Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity. METHODS: A male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing. RESULTS: A novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis. CONCLUSIONS: The study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

Personalized Chronomodulated 5-Fluorouracil Treatment: A Physiologically-Based Pharmacokinetic Precision Dosing Approach for Optimizing Cancer Therapy.

The discovery of circadian clock genes greatly amplified the study of diurnal variations impacting cancer therapy, transforming it into a rapidly growing field of research. Especially, use of chronomodulated treatment with 5-fluorouracil (5-FU) has gained significance. Studies indicate high interindividual variability (IIV) in diurnal variations in dihydropyrimidine dehydrogenase (DPD) activity - a key enzyme for 5-FU metabolism. However, the influence of individual DPD chronotypes on chronomodulated therapy remains unclear and warrants further investigation. To optimize precision dosing of chronomodulated 5-FU, this study aims to: (i) build physiologically-based pharmacokinetic (PBPK) models for 5-FU, uracil, and their metabolites, (ii) assess the impact of diurnal variation on DPD activity, (iii) estimate individual DPD chronotypes, and (iv) personalize chronomodulated 5-FU infusion rates based on a patient's DPD chronotype. Whole-body PBPK models were developed with PK-Sim(R) and MoBi(R). Sinusoidal functions were used to incorporate variations in enzyme activity and chronomodulated infusion rates as well as to estimate individual DPD chronotypes from DPYD mRNA expression or DPD enzymatic activity. Four whole-body PBPK models for 5-FU, uracil, and their metabolites were established utilizing data from 41 5-FU and 10 publicly available uracil studies. IIV in DPD chronotypes was assessed and personalized chronomodulated administrations were developed to achieve (i) comparable 5-FU peak plasma concentrations, (ii) comparable 5-FU exposure, and (iii) constant 5-FU plasma levels via "noise cancellation" chronomodulated infusion. The developed PBPK models capture the extent of diurnal variations in DPD activity and can help investigate individualized chronomodulated 5-FU therapy through testing alternative personalized dosing strategies.

Updated DPYD HapB3 haplotype structure and implications for pharmacogenomic testing.

The DPYD gene encodes dihydropyrimidine dehydrogenase, the rate-limiting enzyme for the metabolism of fluoropyrimidines 5-fluorouracil and capecitabine. Genetic variants in DPYD have been associated with altered enzyme activity, therefore accurate detection and interpretation is critical to predict metabolizer status for individualized fluoropyrimidine therapy. The most commonly observed deleterious variation is the causal variant linked to the previously described HapB3 haplotype, c.1129-5923C>G (rs75017182) in intron 10, which introduces a cryptic splice site. A benign synonymous variant in exon 11, c.1236G>A (rs56038477) is also linked to HapB3 and is commonly used for testing. Previously, these single-nucleotide polymorphisms (SNPs) have been reported to be in perfect linkage disequilibrium (LD); therefore, c.1236G>A is often utilized as a proxy for the function-altering intronic variant. Clinical genotyping of DPYD identified a patient who had c.1236G>A, but not c.1129-5923C>G, suggesting that these two SNPs may not be in perfect LD, as previously assumed. Additional individuals with c.1236G>A, but not c.1129-5923C>G, were identified in the Children's Mercy Data Warehouse and the All of Us Research Program version 7 cohort substantiating incomplete SNP linkage. Consequently, testing only c.1236G>A can generate false-positive results in some cases and lead to suboptimal dosing that may negatively impact patient therapy and prospect of survival. Our data show that DPYD genotyping should include the functional variant c.1129-5923C>G, and not the c.1236G>A proxy, to accurately predict DPD activity.

The burden of rare variants in DPYS gene is a novel predictor of the risk of developing severe fluoropyrimidine-related toxicity.

