Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.

The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Endonuclease Q as a robust enhancer for nucleic acid amplification.

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Machine learning-aided search for ligands of P2Y6 and other P2Y receptors.

The P2Y6 receptor, activated by uridine diphosphate (UDP), is a target for antagonists in inflammatory, neurodegenerative, and metabolic disorders, yet few potent and selective antagonists are known to date. This prompted us to use machine learning as a novel approach to aid ligand discovery, with pharmacological evaluation at three P2YR subtypes: initially P2Y6 and subsequently P2Y1 and P2Y14. Relying on extensive published data for P2Y6R agonists, we generated and validated an array of classification machine learning model using the algorithms deep learning (DL), adaboost classifier (ada), Bernoulli NB (bnb), k-nearest neighbors (kNN) classifier, logistic regression (lreg), random forest classifier (rf), support vector classification (SVC), and XGBoost (XGB) classifier models, and the common consensus was applied to molecular selection of 21 diverse structures. Compounds were screened using human P2Y6R-induced functional calcium transients in transfected 1321N1 astrocytoma cells and fluorescent binding inhibition at closely related hP2Y14R expressed in CHO cells. The hit compound ABBV-744, an experimental anticancer drug with a 6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine scaffold, had multifaceted interactions with the P2YR family: hP2Y6R inhibition in a non-surmountable fashion, suggesting that noncompetitive antagonism, and hP2Y1R enhancement, but not hP2Y14R binding inhibition. Other machine learning-selected compounds were either weak (experimental anti-asthmatic drug AZD5423 with a phenyl-1H-indazole scaffold) or inactive in inhibiting the hP2Y6R. Experimental drugs TAK-593 and GSK1070916 (100 µM) inhibited P2Y14R fluorescent binding by 50% and 38%, respectively, and all other compounds by < 20%. Thus, machine learning has led the way toward revealing previously unknown modulators of several P2YR subtypes that have varied effects.

Can we identify patients carrying targeted deleterious DPYD variants with plasma uracil and dihydrouracil? A GPCO-RNPGx retrospective analysis.

OBJECTIVES: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U) testing. We analyzed the concordance between both approaches. METHODS: This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015-2022). RESULTS: Mean U was 9.9 ± 10.1 ng/mL (median 8.7, range 1.6-856). According to French recommendations, 7.3 % of patients were partially deficient (U 16-150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. CONCLUSIONS: These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency.

Repurposing sodium stibogluconate as an uracil DNA glycosylase inhibitor against prostate cancer using a time-resolved oligonucleotide-based drug screening platform.

Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Final analyses of the prospective controlled trial on the efficacy of uracil and tegafur/leucovorin as an adjuvant treatment for stage II colon cancer with risk factors for recurrence using propensity score-based methods (JFMC46-1201).

BACKGROUND: The efficacy of adjuvant chemotherapy for high-risk stage II colon cancer (CC) has not been well established. Using propensity score matching, we previously reported that the 3-year disease-free survival (DFS) rate was significantly higher in patients treated with uracil and tegafur plus leucovorin (UFT/LV) against surgery alone. We report the final results, including updated 5-year overall survival (OS) rates and risk factor analysis outcomes. METHODS: In total, 1902 high-risk stage II CC patients with T4, perforation/penetration, poorly differentiated adenocarcinoma/mucinous carcinoma, and/or < 12 dissected lymph nodes were enrolled in this prospective, non-randomized controlled study based on their self-selected treatment. Oral UFT/LV therapy was administered for six months after surgery. RESULTS: Of the 1880 eligible patients, 402 in Group A (surgery alone) and 804 in Group B (UFT/LV) were propensity score-matched. The 5-year DFS rate was significantly higher in Group B than in Group A (P = 0.0008). The 5-year OS rates were not significantly different between groups. The inverse probability of treatment weighting revealed significantly higher 5-year DFS (P = 0.0006) and 5-year OS (P = 0.0122) rates in group B than in group A. Multivariate analyses revealed that male sex, age ≥ 70 years, T4, < 12 dissected lymph nodes, and no adjuvant chemotherapy were significant risk factors for DFS and/or OS. CONCLUSION: The follow-up data from our prospective non-randomized controlled study revealed a considerable survival advantage in DFS offered by adjuvant chemotherapy with UFT/LV administered for six months over surgery alone in individuals with high-risk stage II CC. TRIAL REGISTRATION: Japan Registry of Clinical Trials: jRCTs031180155 (date of registration: 25/02/2019), UMIN Clinical Trials Registry: UMIN000007783 (date of registration: 18/04/2012).

