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Resistance to 5-FU reduces its clinical efficacy for the treatment of colorectal cancer. Sphingosine-1-phosphate receptor 2 (S1PR2) has emerged as a potential target to reverse 5-FU-resistance by inhibiting the expression of dihydropyrimidine dehydrogenase (DPD). In this study, 38 novel S1PR2 antagonists based on aryl urea structure were designed and synthesized, and the structure-activity relationship was investigated based on the S1PR2 binding assay. Representative compound

The inter-individual differences in cancer susceptibility are somehow correlated with the genetic differences that are caused by the polymorphisms. These genetic variations in drug-metabolizing enzymes/drug-inactivating enzymes may negatively or positively affect the pharmacokinetic profile of chemotherapeutic agents that eventually lead to pharmacokinetic resistance and toxicity against anti-cancer drugs. For instance, the CYP1B1*3 allele is associated with CYP1B1 overexpression and consequent resistance to a variety of taxanes and platins, while 496TG is associated with lower levels of dihydropyrimidine dehydrogenase, which results in severe toxicities related to 5-fluorouracil. In this context, a pharmacogenomics approach can be applied to ascertain the role of the genetic make-up in a person's response to any drug. This approach collectively utilizes pharmacology and genomics to develop effective and safe medications that are devoid of resistance problems. In addition, recently reported genomics studies revealed the impact of many single nucleotide polymorphisms in tumors. These studies emphasized the importance of single nucleotide polymorphisms in drug-metabolizing enzymes on the effect of anti-tumor drugs. In this review, we discuss the pharmacogenomics aspect of polymorphisms in detail to provide an insight into the genetic manipulations in drug-metabolizing enzymes that are responsible for pharmacokinetic resistance or toxicity against well-known anti-cancer drugs. Special emphasis is placed on different deleterious single nucleotide polymorphisms and their effect on pharmacokinetic resistance. The information provided in this report may be beneficial to researchers, especially those who are working in the field of biotechnology and human genetics, in rationally manipulating the genetic information of patients with cancer who are undergoing chemotherapy to avoid the problem of pharmacokinetic resistance/toxicity associated with drug-metabolizing enzymes.

Fluoropyrimidines, the major chemotherapeutic agents in various malignancies treatment, are metabolized by dihydropyrimidine dehydrogenase (DPD). DPD deficiency can lead to severe and sometimes fatal toxicity. In the present study, we developed a simple protocol to detect the DPYD*2A variant. Common side effects in patients treated with these drugs were also evaluated in a Kurdish population.We established a reverse-transcriptase polymerase chain reaction (RT-PCR) technique for detection of DPYD*2A. Sanger sequencing was used to confirm the results. 121 Kurdish patients receiving fluoropyrimidine derivatives were enrolled, and clinical information regarding the dosage and toxicity was analyzed.Our RT-PCR method was able to detect one patient with heterozygous state for DPYD*2A (0.8%). The most observed adverse drug reactions were tingling, nausea, and hair loss. The frequency of patients with the toxicity of grade 3 or worse was 6.6%.This was the first study that detect DPYD*2A polymorphism in the Kurdish population. Our method was successfully able to detect the DPYD*2A variant and, due to its simplicity and cost-effectiveness, it may be considered as an alternative to the current methods, especially in developing countries. Our detected polymorphism rate at 0.8% is comparable with other studies. Despite the low rate of DPYD*2A polymorphism, pharmacogenetics assessment before beginning the treatment process is highly recommended due to its association with a high risk of severe toxicity.

Several studies have shown an association between 5-fluorouracil toxicity and variations in the dihydropyrimidine dehydrogenase (DPYD) gene.This cross-sectional study aims to elucidate the association between genetic variations in the DPYD gene and 5-fluorouracil toxicity among Jordanians with colorectal cancer (CRC).80 CRC Patients were recruited to screen for mutations in the DPYD gene using the Sanger sequencing technique. Sequencing results were analyzed using Mutation Surveyor software, and mutational effects were predicted by the Mutation Tester bioinformatics tool.Three reported variants (c.85TC, c.1740+40AG, c.1740+39CT) and one novel (g.97515583_97515584insA) variant were identified in this study. Results showed a significant association between these variants and toxicity to 5-Fluorouracil with P-values 0.002, 0.005, 0.019, 0.017, respectively. However, there was no significant association between variants and cancer free survival.The present study identified several variants in the DPYD gene among Jordanians with colorectal cancer, which are associated with toxicity to 5-Fluorouracil treatment.

PURPOSE: Patients who carry reduced-activity DPYD polymorphisms have increased fluoropyrimidine (FP) toxicity risk. Although pretreatment DPYD testing is recommended throughout most of Europe, it is not recommended in the United States, and adoption has been limited. The objective of this survey was to describe the current practice in the United States regarding pretreatment DPYD testing and understand the factors deterring oncologists from ordering testing. METHODS: Survey invitations were e-mailed to 325 medical oncologists practicing in the United States who are members of the SWOG Cancer Research Network Gastrointestinal Cancer, Breast Cancer, or Early Therapeutics Committees. Descriptive statistics were used to evaluate survey responses. RESULTS: Responses were collected from 59 (18.2%) US medical oncologists, of whom 98% strongly or somewhat agree that patients with dihydropyrimidine dehydrogenase (DPD) deficiency have increased toxicity risk and 96% would modify FP dosing for a patient with known DPD deficiency. However, only 32% strongly or somewhat agree that pretreatment DPYD testing is useful to inform FP treatment, 20% have ever ordered pretreatment testing, and 3% order testing for at least 10% of their FP-treated patients. The most important factors that deter oncologists from ordering testing were low prevalence of DPD deficiency (54%) and lack of clinical practice guideline recommendations (48%). CONCLUSION: Clinical adoption of pretreatment DPYD testing is extremely limited in the United States. Utilization may be substantially increased by inclusion in the oncology clinical practice guideline recommendations, coverage through health insurance, and potentially education of medical oncologists regarding available treatment modification guidelines.

