharmacokinetics. The UH2/U value did not demonstrate low DPD activity in the patient. We discuss the advantages and limitations of this approach, particularly concerning the clinical applications of 5-FU pharmacogenetics in the family setting.     

Correlations between antitumor activities of fluoropyrimidines and DPD activity in lung tumor xenografts

The purposes of this study were to evaluate the antitumor activity of S-1 (1 M tegafur, 0.4 M 5-chloro-2,4-dihydroxypyridine and 1 M potassium oxonate) on human lung tumor xenografts, as compared with other fluoro-pyrimidines, and to investigate the relationships between fluoropyrimidine antitumor activities and four distinct enzymatic activities involved in the phosphorylation and degradation pathways of 5-fluorouracil (5-FU) metabolism. S-1, UFT (1 M tegafur-4 M uracil), 5'-deoxy-5-fluorouridine (5'-DFUR), capecitabine and 5-FU were administered for 14 consecutive days to nude mice bearing lung tumor xenografts. S-1 showed stronger tumor growth inhibition in four of the seven tumors than the other drugs. Cluster analysis, on the basis of antitumor activity, indicated that S-1/UFT and 5'-DFUR/capecitabine/5-FU could be classified into another group. We investigated tumor thymidylate synthase content, dihydropyrimidine dehydrogenase (DPD) activity, thymidine phosphorylase (TP) activity and orotate phosphoribosyl transferase activity in seven human lung tumor xenografts and performed regression analyses for the antitumor activities of fluoropyrimidines. There were inverse correlations between antitumor and DPD activities for 5'-DFUR (r=-0.79, P=0.034), capecitabine (r=-0.56, P=0.19) and 5-FU (r=-0.86, P=0.013). However, no such correlations were observed for S-1 and UFT. These findings suggest that S-1 containing a potent DPD inhibitor may have an antitumor effect on lung tumors, with high basal DPD activity, superior to those of other fluoropyrimidines.     

5-fluorouracil and dihydropyrimidine dehydrogenase

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the (fluoro) pyrimidine catabolic pathway, and has an important role in 5-fluorouracil (5-FU) pharmacology. Some studies have demonstrated that the fluoropyrimidine catabolite may be related to the toxicity of 5-FU, and tumoral DPD prescribes the response to 5-FU-based therapy. By controlling DPD activity, a new class of fluorinated pyrimidines have been developed to minimize the variability of 5-FU pharmacodynamics, to decrease 5-FU toxicity, and improve its efficacy. Recently, these drugs, referred to as DIFs (DPD inhibitory fluoropyrimidines), have brought us to a new era of oral 5-FU therapy. The importance of DIF in 5-FU-based therapy is reviewed herein.     

Toxicity and Efficacy of 5-Fluorouracil and Capecitabine in a Patient With TYMS Gene Polymorphism: A Challenge or a Dilemma?

5-Fluorouracil (5-FU) is an antimetabolite that acts during the S phase of the cell cycle. The active metabolite, 5-fluorodeoxyuridine monophosphate inhibits thymidylate synthase (TS), thus preventing DNA synthesis, which leads to imbalanced cell growth and ultimately cell death. 5-FU and its oral prodrug capecitabine are used in the treatment of a number of solid tumors, including colorectal, breast, gastric, pancreatic, prostate, and bladder cancers. Common side effects include leukopenia, diarrhea, stomatitis, nausea, vomiting, and alopecia. Hand-foot syndrome (HFS) is a relatively common side effect of cytotoxic chemotherapy. It is more frequently associated with 5-FU, capecitabine, and cytarabine. This article reports on the case of a 55-year-old black man with metastatic colorectal carcinoma that was refractory to recommended treatment measures who developed grade 3 HFS after treatment with modified FOLFOX6 (leucovorin [LV]/5-FU/oxaliplatin) and bFOL (bolus 5-FU/LV/oxaliplatin) regimens. Treatment was discontinued despite excellent response to chemotherapy. The patient had progression of disease on IROX (irinotecan/oxaliplatin) and irinotecan/cetuximab regimens. He was started on gemcitabine/capecitabine and developed HFS again, which was controlled with aggressive skin care and vitamin B₆ treatment. Full sequencing of the dihydropyrimidine dehydrogenase (DPYD) gene and analysis of the human TS gene (TYMS) promoter region was performed. Pharmacogenetic testing revealed 2R/2R genotype of TYMS gene, which is associated with up to a 2.5-fold risk of toxicity to 5-FU therapy. Hand-foot syndrome has proven to be a dose-limiting toxicity of 5-FU, especially of capecitabine, leading to significant morbidity. Hand-foot syndrome seems to be dose dependent, and both peak drug concentration and total cumulative dose determine its occurrence. Genetic variations such as polymorphic abnormality of TYMS are potential causative factors for a significant portion of serious adverse reactions to 5-FU–based therapy.     