Synthesis and biological evaluation of novel salicylidene uracils: Cytotoxic activity on human cancer cell lines and inhibitory action on enzymatic activity.

A series of salicylidene uracil (1-18) derived from 5-aminouracil and substituted salicylaldehydes were analyzed for cytotoxic activity and enzyme inhibitory potency. Nine out of eighteen derivatives (6-8, 10, 12-15, 18) are novel molecules synthesized for the first time in this work, and other derivatives were previously synthesized by our group. The compounds were characterized by Proton nuclear magnetic resonance, carbon nuclear magnetic resonance, fourier transform infrared spectroscopy, and elemental analysis. All compounds were tested for their in vitro cytotoxicity against PC-3 (human prostate adenocarcinoma), A549 (human alveolar adenocarcinoma), and SHSY-5Y (human neuroblastoma) cancer cell lines and the nontumorigenic HEK293 (human embryonic kidney cells) cell line. The 3,5-di-tert-butylsalicylaldehyde derived compound (8) was toxic to PC-3 human prostate adenocarcinoma cells, showing a promising IC50 value at 7.05 ± 0.76 µM. The present study also aimed to evaluate the inhibitory effects of the compounds against several key enzymes, namely carbonic anhydrase I and II (CA I and CA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and glutathione reductase (GR), which are implicated in various global disorders, such as Alzheimer's disease, epilepsy, cancer, malaria, diabetes, and glaucoma. The inhibitory profiles of the tested compounds were assessed by determining their Ki values, which ranged from 2.96 to 9.24 nM for AChE, 3.78 to 12.57 nM for BChE, 8.42 to 25.74 nM for CA I, 7.24 to 19.74 nM for CA II, and 0.541 to 1.124 µM for GR. Molecular docking studies were also performed for all compounds. Most derivatives exhibited much more effective inhibitory action compared with clinically used standards. Thus, our findings indicate that the salicylidene derivatives presented in this study are promising drug candidates that need further evaluation.

Facile synthesis and cytotoxicity of substituted uracil-1'(N)-acetic acid and 4-pyridone-1'(N)-acetic acid esters of 20(S)-camptothecins.

A series of novel substituted uracil-1'(N)-acetic acid esters (5-9) and 4-pyridone-1'(N)-acetic acid esters (10-11) of 20(S)-camptothecins (CPTs) have been synthesized by the acylation method. All of these new esters were assayed for in vitro cytotoxicity against five human cancer cell lines A549, Bel7402, BGC-823, HCT-8 and A2780. The in vitro bioassay results showed that all the synthesized compounds 5-11 had cytotoxities that were higher than TPT and comparable to CPT on these five tumor cell lines, some of them even showed comparable or superior cytotoxic activity to CPT. The in vitro data exhibited the cytotoxicity of the ester depended on that of its parent compound. The ester 5, 6, 8, 10, 11 even possessed the cytotoxity activity comparable to or even a little better than CPT on A549, HCT-8 and A2780. The compound 11 had the same level of cytoxity on Bel7402 as that of CPT. Here the synthesis and the in vitro antitumor evaluation of a series of novel 20-O-linked substituted uracil-1'(N)-acetic acid and 4-pyridone-1'(N)-acetic acid esters derivatives of CPTs are reported.

Aquaporin water channels affect the response of conventional anticancer therapies of 3D grown breast cancer cells.

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.

Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation.

DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA-dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins.

Using oral tegafur/uracil (UFT) plus leucovorin as adjuvant chemotherapy in stage II colorectal cancer: a propensity score matching study from Taiwan.

