暂无摘要。

BACKGROUND: Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5-fluorouracil (5-FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5-FU with Lut in PDACs.
RESULTS: PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5-FU. The xenograft tumors of DPYD-overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA-seq analysis of the DPYD-overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity-MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5-FU on DPYD-overexpressing xenograft tumors and PDAC of Pdx1-Cre; LSL-KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5-FU nor Lut showed significant inhibitory effects; however, the combined administration of 5-FU and Lut exhibited a significant tumor-suppressive effect in both the xenograft tumors and KPPC models.
CONCLUSIONS: We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5-FU resistance, in PDACs. The combination therapy of Lut and 5-FU holds the potential for enhanced efficacy against PDACs.

暂无摘要。

BACKGROUND: The Alpe-DPD study (NCT02324452) demonstrated that prospective genotyping and dose-individualization using four alleles in DPYD (DPYD*2A/rs3918290, c.1236G > A/rs75017182, c.2846A > T/rs67376798 and c.1679 T > G/rs56038477) can mitigate the risk of severe fluoropyrimidine toxicity. However, this could not prevent all toxicities. The goal of this study was to identify additional genetic variants, both inside and outside DPYD, that may contribute to fluoropyrimidine toxicity.
METHODS: Biospecimens and data from the Alpe-DPD study were used. Exon sequencing was performed to identify risk variants inside DPYD. In silico and in vitro analyses were used to classify DPYD variants. A genome-wide association study (GWAS) with severe fluoropyrimidine-related toxicity was performed to identify variants outside DPYD. Association with severe toxicity was assessed using matched-pair analyses for the exon sequencing and logistic, Cox, and ordinal regression analyses for GWAS.
RESULTS: Twenty-four non-synonymous, frameshift, and splice site DPYD variants were detected in ten of 986 patients. Seven of these variants (c.1670C > T, c.1913 T > C, c.1925 T > C, c.506delC, c.731A > C, c.1740 + 1G > T, c.763 - 2A > G) were predicted to be deleterious. The carriers of either of these variants showed a trend towards a 2.14-fold (95% CI, 0.41-11.3, P = 0.388) increased risk of severe toxicity compared to matched controls (N = 30). After GWAS of 942 patients, no individual single nucleotide polymorphisms achieved genome-wide significance (P ≤ 5 × 10-8), however, five variants were suggestive of association (P < 5 × 10-6) with severe toxicity.
CONCLUSIONS: Results from DPYD exon sequencing and GWAS analysis did not identify additional genetic variants associated with severe toxicity, which suggests that testing for single markers at a population level currently has limited clinical value. Identifying additional variants on an individual level is still promising to explain fluoropyrimidine-related severe toxicity. In addition, studies with larger samples sizes, in more diverse cohorts are needed to identify potential clinically relevant genetic variants related to severe fluoropyrimidine toxicity.

To reduce severe fluoropyrimidine-related toxicity, pharmacogenetic guidelines recommend a dose reduction for carriers of four high-risk variants in the DPYD gene (*2A, *13, c.2846A>T, HapB3). The polymorphism in the MIR27A gene has been shown to enhance the predictive value of these variants. Our study aimed to explore whether rs895819 in the MIR27A gene modifies the effect of five common DPYD variants: c.1129-5923C>G (rs75017182, HapB3), c.2194G>A (rs1801160, *6), c.1601G>A (rs1801158, *4), c.496A>G (rs2297595), and c.85T>C (rs1801265, *9A). The study included 370 Caucasian patients with gastrointestinal tumors who received fluoropyrimidine-containing chemotherapy. Genotyping was performed using high-resolution melting analysis. The DPYD*6 allele was associated with overall severe toxicity and neutropenia with an increased risk particularly pronounced in patients carrying the MIR27A variant. All carriers of DPYD*6 exhibited an association with asthenia regardless of their MIR27A status. The increased risk of neutropenia in patients with c.496G was only evident in those co-carrying the MIR27A variant. DPYD*4 was also significantly linked to neutropenia risk in co-carriers of the MIR27A variant. Thus, we have demonstrated the predictive value of the *6, *4, and c.496G alleles of the DPYD gene, considering the modifying effect of the MIR27A polymorphism.