Fluorouracil/5-flourouracil (5FU) is a first-line chemotherapy drug for many cancer types; however, its associated toxicities contribute to poor quality of life and reduced dose intensities negatively impacting patient prognosis. While obesity remains a critical risk factor for most cancers, our understanding regarding how obesity may impact chemotherapy's toxicities is extremely limited. C56BL/6 mice were given high fat (Obese) or standard diets (Lean) for 4 months and then subjected to three cycles of 5FU (5d-40 mg/kg Lean Mass, 9d rest) or PBS vehicle control. Shockingly, only 60% of Obese survived 3 cycles compared to 100% of Lean, and Obese lost significantly more body weight. Dihydropyrimidine dehydrogenase (DPD), the enzyme responsible for 5FU catabolism, was reduced in obese livers. Total white blood cells, neutrophils, and lymphocytes were reduced in Obese 5FU compared to Lean 5FU and PBS controls. While adipocyte size was not affected by 5FU in Obese, skeletal muscle mass and myofibrillar cross section area were decreased following 5FU in Lean and Obese. Although adipose tissue inflammatory gene expression was not impacted by 5FU, distinct perturbations to skeletal muscle inflammatory gene expression and immune cell populations (CD45

Fluoropyrimidines (FP; 5-fluorouracil, capecitabine, and tegafur) are a commonly prescribed class of antimetabolite chemotherapies, used for various solid organ malignancies in over 2 million patients globally per annum. Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the critical enzyme implicated in FP metabolism. DPYD variant genotypes can result in decreased DPD production, leading to the development of severe toxicities resulting in hospitalization, intensive care admission, and even death. Management of toxicity incurs financial burden on both patients and healthcare systems alike. Upfront DPYD genotyping to identify variant carriers allows an opportunity to identify patients who are at high risk to suffer from serious toxicities and allow prospective dose adjustment of FP treatment. This approach has been shown to reduce patient morbidity, as well as improve the cost-effectiveness of managing FP treatment. Upfront DPYD genotyping has been recently endorsed by several countries in Europe and the United Kingdom. This review summarizes current knowledge about DPD deficiency and upfront DPYD genotyping, including clinical and cost-effectiveness outcomes, with the intent of supporting implementation of an upfront DPYD genotyping service with individualized dose-personalization.

BACKGROUND: Serious and potentially life-threatening toxicities can occur following 5-fluorouracil/capecitabine exposure. Patients carrying Dihydropyrimidine Dehydrogenase (DPYD) variant alleles associated with decreased enzymatic function are at a greater risk of early/severe 5-fluorouracil/capecitabine toxicity. The objective of this systematic review/meta-analysis was to evaluate treatment outcomes between Pharmacogenetics Guided Dosing (PGD) versus non-PGD and within PGD (DPYD variant allele carriers versus wild type). METHODS: A systematic review/meta-analysis of original publications indexed in Ovid Medline, Ovid Embase, and the Cochrane CENTRAL (Wiley) library from inception to 7-Dec-2020. Eligible studies evaluated at least one pre-defined treatment outcome measures (toxicity/hospitalisations/survival/overall response/quality of life). RESULTS: Of 1090 identified publications, 17 met predefined eligibility criteria. The meta-analysis observed reduced incidence of grade 3/4 overall toxicity (Risk Ratio [RR] 0.32 [95% Cl 0.27–0.39], p

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) predict the effects of fluoropyrimidine. However, the effects of FOLFOX therapy from the perspective of fluorouracil plus leucovorin (FL) remain underexplored. Hence, the relationship between mFOLFOX6 therapy (mFOLFOX6) and therapeutic efficacy was evaluated in patients with advanced/recurrent colorectal cancer (CRC).Correlations between TS and DPD and primary and metastatic lesions in recurrent CRC were analyzed. Univariate and multivariate analyses of TS and DPD in combination with response rate (RR), progression-free survival (PFS), and overall survival (OS) were performed.A positive correlation between DPD and primary and metastatic lesions; correlations between TS and RR, DPD and RR, and PFS and OS; and significant differences for RR and DPD and TS, PFS and DPD, and OS and DPD were obtained.Nucleic acid metabolizing enzymes in primary lesions can be used to predict mFOLFOX6 efficacy in patients with recurrent CRC.

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.

5-Fluorouracil (5-FU) and oral fluoropyrimidines, such as capecitabine, are widely used in the treatment of cancer, especially gastrointestinal tumors and breast cancer, but their administration can produce serious and even lethal toxicity. This toxicity is often related to the partial or complete deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme, which causes a reduction in clearance and a longer half-life of 5-FU. It is advisable to determine if a DPD deficiency exists before administering these drugs by genotyping DPYD gene polymorphisms. The objective of this consensus of experts, in which representatives from the Spanish Pharmacogenetics and Pharmacogenomics Society and the Spanish Society of Medical Oncology participated, is to establish clear recommendations for the implementation of genotype and/or phenotype testing for DPD deficiency in patients who are candidates to receive fluoropyrimidines. The genotyping of DPYD previous to treatment classifies individuals as normal, intermediate, or poor metabolizers. Normal metabolizers do not require changes in the initial dose, intermediate metabolizers should start treatment with fluoropyrimidines at doses reduced to 50%, and poor metabolizers are contraindicated for fluoropyrimidines, This project has been financed with SEOM and SEFF resources