BACKGROUND: Despite a growing number of publications highlighting the potential impact on the therapy outcome, rare genetic variants (minor allele frequency < 1%) in genes associated to drug adsorption, distribution, metabolism, and elimination are poorly studied. Previously, rare germline DPYD missense variants were shown to identify a subset of fluoropyrimidine-treated patients at high risk for severe toxicity. Here, we investigate the impact of rare genetic variants in a panel of 54 other fluoropyrimidine-related genes on the risk of severe toxicity. METHODS: The coding sequence and untranslated regions of 54 genes related to fluoropyrimidine pharmacokinetics/pharmacodynamics were analyzed by next-generation sequencing in 120 patients developing grade 3-5 toxicity (NCI-CTC vs3.0) and 104 matched controls. Sequence Kernel Association Test (SKAT) analysis was used to select genes with a burden of genetic variants significantly associated with risk of severe toxicity. The statistical association of common and rare genetic variants in selected genes was further investigated. The functional impact of genetic variants was assessed using two different in silico prediction tools (Predict2SNP; ADME Prediction Framework). RESULTS: SKAT analysis highlighted DPYS and PPARD as genes with a genetic mutational burden significantly associated with risk of severe fluoropyrimidine-related toxicity (Bonferroni adjusted P = 0.024 and P = 0.039, respectively). Looking more closely at allele frequency, the burden of rare DPYS variants was significantly higher in patients with toxicity compared with controls (P = 0.047, Mann-Whitney test). Carrying at least one rare DPYS variant was associated with an approximately fourfold higher risk of severe cumulative (OR = 4.08, P = 0.030) and acute (OR = 4.21, P = 0.082) toxicity. The burden of PPARD rare genetic variants was not significantly related to toxicity. Some common variants with predictive value in DPYS and PPARD were also identified: DPYS rs143004875-T and PPARD rs2016520-T variants predicted an increased risk of severe cumulative (P = 0.002 and P = 0.001, respectively) and acute (P = 0.005 and P = 0.0001, respectively) toxicity. CONCLUSION: This work demonstrated that the rare mutational burden of DPYS, a gene strictly cooperating with DPYD in the catabolic pathway of fluoropyrimidines, is a promising pharmacogenetic marker for precision dosing of fluoropyrimidines. Additionally, some common genetic polymorphisms in DPYS and PPARD were identified as promising predictive markers that warrant further investigation.

The unusual chemical sequences of mammalian dihydropyrimidine dehydrogenase revealed by transient-state analysis.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the 5,6-vinylic bond of uracil and thymine with electrons from NADPH. The complexity of the enzyme belies the simplicity of the reaction catalyzed. To accomplish this chemistry DPD has two active sites that are ∼60Šapart, both of which house flavin cofactors, FAD and FMN. The FAD site interacts with NADPH, while the FMN site with pyrimidines. The distance between the flavins is spanned by four Fe4S4 centers. Though DPD has been studied for nearly 50years, it is only recently that the novel apects of its mechanism have been described. The primary reason for this is that the chemistry of DPD is not portrayed adequately by known descriptive steady-state mechanism categories. The highly chromophoric nature of the enzyme has recently been exploited in transient-state to document unexpected reaction sequences. Specifically, DPD undergoes reductive activation prior to catalytic turnover. Two electrons are taken up from NADPH and transmitted via the FAD and Fe4S4 centers to form the FAD•4(Fe4S4)•FMNH2 form of the enzyme. This form of the enzyme will only reduce pyrimidine substrates in the presence NADPH, establishing that hydride transfer to the pyrimidine precedes reductive reactivation that reinstates the active form of the enzyme. DPD is therefore the first flavoprotein dehydrogenase known to complete the oxidative half-reaction prior to the reductive half-reaction. Here we describe the methods and deduction that led to this mechanistic assignment.

Addressing barriers to increased adoption of DPYD genotyping at a large multisite cancer center.