Upfront DPYD Genotyping and Toxicity Associated with Fluoropyrimidine-Based Concurrent Chemoradiotherapy for Oropharyngeal Carcinomas: A Work in Progress

Simple SummaryThe combination of carboplatin and 5-fluorouracil (5-FU) is effective when used concurrently with radiotherapy for locoregionally advanced oropharyngeal carcinomas. DPYD polymorphisms can be associated with an increased risk of severe toxicity to fluoropyrimidines. Upfront screening for the DPYD*2A allele has been available in the province of Québec, Canada, since March 2017. This study aimed to determine the effect of upfront genotyping on the incidence of grade ≥3 toxicities. We included 181 patients in the analysis. Extended screening for three supplemental at-risk DPYD variants was also retrospectively performed in August 2019. The DPYD*2A, c.2846A>T and c.1236G>A polymorphisms were associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can thus identify patients in whom 5-FU-related toxicity should be avoided.AbstractBackground: 5-FU-based chemoradiotherapy (CRT) could be associated with severe treatment-related toxicities in patients harboring at-risk DPYD polymorphisms. Methods: The studied population included consecutive patients with locoregionally advanced oropharyngeal carcinoma treated with carboplatin and 5-FU-based CRT one year before and after the implementation of upfront DPYD*2A genotyping. We aimed to determine the effect of DPYD genotyping on grade ≥3 toxicities. Results: 181 patients were analyzed (87 patients before and 94 patients following DPYD*2A screening). Of the patients, 91% (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of six additional patients with alternative DPYD variants (two c.2846A>T and four c.1236G>A mutations). Grade ≥3 toxicities occurred in 71% of the patients before DPYD*2A screening versus 62% following upfront genotyping (p = 0.18). When retrospectively analyzing additional non-DPYD*2A variants, the relative risks for mucositis (RR 2.36 [1.39–2.13], p = 0.0063), dysphagia (RR 2.89 [1.20–5.11], p = 0.019), and aspiration pneumonia (RR 13 [2.42–61.5)], p = 0.00065) were all significantly increased. Conclusion: The DPYD*2A, c.2846A>T, and c.1236G>A polymorphisms are associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can identify patients in whom 5-FU-related toxicity should be avoided.     

Antimetastatic and anticancer activity of S-1, a new oral dihydropyrimidine-dehydrogenase-inhibiting fluoropyrimidine, alone and in combination with paclitaxel in an orthotopically implanted human breast cancer model

To evaluate the antitumor and antimetastatic efficacy of oral fluoropyrimidines, alone and combined with taxane on human breast cancer xenografts model, we developed a breast cancer model that spontaneously metastasizes to the lung by orthotopic implantation of MDA-MB-435S-HM tumors into the mammary fat pad (mfp) of SCID mice. The activity of the 5-fluorouracil (5-FU)-degrading enzyme dihydropyrimidine dehydrogenase (DPD) was significantly higher in the metastatic tumors than in the primary tumors. Based on this enzymatic characteristic of pulmonary metastases of breast cancer in regard to 5-FU metabolism, we investigated the antitumor activity of two types of oral 5-FU prodrugs, with and without paclitaxel, on both orthotopically implanted breast tumors and metastatic lung tumors in mice. The drugs and doses used were: S-1, a new oral DPD-inhibiting fluoropyrimidine (DIF) 8.3 mg/kg/day, capecitabine 360 mg/kg/day as a non-DIF, and paclitaxel 50 mg/kg, all of which display minimal toxicity in mice. In the primary tumors, paclitaxel and S-1 displayed a significant antitumor activity, with 57 and 41%, respectively inhibition of tumor growth (p < 0.01), but capecitabine had no effect. When S-1 and paclitaxel were combined, they synergistically caused tumor regression (tumor growth inhibition ratio 94%, p < 0.01) in mice compared to capecitabine plus paclitaxel, without any toxicity. In the pulmonary metastasis model, paclitaxel, and both S-1 alone and combined with paclitaxel, but not capecitabine alone or combined with paclitaxel, diaplayed almost complete antimetastatic activity. These results strongly suggest that combination of S-1, as a DIF with taxanes will show a potent high antitumor and antimetastatic effect on refractory human breast cancers, especially those expressing strong DPD activity.     

Impact of DPYD,DPYS, and UPB1 gene variations on severe drug‐related toxicity in patients with cancer

Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype‐guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP‐related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP‐related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss‐of‐function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP‐related high toxicity (P = .003). Although the availability of screening of these rare loss‐of‐function variants is still unknown, our data provide useful information that may help to alleviate FP‐related toxicity in Japanese patients with cancer.     

Novel 5-fluorouracil-resistant human esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase overexpression

5-Fluorouracil (5-FU) is a key drug for the treatment of esophageal squamous cell carcinoma (ESCC); however, resistance to it remains a critical limitation to its clinical use. To clarify the mechanisms of 5-FU resistance of ESCC, we originally established 5-FU-resistant ESCC cells, TE-5R, by step-wise treatment with continuously increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold more resistant to 5-FU in comparison with parental TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation. 5-FU treatment resulted in a significant decrease of the intracellular 5-FU concentration and increase of the concentration of α-fluoro-ureidopropionic acid (FUPA), a metabolite of 5-FU, in TE-5R compared with TE-5 cells in vitro. Conversely, gimeracil, a DPD inhibitor, markedly increased the intracellular 5-FU concentration, decreased the intracellular FUPA concentration, and attenuated 5-FU resistance of TE-5R cells. These results indicate that 5-FU resistance of TE-5R cells is due to the rapid degradation of 5-FU by DPD overexpression. The investigation of 5-FU-resistant ESCC with DPYD gene copy number amplification and consequent DPD overexpression may generate novel biological evidence to explore strategies against ESCC with 5-FU resistance.