Introduction: Colorectal cancer is the third most common cancer type worldwide. Fluoropyrimidines and their prodrug-based regimens are widely applied as primary medications. The main enzyme responsible for the rate-limiting step in pyrimidine and for the 5-fluorouracil catabolism is dihydropyrimidine dehydrogenase (DPD). Aim: We aimed to screen DPD level and the changes of plasma antioxidant capacity of colorectal cancer patients on 5-fluorouracil regimen. Materials and methods: Human DPD Elisa Kit based on sandwich enzyme-linked immune-sorbent assay and spectrophotometric methods (FRAP and ABTS) were used in the study. Results: No statistically significant changes in plasma scavenging activity according to the results obtained in the ABTS system have been observed after evaluating all patients and considering DPD concentration. A decrease of the ferric reducing ability of patients’ plasma taken after the administered treatment was found. The increase of DPD level is accompanied by a decrease in the p values and therefore the statistical significance of the differences increases. Conclusions: Based on the aforementioned observations, it could be concluded that some aspects of plasma antioxidant capacity and individuals’ antioxidant status might be involved in the pathogenesis of the disease and could be altered by the activity of some enzymes. The cancer therapy in question, by the specificity of its mechanism of action, can modify patient’s oxidative status.

Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD deficiency in End Stage Renal Disease (ESRD) patients is unknown. We assessed the characteristics of both DPD phenotypes and DPYD genotypes in 20 dialyzed patients before and after dialysis session. The extent to which the concentrations of uracil [U] and dihydrouracil [UH2] were affected by dialysis was evaluated. Mean [U] was 14 ± 3.3 ng/ml before the dialysis session, and 7.9 ± 2.7 ng/ml after. Notably, mean [U] in 119 non-ESRD patients during the same timeline was 8.7 ± 3.9 ng/ml, which is similar to [U] values after dialysis session (p = 0.38). [U] values > 16 ng/ml were measured in 4 ESRD patients (20%), whereas the rate was 3.3% in the non-ESRD cohort. Whole gene sequencing did not reveal DPYD deleterious allelic variants in the 4 ESRD patients with [U] values > 16 ng/ml. The profile of [UH2] values during dialysis was similar to that of [U]: 385 ± 86 ng/ml before, and 185 ± 62 ng/ml after (mean reduction rate 42.5%). Thus, [UH2]:[U] ratio remained unaffected by dialysis, and was similar to the values in non-ESRD patients (22.4 ± 7.1). Phenotyping based on measuring plasma [U] before a dialysis sessions in ESRD patients is associated with an unacceptable high rate of false positives. The optimal strategy for the identification of patients with DPD deficiency in this population would be the monitor the [UH2]:[U] ratio, which remains unaffected.

Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity. In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed. Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27-52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U. In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.

Deficiency of dihydropyrimidine dehydrogenase (DPD), encoded by the

Patients who carry pathogenic variants inThis special article uses previously published frameworks for assessing the clinical utility of cancer biomarker tests, including for germline indicators of toxicity risk, to assess the clinical utility of pretreatmentThere is no direct evidence of efficacy reduction, and the available indirect evidence demonstrates thatThis article should serve as a call to action for clinicians and clinical guidelines committees in the United States to re-evaluate the clinical utility of pretreatment

MiRNAs regulate the expression of hepatic genes involved in pharmacokinetics and pharmacodynamics. Genetic variants affecting miRNA binding (mirSNPs) have been associated with altered drug response, but previously used methods to identify miRNA binding sites and functional mirSNPs in pharmacogenes are indirect and limited by low throughput. We utilized the high-throughput chimeric-eCLIP assay to directly map thousands of miRNA-mRNA interactions and define the miRNA binding sites in primary hepatocytes. We then used the high-throughput PASSPORT-seq assay to functionally test 262 potential mirSNPs with coordinates overlapping the identified miRNA binding sites. Using chimeric-eCLIP, we identified a network of 448 miRNAs that collectively target 11,263 unique genes in primary hepatocytes pooled from 100 donors. Our data provide an extensive map of miRNA binding of each gene, including pharmacogenes, expressed in primary hepatocytes. For example, we identified the hsa-mir-27b-DPYD interaction at a previously validated binding site. A second example is our identification of 19 unique miRNAs that bind to CYP2B6 across 20 putative binding sites on the transcript. Using PASSPORT-seq, we then identified twenty-four mirSNPs that functionally impacted reporter mRNA levels. To our knowledge this is the most comprehensive identification of miRNA binding sites in pharmacogenes. Combining chimeric-eCLIP with PASSPORT-seq successfully identified functional mirSNPs in pharmacogenes that may affect transcript levels through altered miRNA binding. These results provide additional insights into potential mechanisms contributing to interindividual variability in drug response.

Consensus guidelines exist for genotype‐guided fluoropyrimidine dosing based on variation in the gene dihydropyrimidine dehydrogenase (DPYD). However, these guidelines have not been widely implemented in North America and most studies of pretreatment DPYD screening have been conducted in Europe. Given regional differences in treatment practices and rates of adverse events (AEs), we investigated the impact of pretreatment DPYD genotyping on AEs in a Canadian context. Patients referred for DPYD genotyping prior to fluoropyrimidine treatment were enrolled from December 2013 through November 2019 and followed until completion of fluoropyrimidine treatment. Patients were genotyped for DPYD c.1905+1G>A, c.2846A>T, c.1679T>G, and c.1236G>A. Genotype‐guided dosing recommendations were informed by Clinical Pharmacogenetics Implementation Consortium guidelines. The primary outcome was the proportion of patients who experienced a severe fluoropyrimidine‐related AE (grade ≥3, Common Terminology Criteria for Adverse Events version 5.0). Secondary outcomes included early severe AEs, severe AEs by toxicity category, discontinuation of fluoropyrimidine treatment due to AEs, and fluoropyrimidine‐related death. Among 1394 patients, mean (SD) age was 64 (12) years, 764 (54.8%) were men, and 47 (3.4%) were DPYD variant carriers treated with dose reduction. Eleven variant carriers (23%) and 418 (31.0%) noncarriers experienced a severe fluoropyrimidine‐related AE (p = 0.265). Six carriers (15%) and 284 noncarriers (21.1%) experienced early severe fluoropyrimidine‐related AEs (p = 0.167). DPYD variant carriers treated with genotype‐guided dosing did not experience an increased risk for severe AEs. Our data support a role for DPYD genotyping in the use of fluoropyrimidines in North America.