Fluoropyrimidines (FLs) [5-Fluorouracil, Capecitabine] are used in the treatment of several solid tumors. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for FL detoxification, and its deficiency could lead to severe, life-threatening or fatal toxicity after FL administration. Testing with a pharmacogenetic panel of four deleterious variants in the dihydropyrimidine dehydrogenase gene (DPYD) (DPYD*2A, DPYD*13, c.2846A > T, c.1129-5923C > G) prior to FL treatment, is recommended by scientific consortia (e.g., CPIC, DPWG) and drug regulatory agencies (e.g., EMA). However, this panel identifies < 20% of patients at risk of severe FL-related toxicity. Cumulative recent evidence highlights the potential clinical value of rare (minor allele frequency < 1%) and novel DPYD genetic variants for identifying an additional fraction of DPD-deficient patients at increased risk of severe FL-related toxicity. In this review, we aimed to comprehensively describe the available evidence regarding the potential clinical predictive role of novel and rare DPYD variants as toxicity markers in FL-treated patients, and to discuss the challenges and opportunities in tailoring FL treatment based upon clinical application of such markers. Although we must overcome existing barriers to the clinical implementation, the available data support that comprehensive assessment of the DPYD sequence, including rare and novel genetic variants, may significantly enhance the pre-emptive identification of at-risk patients, compared to the current targeted approach.

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60-65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

OBJECTIVE: Patients with dihydropyrimidine dehydrogenase (DPD) deficiency are at high risk for severe and fatal toxicity from fluoropyrimidine (FP) chemotherapy. Pre-treatment DPYD testing is standard of care in many countries, but not the United States (US). This survey assessed pre-treatment DPYD testing approaches in the US to identify best practices for broader adoption.
METHODS: From August to October 2023, a 22-item QualtricsXM survey was sent to institutions and clinicians known to conduct pre-treatment DPYD testing and broadly distributed through relevant organizations and social networks. Responses were analyzed using descriptive analysis.
RESULTS: Responses from 24 unique US sites that have implemented pre-treatment DPYD testing or have a detailed implementation plan in place were analyzed. Only 33% of sites ordered DPYD testing for all FP-treated patients; at the remaining sites, patients were tested depending on disease characteristics or clinician preference. Almost 50% of sites depend on individual clinicians to remember to order testing without the assistance of electronic alerts or workflow reminders. DPYD testing was most often conducted by commercial laboratories that tested for at least the four or five DPYD variants considered clinically actionable. Approximately 90% of sites reported receiving results within 10 days of ordering.
CONCLUSIONS: Implementing DPYD testing into routine clinical practice is feasible and requires a coordinated effort among the healthcare team. These results will be used to develop best practices for the clinical adoption of DPYD testing to prevent severe and fatal toxicity in cancer patients receiving FP chemotherapy.

Determination of plasma uracil was reported as a method for evaluation of Dihydropyrimidine dehydrogenase (DPD) activity that is highly demanded to ensure the safe administration of 5-fluorouracil (5-FU)-based therapies to cancer patients. This work reports the development of a simple electroanalytical method based on adsorptive stripping square wave voltammetry (AdSWV) at mercury film-coated glassy carbon electrode (MF/GCE) for the highly sensitive determination of uracil in biological fluids that can be used for diagnosis of decreased DPD activity. Due to the formation of the HgII-Uracil complex at the electrode surface, the accuracy of the measurement was not affected by the complicated matrices in biological fluids including human serum, plasma, and urine. The high sensitivity of the developed method results in a low limit of detection (≈1.3 nM) in human plasma samples, falling below the practical cut-off level of 15 ng mL-1 (≈0.14 μM). This threshold concentration is crucial for predicting 5-FU toxicity, as reported in buffer, and ≤1.15% in biological samples), and accuracy (recovery percentage close to 100%).