PURPOSE: To describe the implementation of an in-house genotyping program to detect genetic variants linked to impaired dihydropyrimidine dehydrogenase (DPD) metabolism at a large multisite cancer center, including barriers to implementation and mechanisms to overcome barriers to facilitate test adoption. SUMMARY: Fluoropyrimidines, including fluorouracil and capecitabine, are commonly used chemotherapy agents in the treatment of solid tumors, such as gastrointestinal cancers. DPD is encoded by the DPYD gene, and individuals classified as DPYD intermediate and poor metabolizers due to certain genetic variations in DPYD can experience reduced fluoropyrimidine clearance and an increased risk of fluoropyrimidine-related adverse events. Although pharmacogenomic guidelines provide evidence-based recommendations for DPYD genotype-guided dosing, testing has not been widely adopted in the United States for numerous reasons, including limited education/awareness of clinical utility, lack of testing recommendations by oncology professional organizations, testing cost, lack of accessibility to a comprehensive in-house test and service, and prolonged test turnaround time. Based on stakeholder feedback regarding barriers to testing, we developed an in-house DPYD test and workflow to facilitate testing in multiple clinic locations at Levine Cancer Institute. Across 2 gastrointestinal oncology clinics from March 2020 through June 2022, 137 patients were genotyped, and 13 (9.5%) of those patients were heterozygous for a variant and identified as DPYD intermediate metabolizers. CONCLUSION: Implementation of DPYD genotyping at a multisite cancer center was feasible due to operationalization of workflows to overcome traditional barriers to testing and engagement from all stakeholders, including physicians, pharmacists, nurses, and laboratory personnel. Future directions to scale and sustain testing in all patients receiving a fluoropyrimidine across all Levine Cancer Institute locations include electronic medical record integration (eg, interruptive alerts), establishment of a billing infrastructure, and further refinement of workflows to improve the rate of pretreatment testing.

Dihydropyrimidine Dehydrogenase (DPD) as a Bridge between the Immune Microenvironment of Colon Cancers and 5-FU Resistance.

BACKGROUND: The purpose of the present study was to investigate the role of the 5-Fluorouracil (5-FU) resistance-related factor dihydropyrimidine dehydrogenase (DPD) in tumor immunity and prognosis and to study the relationship between drug resistance and the immune microenvironment of colon cancer. METHODS: Bioinformatics methods were used to analyze the expression of DPD associated with prognosis, immunity, microsatellite instability, and tumor mutational burden in colon cancer. Immunohistochemistry (IHC) was used to detect DPD, MLH1, MSH2, MSH6, and PMS2 in 219 colon cancer tissue samples. Additional IHC analyses were conducted to detect CD4, CD8, CD20, and CD163 in 30 colon cancer tissue samples with the most extensive immune infiltration. The significance of the correlations and clinical significance of DPD with immune infiltration, immune-related markers, microsatellite instability-related indicators, and prognosis were evaluated. RESULTS: The major findings of the present study are as follows: (1) DPD was expressed in tumor and immune cells and associated with certain immune cell-related markers, particularly M2 macrophages that expressed CD163. (2) DPD expression significantly and positively correlated with immune cell markers and immune checkpoints PD-1 and PD-L1. High expression of DPD in immune cells, but not tumor cells, led to increased immune infiltration. (3) High expression of DPD in immune and tumor cells induced 5-FU resistance and was associated with unfavorable prognosis. (4) DPD expression closely correlated with microsatellite instability and tumor mutational burden and led to resistance to 5-FU in patients with microsatellite instability. (5) Bioinformatics analyses revealed that DPD was enriched in immune-related functions and pathways such as activation of T cells and macrophages. CONCLUSIONS: DPD plays an important role in the immune microenvironment and drug resistance of colon cancers and their functional association.

Precision fluoropyrimidines dosing in a compound heterozygous variant carrier of the DPYD gene: a case report.

BACKGROUND: Fluoropyrimidines (FPs) form still nowadays the backbone of chemotherapic schemes in colorectal cancer (CRC). Inter-patient variability of the toxicity profile of FPs may be partially accounted for by variable expression of dihydropyrimidine dehydrogenase (DPD). DPD rate activity is genetically determined by its extremely polymorphic coding gene DPYD. In spite of pharmacogenetic guideline-directed-dosing of FPs based regimens treating carrier of multiple variants of DPYD gene remains still challenging. CASE PRESENTATION: We present a case of a 48-year-old Caucasian man, compound heterozygous variant carrier of the DPYD gene (HapB3 and c.2194G>A) who had a diagnosis of adenocarcinoma of the left colon and was safely treated with a pharmacogenetic-guided 25% dose reduction of the standard CAP adjuvant treatment. Compound heterozygosis may have been responsible for an earlier over exposure to CAP resulting into low-grade toxicity with an anticipated median time to toxicity of the c.2194G>A variant to the 4th vs. 6th cycles. Some haplotypes of DPYD variants may have an advantage in terms of survival compared to wild-type patients. Our patient may also have benefitted from compound heterozygosis, as shown by no evidence of disease (NED) at 6-month follow-up. CONCLUSION: Pharmacogenetic-guided dosing of DPYD intermediate metabolizer compound heterozygous HapB3 and c.2194G>A variant carries should be managed by a multidisciplinary team with a dose reduction ranging from 25 to 50% to maintain effectiveness and close clinical monitoring for early detection of ADRs.