The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 A that are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.

Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of ∼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 A across the protein.

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil administration. In an in vitro model of fatty acid-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.

To the Editor: We read with interest the article by de With et al. reporting the results of a prospective multicenter study where pretreatment uracil blood samples were collected and handled in 17 Dutch hospitals but analyzed in the reference hospital. [...]

5-fluorouracil (5-FU) is a widely used drug for chemotherapy in colorectal cancer. In this study, we investigated the relationship between the severity of 5-FU induced adverse events and several variations in

The 5-year survival rate of pancreatic ductal carcinoma (PDAC) patients is, This study describes the chemopreventive effects of luteolin on pancreatic carcinogenesis via STAT3 inactivation caused by DPYD down-regulation, resulting in suppression of cell proliferation. This is consistent with the association of high DPYD expression with the poor prognosis of pancreatic cancer.

Aim: To evaluate toxicity risk in carriers of four DPYD variants using an institutional genetic repository. Materials & methods: Of over 65,000 patients in the repository, 582 were evaluated for the primary composite end point of grade 3 or higher toxicity or treatment modification due to toxicity. Results: The primary end point was more common in DPYD variant carriers (36.5 vs 18.1%, adjusted odds ratio 2.42, 95% CI: 1.05–5.55, p = 0.04), and in patients with decreased DPD activity (≤1 vs 2) (75.6 vs 17.0%, adjusted odds ratio 16.31, 95% CI: 2.64–100.68, p = 0.003). Conclusion: Patients carrying any of the four DPYD variants are at increased risk of severe toxicity or subsequent treatment modifications, suggesting such patients may benefit from genotype-informed treatment.

Background Fluoropyrimidines are used in chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. DPYD*2A polymorphism is one of the most studied variants. The study objective was to document the impact of implementing this test in routine clinical practice. Methods We retrospectively performed chart reviews of all patients who tested positive for a heterozygous or homozygous DPYD*2A mutation in samples obtained from patients throughout the province of Quebec, Canada. Results During a period of 17 months, 2,617 patients were tested: 25 patients tested positive. All were White. Twenty-four of the 25 patients were heterozygous (0.92%), and one was homozygous (0.038%). Data were available for 20 patients: 15 were tested upfront, whereas five were identified after severe toxicities. Of the five patients confirmed after toxicities, all had grade 4 cytopenias, 80% grade ≥3 mucositis, 20% grade 3 rash, and 20% grade 3 diarrhea. Eight patients identified with DPYD*2A mutation prior to treatment received fluoropyrimidine-based chemotherapy at reduced initial doses. The average fluoropyrimidine dose intensity during chemotherapy was 50%. No grade ≥3 toxicities were observed. DPYD*2A test results were available in an average of 6 days, causing no significant delays in treatment initiation. Conclusion Upfront genotyping before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. The administration of chemotherapy at reduced doses appears to be safe in patients heterozygous for DPYD*2A. Implications for Practice Fluoropyrimidines are part of chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. This retrospective analysis demonstrates that upfront genotyping of DPYD before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. This approach was reported previously, but insufficient data concerning its application in real practice are available. This is likely the first reported experience of systematic DPYD genotyping all over Canada and North America as well.

Uveal melanoma (UM) is the most common intraocular tumor in adults and has a high incidence of metastases. Possible treatments remain limited in UM with enucleation and radiation, leading to poor prognosis in this chemo-resistant carcinoma. Thus, urging demand for novel treatment is needed. We examined the antitumor efficacy of a new recombinant oncolytic herpes simplex virus type 1 (oHSV-1) armed with E.coli cytosine deaminase (CD). We determined the efficacy of the oncolytic virus in UM cell lines. In vivo experiments showed that oHSV-CD/5-fluorocytosine (5-FC) treatment reduce tumor volume and prolonged survival. We further demonstrated the molecular mechanisms of oHSV-CD/5-FC treatment. The oncolytic virus down-regulated IL-6 expression and thereby reversed the epithelial-mesenchymal transition (EMT) phenotype. Dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-fluorouracil (5-FU) metabolism, was also down-regulated. Therefore, the efficacy of oHSV-CD/5-FC was synergistically enhanced by DPD down-regulation and EMT inhibition. This study provides solid evidence for the antitumor efficacy of oHSV-CD/5-FC treatment in vitro and in vivo. The molecular mechanisms of this treatment may bring a new therapeutic approach for future treatment of UM.

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.

Background Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive. Methods Among a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated. Results When [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] 150 ng/ml), and [UH2]:[U] DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant. Conclusions Frequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Background 5-Fluorouracil (5-FU) and capecitabine are fluoropyrimidine derivatives that mainly metabolized with dihydropyrimidine dehydrogenase enzyme (DPD). The genetic polymorphism in the genes encoding this enzyme may result in a decrease or loss of enzyme activity which may lead to the accumulation of medicines, their metabolites and potential toxicity. Method This cross-sectional study was conducted on 88 participants with colorectal cancer (CRC). After DNA extraction, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the DPD gene (DPYD) polymorphisms including IVS 14 + 1 G > A, 2846 A > T and 2194 G > A. Chemotherapy-induced side effects were evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE Version 5.0). Result Data were collected from 227 chemotherapy cycles of 88 patients with CRC. In a comparison of FOLFOX and FOLFIRI regimens, there was no significant difference in the occurrence of chemotherapy-induced diarrhea, nausea, vomiting and oral mucositis. However, the peripheral neuropathy was more frequent in patients who were treated with FOLFOX (P P = 0.048). Incidence of the DPD IVS14 + 1 G > A polymorphism was observed in four patients (5.5%). There was no association between IVS14 + 1 G > A polymorphism and the occurrence of adverse reactions. Conclusion FOLFOX and FOLFIRI were the most common regimens in CRC patients and their toxicity profile was different in some adverse reactions. Prevalence of IVS14 + 1G > A variant was relatively higher than other similar studies. Trial registration Approval code; IR.MAZUMS.REC.95.2480.

International audience; Detecting patients with dihydropyrimidine dehydrogenase (DPD) deficiency is becoming a major concern in clinical oncology. Monitoring physiologic plasma uracil and/or plasma uracil-to-dihydrouracil metabolic ratio is a common surrogate frequently used to determine DPD phenotype without direct measurement of the enzymatic activity. With respect to the increasing number of patients rquiring analysis, it is critical to develop simple, rapid, and affordable methods suitable for routine screening. We have developed and validated a simple and robust ultraperformance liquid chromatography-ultraviolet (UPLC-UV) method with shortened (i.e., 12 minutes) analytical run-times, compatible with the requirements of large-scale upfront screening. The method enables detection of uracil (U) over a range of 5-500 ng/ml (265 nm) and of dihydrouracil (UH2) over a range of 40-500 ng/ml (210 nm) in plasma with no chromatographic interference. When used as part of routine screening for DPD deficiency, this method was fully able to discriminate nondeficient patients (i.e., with U levels < 16 ng/ml) from deficient patients at risk of severe toxicity (i.e., U > 16 ng/ml). Results from 1 month of routine testing are presented and, although no complete deficits were detected, 10.7% of the screened patients presented DPD deficiency and would thus require s decresed dose. Overall, this new method, using a simple preanalytical solid-phase extraction procedure, and based on use of a standard UPLC apparatus, is both cost- and time-effective and can be easily implemented in any laboratory aiming to begin routine DPD testing.

Capecitabine is an oral prodrug of 5-fluorouracil with a relevant role in the treatment of breast cancer. Severe and unexpected toxicities related to capecitabine are not rare, and the identification of biomarkers is challenging. We evaluate the relationship between dihydropyrimidine dehydrogenase, thymidylate synthase enhancer region and methylenetetrahydrofolate reductase polymorphisms, 5-fluorouracil degradation rate and the onset of G3–4 toxicities in breast cancer patients. Genetic polymorphisms and the 5-fluorouracil degradation rate of breast cancer patients treated with capecitabine were retrospectively studied. Genetic markers and the 5-fluorouracil degradation rate were correlated with the reported toxicities. Thirty-seven patients with a median age of 58 years old treated with capecitabine for stages II–IV breast cancer were included in this study. Overall, 34 (91.9%) patients suffered from at least an episode of any grade toxicity while nine patients had G3–4 toxicity. Homozygous methylenetetrahydrofolate reductase 677TT was found to be significantly related to haematological toxicity (OR = 6.5 [95% IC 1.1–37.5], P = 0.04). Three patients had a degradation rate less than 0.86 ng/mL/106 cells/min and three patients greater than 2.1 ng/mL/106 cells/min. At a univariate logistic regression analysis, an altered value of 5-fluorouracil degradation rate (values 2.10 ng/mL/106 cells/min) increased the risk of G3–4 adverse events (OR = 10.40 [95% IC: 1.48–7.99], P = 0.02). A multivariate logistic regression analysis, adjusted for age, comorbidity and CAPE-regimen, confirmed the role of 5-fluorouracil degradation rate as a predictor of G3–4 toxicity occurrence (OR = 10.9 [95% IC 1.2–96.2], P = 0.03). The pre-treatment evaluation of 5-fluorouracil degradation rate allows to identify breast cancer patients at high risk for severe 5-FU toxicity.

In clinical practice, 25–30% of the patients treated with fluoropyrimidines experience severe fluoropyrimidine-related toxicity. Extensively clinically validated DPYD genotyping tests are available to identify patients at risk of severe toxicity due to decreased activity of dihydropyrimidine dehydrogenase (DPD), the rate limiting enzyme in fluoropyrimidine metabolism. In April 2020, the European Medicines Agency recommended that, as an alternative for DPYD genotype-based testing for DPD deficiency, also phenotype testing based on pretreatment plasma uracil levels is a suitable method to identify patients with DPD deficiency. Although the evidence for genotype-directed dosing of fluoropyrimidines is substantial, the level of evidence supporting plasma uracil levels to predict DPD activity in clinical practice is limited. Notwithstanding this, uracil-based phenotyping is now used in clinical practice in various countries in Europe. We aimed to determine the value of pretreatment uracil levels in predicting DPD deficiency and severe treatment-related toxicity. To this end, we determined pretreatment uracil levels in 955 patients with cancer, and assessed the correlation with DPD activity in peripheral blood mononuclear cells (PBMCs) and fluoropyrimidine-related severe toxicity. We identified substantial issues concerning the use of pretreatment uracil in clinical practice, including large between-center study differences in measured pretreatment uracil levels, most likely as a result of pre-analytical factors. Importantly, we were not able to correlate pretreatment uracil levels with DPD activity nor were uracil levels predictive of severe treatment-related toxicity. We urge that robust clinical validation should first be performed before pretreatment plasma uracil levels are used in clinical practice as part of a dosing strategy for fluoropyrimidines.