Energy decomposition and waterswapping analysis to investigate the SNP associated DPD mediated 5-FU resistance.

5-fluorouracil is an essential component of systemic chemotherapy for colon, breast, head, and neck cancer patients. However, tumoral overexpression of the dihydropyrimidine dehydrogenase has rendered 5-FU clinically ineffective by inactivating it to 5'-6'-dihydro fluorouracil. The responses to 5-FU in terms of efficacy and toxicity greatly differ depending upon the population group, because of variability in the DPD activity levels. In the current study, key active site amino acids involved in the 5-FU inactivation were investigated by modelling the 3D structure of human DPD in a complex with 5-FU. The identified amino acids were analyzed for their possible missense mutations available in dbSNP database. Out of 12 missense SNPs, four were validated either by sequencing in the 1000 Genomes project or frequency/genotype data. The recorded validated missense SNPs were further considered to analyze the effect of their respective alterations on 5-FU binding. Overall findings suggested that population bearing the Glu611Val DPD mutation (rs762523739) is highly vulnerable to 5-FU resistance. From the docking, electrostatic complementarity, dynamics, and energy decomposition analyses it was found that the above mutation showed superior scores than the wild DPD -5FU complex. Therefore, prescribing prodrug NUC-3373 or DPD inhibitors (Gimeracil/3-Cyano-2,6-Dihydroxypyridines) as adjuvant therapy may overcome the 5-FU resistance.

Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidine drugs (FP) are the backbone of many chemotherapy protocols for treating solid tumours. The rate-limiting step of fluoropyrimidine catabolism is dihydropyrimidine dehydrogenase (DPD), and deficiency in DPD activity can result in severe and even fatal toxicity. In this review, we survey the evidence-based pharmacogenetics and therapeutic recommendations regarding DPYD (the gene encoding DPD) genotyping and DPD phenotyping to prevent toxicity and optimize dosing adaptation before FP administration. The French experience of mandatory DPD-deficiency screening prior to initiating FP is discussed.

Design, synthesis, and evaluation of JTE-013 derivatives as novel potent S1PR2 antagonists for recovering the sensitivity of colorectal cancer to 5-fluorouracil.

Targeting sphingosine-1-phosphate receptor 2 (S1PR2) has been proved as a promising strategy to reverse 5-fluorouracil (5-FU) resistance. Here, we report the discovery of the novel JTE-013 derivative compound 37 h as a more effective S1PR2 antagonist to reverse 5-FU resistance in SW620/5-FU and HCT116DPD cells than JTE-013 and previously reported compound 5. Compound 37 h could effectively bind S1PR2 and reduce its expression, thus leading to decreased expression of JMJD3 and dihydropyrimidine dehydrogenase (DPD), while also increasing the level of H3K27me3 to decrease the degradation of 5-FU and thereby increase its intracellular concentration in SW620/5-FU, HCT116DPD, and L02 cells. Furthermore, compound 37 h showed good selectivity to other S1PRs and normal colon cell line NCM460. Western blot analysis demonstrated that compound 37 h could abrogate the FBAL-stimulated upregulation of DPD expression by S1PR2. Importantly, compound 37 h also showed favorable metabolic stability with a long half-life (t1/2) of 7.9 h. Moreover, compound 37 h significantly enhanced the antitumor efficacy of 5-FU in the SW620/5-FU animal model. Thus, the JTE-013-based derivative compound 37 h represents a promising lead compound for the development of novel 5-FU sensitizers for colorectal cancer (CRC) therapy.

Fluoropyrimidine-associated toxicity and DPYD variants c.85T>C, c.496A>G, and c.1236G>A: impact of haplotype.

Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting step in fluoropyrimidines metabolism. Currently, genotype-guided fluoropyrimidine dosing is recommended for four DPYD single nucleotide variants (SNVs). However, the clinical impact of additional DPYD SNVs on fluoropyrimidine-related toxicity remains controversial. We assessed common DPYD SNVs c.85T>C, and c.496A>G which are often in linkage disequilibrium with c.1236G>A, a variant currently recommended for DPYD genotyping, in a retrospective cohort of cancer patients who had received fluoropyrimidines (N = 1371). When assessing individual SNVs, during the total chemotherapy treatment period, a significant increased risk of severe grade ≥ 3 toxicity was seen in carriers of c.496A>G (OR = 1.38, 95% CI 1.01-1.88, p = 0.0405) after adjusting for age, sex and treatment drug (capecitabine or 5-Fluorouracil). No association with fluoropyrimidine-related toxicity was seen in patients given standard dosing among those carrying one allele of DPYD c.1236G>A (OR = 1.19, 95% CI 0.59-2.27, p = 0.6147) or c.85T>C (OR = 1.04, 95% CI 0.80-1.62, p = 0.7536). Haplotype analysis confirmed a high linkage disequilibrium of these three variants. Toxicity was not significantly increased in haplotypes containing only one of c.85T>C or c.496A>G or c.1236G>A alleles. However, the haplotype containing both c.85T>C and c.496A>G alleles, which had a predicted frequency of 7.1%, was associated with an increased risk of fluoropyrimidine toxicity (OR = 1.57, 95% CI 1.15-2.13, p = 0.0041). This study suggests DPYD haplotype structure may help explain previous conflicting studies concerning the impact of these variants. Our findings suggest patients with both DPYD c.85T>C and c.496A>G variants have a significant increased risk for toxicity and may potentially benefit from genotype-guided fluoropyrimidine dosing.

Predicting Dihydropyrimidine Dehydrogenase Deficiency and Related 5-Fluorouracil Toxicity: Opportunities and Challenges of DPYD Exon Sequencing and the Role of Phenotyping Assays.

Deficiency of dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is associated with severe toxicity induced by the anti-cancer drug 5-Fluorouracil (5-FU). DPYD genotyping of four recommended polymorphisms is widely used to predict toxicity, yet their prediction power is limited. Increasing availability of next generation sequencing (NGS) will allow us to screen rare variants, predicting a larger fraction of DPD deficiencies. Genotype−phenotype correlations were investigated by performing DPYD exon sequencing in 94 patients assessed for DPD deficiency by the 5-FU degradation rate (5-FUDR) assay. Association of common variants with 5-FUDR was analyzed with the SNPStats software. Functional interpretation of rare variants was performed by in-silico analysis (using the HSF system and PredictSNP) and literature review. A total of 23 rare variants and 8 common variants were detected. Among common variants, a significant association was found between homozygosity for the rs72728438 (c.1974+75A>G) and decreased 5-FUDR. Haplotype analysis did not detect significant associations with 5-FUDR. Overall, in our sample cohort, NGS exon sequencing allowed us to explain 42.5% of the total DPD deficiencies. NGS sharply improves prediction of DPD deficiencies, yet a broader collection of genotype−phenotype association data is needed to enable the clinical use of sequencing data.

Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population.

PURPOSE: 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. METHOD: 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. RESULTS: 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%. CONCLUSION: 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.

Quantification of uracil, dihydrouracil, thymine and dihydrothymine for reliable dihydropyrimidine dehydrogenase (DPD) phenotyping critically depend on blood and plasma storage conditions.

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.

Dihydropyrimidine Dehydrogenase Deficiency and Implementation of Upfront DPYD Genotyping.

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.

Dihydropyrimidine Dehydrogenase Phenotyping Using Pretreatment Uracil: A Note of Caution Based on a Large Prospective Clinical Study.

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.

Nucleic Acid Metabolizing Enzyme Levels Predict Chemotherapy Effects in Advanced and Recurrent Colorectal Cancer.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: Nucleic acid metabolizing enzymes in primary lesions can be used to predict mFOLFOX6 efficacy in patients with recurrent CRC.