To determine the prevalence of loss-of-function variants in the dihydropyrimidine dehydrogenase gene in patients with gastrointestinal neoplasms, assess their clinical relevance, and evaluate the implementation of a multidisciplinary circuit at three months from its implementation.This is a descriptive, observational and retrospective study, which included adult patients with gastrointestinal cancer treated at a tertiary university hospital who underwent dihydropyrimidine dehydrogenase genotyping between September 2019 and December 2020. The variables collected were sex, age, type of cancer, location, stage, treatment received, indication of treatment and degree of toxicity developed during the first three cycles. The genotyped variants were rs3918290 (c.1905+1Ggt;A), rs55886062 (c.1679Tgt;G), rs67376798 (c.2846Agt;T) and rs75017182 (c.1129-5923Cgt;G).A total of 115 patients were included. The frequency of heterozygous dihydropyrimidine dehydrogenase variant carriers was 9.6% (11 patients). The most frequently identified variant was rs75017182 (6 patients). The second most common variant was rs67376798 (3 patients), followed by rs3918290 (2 patients). No patients presented with the rs55886062 variant. Two of the dihydropyrimidine dehydrogenase carriers developed grade 3-5 toxicity after the first cycle of a regimen that included fluoropyrimidines. Both received full doses of fluoropyrimidine, since their dihydropyrimidine dehydrogenase genotype was unknown before treatment initiation. None of the dihydropyrimidine dehydrogenase carriers who began treatment with a reduced dose of fluoropyrimidine experienced grade 3-5 toxicity. Since the creation in October 2020 of a multidisciplinary team, with the active participation of hospital pharmacists, the monthly average of dihydropyrimidine dehydrogenase genotyping studies has increased from 6.4 (January-October) to 17.5 (November-December).The present study shows a relatively high prevalence of loss-of- function variants in the dihydropyrimidine dehydrogenase gene as well as the importance of genotyping such variants before starting a treatment with fluoropyrimidines. Hospital pharmacists can contribute to the implementation of pharmacogenetics in daily clinical practice in a tertiary hospital.Determinar la prevalencia de variantes de pérdida de función en el gen de la dihidropirimidina deshidrogenasa (DPYD) en pacientes con tumores digestivos, valorar su relevancia clínica y evaluar la implementación de un circuito multidisciplinar tras tres meses de funcionamiento.Método: Estudio descriptivo, observacional y retrospectivo donde se incluyeron los pacientes adultos afectos de tumores digestivos, atendidos en un hospital universitario de tercer nivel, a los que se había afectuado el genotipado de DPYD entre septiembre de 2019 y diciembre de 2020. Las variables recogidas fueron sexo, edad, tipo de cáncer, localización, estadio, tratamiento recibido, indicación del tratamiento y grado de toxicidad desarrollado durante los tres primeros ciclos. Se genotiparon las variantes rs3918290 (c.1905+1Ggt;A), rs55886062 (c.1679Tgt;G), rs67376798 (c.2846Agt;T) y rs75017182 (c.1129-5923Cgt;G).Se incluyeron 115 pacientes. La frecuencia de portadores en heterocigosis de variantes del gen DPYD fue del 9,6% (11 pacientes). La variante más frecuentemente identificada fue el rs75017182 (6 pacientes). La segunda variante más frecuente fue el rs67376798 (3 pacientes), seguida del rs3918290 (2 pacientes). Ningún paciente presentó la variante rs55886062. Dos de los pacientes portadores desarrollaron toxicidad grados 3-5 tras el primer ciclo de un esquema que incluía fluoropirimidinas. Ambos recibieron dosis plenas de fluoropirimidina, puesto que no se conocía el genotipo de DPYD antes de iniciar el tratamiento. Ninguno de los pacientes portadores que tmpezó el tratamiento con una dosis reducida de fluoropirimidina experimentó toxicidad grados 3-5. Desde la creación en octubre de 2020 de un equipo multidisciplinar, con participación activa del farmacéutico hospitalario, se ha incrementado el número de estudios de genotipado de DPYD de una media de 6,4 estudios mensuales (enero-octubre) a 17,5 (noviembre-diciembre).Nuestro estudio muestra la relativamente elevada prevalencia de variantes de pérdida de función en el gen DPYD, así como la importancia de genotiparlas antes de empezar un esquema de tratamiento que contenga fluoropirimidinas. El farmacéutico hospitalario puede contribuir a la implementación de la farmacogenética en la práctica clínica diaria en un hospital de tercer nivel.

In response to the recently published article by Sharma et al, this letter to the editor presents data about the concordance of blood and normal tissue-based evaluation of DPYD genotyping that suggests that pharmacogenetic screening could be contextual to tumor molecular profiling.

Adjuvant 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.We conducted a cost-effectiveness analysis of DPYD genotyping prior to fluoropyrimidine-based adjuvant chemotherapy for stage 3 colon cancer, covering the c.1129-5923CG (HapB3), c.1679TG (*13), c.1905+1GA (*2A), and c.2846AT 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.Compared 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.Among 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.

Chemotherapy is a standard therapy for muscle-invasive bladder cancer (MIBC). However, genomic alterations associated with chemotherapy sensitivity in MIBC have not been fully explored. This study aimed to investigate the genomic landscape of MIBC in association with the response to chemotherapy and to explore the biological role of genomic alterations. Genomic alterations in MIBC were sequenced by targeted exome sequencing of 409 genes. Gene expression in MIBC tissues was analyzed by western blotting, immunohistochemistry, and RNA microarray. Cellular sensitivity to gemcitabine and gemcitabine metabolite was examined in bladder cancer cells after modulation of candidate gene. Targeted exome sequencing in 20 cases with MIBC revealed various genomic alterations including pathogenic missense mutation of DPYD gene encoding dihydropyrimidine dehydrogenase (DPD). Conversely, high DPYD and DPD expression were associated with poor response to gemcitabine-containing chemotherapy among patients with MIBC, as well as gemcitabine resistance in bladder cancer cells. DPD suppression rendered cells sensitive to gemcitabine, while DPD overexpression made cells gemcitabine-resistant through reduced activity of the cytotoxic gemcitabine metabolite difluorodeoxycytidine diphosphate. This study revealed the novel role of DPD in gemcitabine metabolism. It has been suggested that DPYD genomic alterations and DPD expression are potential predictive biomarkers in gemcitabine treatment.

International audience; AIMS: Determining dihydropyrimidine dehydrogenase (DPD) activity by measuring patient’s uracil (U) plasma concentration is mandatory before fluoropyrimidines (FP) administration in France. In this study, we aimed to refine the pre-analytical recommendations for determining U and dihydrouracil (UH(2) ) concentrations, since they are essential in reliable DPD deficiency testing.METHODS: U and UH(2) concentrations were collected from 14 hospital laboratories. Stability in whole blood and plasma after centrifugation, the type of anticoagulant and long-term plasma storage were evaluated. The variation induced by time and temperature was calculated and compared to an acceptability range of ± 20%. Inter-occasion variability (IOV) of U and UH(2) was assessed in 573 patients double sampled for DPD-deficiency testing. RESULTS: Storage of blood samples before centrifugation at room temperature (RT) should not exceed 1 h, whereas cold (+4°C) storage maintains U stable after 5 hours. For patients correctly double sampled, IOV of U reached 22.4% for U (SD = 17.9%, range = [0-99%]). Notably, 17% of them were assigned with a different phenotype (normal or DPD deficient) based on the analysis of their two samples. For those having at least one non-compliant sample, this percentage increased up to 33.8%. The moment of blood collection did not affect the DPD phenotyping result.CONCLUSION: Caution should be taken when interpreting U concentrations if the time before centrifugation exceeds 1 h at RT, since it rises significantly afterwards. Not respecting the pre-analytical conditions for DPD phenotyping increases the risk of DPD status misclassification.

Preemptive targeted pharmacogenetic testing of candidate variations in DPYD is currently being used to limit toxicity associated with fluoropyrimidines. The use of innovative next generation sequencing (NGS) approaches could unveil additional rare (minor allele frequency 1%) genetic risk variants. However, their predictive value and management in clinical practice are still controversial, at least partly due to the challenges associated with functional analyses of rare variants. The aim of this study was to define the predictive power of rare DPYD variants burden on the risk of severe fluoropyrimidine-related toxicity. The DPYD coding sequence and untranslated regions were analyzed by NGS in 120 patients developing grade 3-5 (NCI-CTC vs3.0) fluoropyrimidine-related toxicity and 104 matched controls (no-toxicity). The functional impact of rare variants was assessed using two different in silico predictive tools (i.e., Predict2SNP and ADME Prediction Framework) and structural modeling. Plasma concentrations of uracil (U) and dihydrouracil (UH2) were quantified in carriers of the novel variants. Here, we demonstrate that the burden of rare variants was significantly higher in patients with toxicity compared to controls (p = 0.007, Mann-Whitney test). Carriers of at least one rare missense DPYD variant had a 16-fold increased risk in the first cycle and an 11-fold increased risk during the entire course of chemotherapy of developing a severe adverse event compared to controls (p = 0.013 and p = 0.0250, respectively by multinomial regression model). Quantification of plasmatic U/UH2 metabolites and in silico visualization of the encoded protein were consistent with the predicted functional effect for the novel variations. Analysis and consideration of rare variants by DPYD-sequencing could improve prevention of severe toxicity of fluoropyrimidines and improve patients' quality of life.

Aim To independently assess the impact of mandatory testing using an extended DPYD variant panel (ToxNav®) and consequent dose adjustment of Capecitabine/5-FU on recorded quantitative toxicity, symptoms of depression, and hospital costs. Methods We used propensity score matching (PSM) to match 466 patients tested with ToxNav® with 1556 patients from a historical cohort, and performed regression analysis to estimate the impact of ToxNav®on toxicity, depression, and hospital costs. Results ToxNav® appeared to reduce the likelihood of experiencing moderate (OR: 0.59; 95%CI: 0.45–0.77) and severe anaemia (OR: 0.55; 95%CI: 0.33–0.90), and experience of pain for more than 4 days a week (OR: 0.50; 95%CI: 0.30–0.83), while it increased the likelihood of mild neutropenia (OR: 1.73; 95%CI: 1.27–2.35). It also reduced the cost of chemotherapy by 12% (95%CI: 3–31) or £9765, the cost of non-elective hospitalisation by 23% (95%CI: 8–36) or £2331, and the cost of critical care by 21% (95%CI: 2–36) or £1219 per patient. For the DPYD variant associated with critical risk of toxicity (rs3918290), the improved non-elective hospital costs were > £20,000, whereas variants associated with hand-foot syndrome toxicity had no detectable cost improvement. Conclusion Upfront testing of DPYD variants appears to reduce the toxicity burden of Capecitabine and 5-FU in cancer patients and can lead to substantial hospital cost savings, only if the dose management of the drugs in response to variants detected is standardised and regulated.

Background: In clinical oncology, systemic 5-fluorouracil (5-FU) and its oral pro-drugs are used to treat a broad group of solid tumours. Patients with dihydropyrimidine dehydrogenase (DPD) enzyme deficiency are at elevated risk of toxicity if treated with standard doses of 5-FU. DPYD genotyping and measurements of plasma uracil concentration (DPD phenotyping) can be applied as tests for DPD deficiency. In April 2020, the European Medicines Agency recommended pre-treatment DPD testing to reduce the risk of 5-FU-related toxicity. Objectives: The objective of this study is to present the current evidence for DPD testing in routine oncological practice. Methods: Two systematic literature searches were performed following the PRISMA guidelines. We identified studies examining the possible benefit of DPYD genotyping or DPD phenotyping on the toxicity risk. Findings: Nine and 12 studies met the criteria for using DPYD genotyping and DPD phenotyping, respectively. Conclusions: The evidence supporting either DPYD genotyping or DPD phenotyping as pre-treatment tests to reduce 5-FU toxicity is poor. Further evidence is still needed to fully understand and guide clinicians to dose by DPD activity.

This letter to the editor responds to Morelli et al, continuing the discussion on the implementation of DPYD genotyping.

To investigate if dihydropyrimidine dehydrogenase phenotyping has added value when combined withRetrospective cohort study in which treatment and toxicity data were collected of 228 patients genotyped for fourSevere toxicity occurred in 25% of patients with a variant and normal dihydropyrimidine dehydrogenase activity, in 21% of patients without a variant and with decreased dihydropyrimidine dehydrogenase activity, and in 29% of patients without a variant and with normal dihydropyrimidine dehydrogenase activity (controls). The majority of patients with a variant or a decreased dihydropyrimidine dehydrogenase activity received an initial dose reduction (68% and 53% vs 19% in controls) and had a lower mean dose intensity (75% and 81% vs 91% in controls). Fifty percent of patients with a variant and decreased enzyme activity experienced severe toxicity, despite the lowest initial dose and whole treatment dose intensity. They also experienced more grade 4/5 toxicities.Our results indicate that a combined genotype-phenotype approach could be useful to identify patients at increased risk for fluoropyrimidine-associated toxicity (e.g. patients with a variant and decreased dihydropyrimidine dehydrogenase activity). Because the group sizes are too small to demonstrate statistically significant differences, this warrants further research in a prospective study in a larger cohort.

Fluoropyrimidines are chemotherapy drugs that may cause severe adverse events, and their metabolism occurs by dihydropyrimidine deydrogenase (DPD), coded by DPYD. Variants in the DPYD were associated to a greater risk of toxicity. Our aim was to determine the frequency of the most relevant DPYD alleles according to CPIC guidelines (DPYD*2A-rs3918290, DPYD*13-rs55886062, rs67376798, and HapB3-rs75017182) in a sample of 800 healthy Southern Brazilians. Frequencies for rs3918290, rs75017182, and rs67376798 were 0.25%, 1.06%, and 0.38%, respectively. No rs55886062 allele was detected. In total, 3.4% of individuals were classified as intermediate metabolizers. Frequencies for rs3918290, rs55886062, and rs67376798 were similar to those found in non-Finnish Europeans; however, rs75017182 was less frequent when compared to non-Finnish Europeans, but more frequent than in Africans and East Asians. rs3918290 and rs67376798 also presented higher frequency when compared to Africans. The Latino population was the only one that did not differ from our sample in any variant analyzed. The frequencies for all the other populations (non-Finnish European, African, South Asian, and East Asian) presented differences from our sample in at least one variant. rs115232898 was not analyzed in the present study. Cost-effective studies should be performed to evaluate the implementation of these tests in the clinical practice in the Southern Brazil.

Cancer patients with reduced dihydropyrimidine dehydrogenase (DPD) activity are at increased risk of severe fluoropyrimidine (FP)-related adverse events (AE). Guidelines recommend FP dosing adjusted to genotype-predicted DPD activity based on four DPYD variants (rs3918290, rs55886062, rs67376798 and rs56038477). We evaluated the relationship between three further DPYD polymorphisms: c.496AG (rs2297595), *6 c.2194GA (rs1801160) and *9A c.85TC (rs1801265) and the risk of severe AEs.Consecutive FP-treated adult patients were genotyped for "standard" and tested DPYD variants, and for UGT1A1*28 if irinotecan was included, and were monitored for the occurrence of grade ≥3 (National Cancer Institute Common Terminology Criteria) vs. grade 0-2 AEs. For each of the tested polymorphisms, variant allele carriers were matched to respective wild type controls (optimal full matching combined with exact matching, in respect to: age, sex, type of cancer, type of FP, DPYD activity score, use of irinotecan/UGT1A1, adjuvant therapy, radiotherapy, biological therapy and genotype on the remaining two tested polymorphisms).Of the 503 included patients (82.3% colorectal cancer), 283 (56.3%) developed grade ≥3 AEs, mostly diarrhoea and neutropenia. Odds of grade ≥3 AEs were higher in c.496AG variant carriers (n = 127) than in controls (n = 376) [OR = 5.20 (95% CI 1.88-14.3), Bayesian OR = 5.24 (95% CrI 3.06-9.12)]. Odds tended to be higher in c.2194GA variant carriers (n = 58) than in controls (n = 432) [OR = 1.88 (0.95-3.73), Bayesian OR = 1.90 (1.03-3.56)]. c.85TC did not appear associated with grade ≥3 AEs (206 variant carriers vs. 284 controls).DPYD c.496AG and possibly c.2194GA variants might need to be considered for inclusion in the DPYD genotyping panel.