BACKGROUND: Early-stage colorectal cancer had excellent outcomes after curative resection, typically. However, a perplexing survival paradox between stage II and stage III was noted. This paradox could be influenced by the administration of routine postoperative adjuvant chemotherapy and the presence of high-risk factors in stage II CRC. The objective of the study was to investigate the influence of high-risk factors on patients with stage II CRC and assess the efficacy of oral tegafur/uracil (UFT) plus leucovorin as adjuvant chemotherapy for stage II CRC patients. METHODS: A retrospective study was conducted using propensity score matching at a single medical institution. A total of 1544 patients with stage II colorectal cancer who underwent radical surgery between January 2004 and January 2009 were included. The intervention used was tegafur/uracil plus leucovorin as adjuvant chemotherapy. The main outcome measures were disease-free survival and overall survival. RESULTS: After propensity score matching, 261 patients were included in three groups: no-treatment, half-year treatment, and one-year treatment. The clinical characteristics of each group tended to be more consistent. The Cox proportional hazard models showed that tegafur/uracil treatment or not was a significant independent factor for oncological outcome. Kaplan-Meier analysis also showed significantly better disease-free survival and overall survival. Further investigation revealed that tegafur/uracil duration was an independent factor for oncological outcome. While the survival curve did not reach statistical significance, the one-year UFT treatment group demonstrated the best treatment trend. CONCLUSIONS: This study suggests that tegafur/uracil plus leucovorin is a feasible adjuvant chemotherapy regimen for patients with stage II colorectal cancer after curative surgical treatment. Prolonged tegafur/uracil plus leucovorin treatment for 12 months showed a trend towards better outcomes in patients with stage II colorectal cancer.

An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1.

2'-Deoxynucleoside 5'-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2'-deoxyuridine 5'-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

Discrepancies between dihydropyrimidine dehydrogenase phenotyping and genotyping: What are the explanatory factors?

AIM: Dihydropyrimidine dehydrogenase (DPD) deficiency can be detected by phenotyping (measurement of plasma uracil [U], with U ≥ 16 µg/L defining a partial deficiency) and/or by genotyping (screening for the four most frequent DPYD variants). We aimed to determine the proportion of discrepancies between phenotypic and genotypic approaches and to identify possible explanatory factors. METHODS: Data from patients who underwent both phenotyping and genotyping were retrospectively collected. Complementary genetic analyses (genotyping of the variant c.557A>G and DPYD sequencing) were performed for patients with U ≥ 16 µg/L without any common variants. The characteristics of patients classified according to the congruence of the phenotyping and genotyping approaches were compared (Kruskal-Wallis test), and determinants of U levels were studied in the whole cohort (linear model). RESULTS: Among the 712 included patients, phenotyping and genotyping were discordant for 12.5%, with 63 (8.8%) having U ≥ 16 µg/L in the absence of a common variant. Complementary genetic investigations marginally reduced the percentage of discrepancies to 12.1%: Among the nine additional identified variants, only the c.557A>G variant, carried by three patients, had been previously reported to be associated with DPD deficiency. Liver dysfunction could explain certain discordances, as ASAT, ALP, GGT and bilirubin levels were significantly elevated, with more frequent liver metastases in patients with U ≥ 16 µg/L and the absence of a DPYD variant. The impact of cytolysis was confirmed, as ASAT levels were independently associated with increased U (p < 0.001). CONCLUSION: The frequent discordances between DPD phenotyping and genotyping approaches highlight the need to perform these two approaches to screen for all DPD deficiencies.

Quantitative impact of pre-analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency.

AIMS: Determining dihydropyrimidine dehydrogenase (DPD) activity by measuring patient's uracil (U) plasma concentration is mandatory before fluoropyrimidine (FP) administration in France. In this study, we aimed to refine the pre-analytical recommendations for determining U and dihydrouracil (UH2 ) concentrations, as they are essential in reliable DPD-deficiency testing. METHODS: U and UH2 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 UH2 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 the stability of uracil 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 hour at RT, since it rises significantly afterwards. Not respecting the pre-analytical conditions for DPD phenotyping increases the risk of DPD status misclassification.

Short-term biological variation of plasma uracil in a Caucasian healthy population.

OBJECTIVES: Plasma uracil is a new biomarker to assess the activity of dihydropyrimidine dehydrogenase before cancer treatment with fluoropyrimidine drugs. Knowledge on the biological variation of plasma uracil is important to assess the applicability of plasma uracil as a biomarker of drug tolerance and efficacy. METHODS: A total of 33 apparently healthy individuals were submitted to sequential blood draws for three days. On the second day, blood draws were performed every third hour for 12 h. Plasma uracil was quantified by LC-MS/MS. The within-subject (CVI) and between-subject (CVG) biological variation estimates were calculated using linear mixed-effects models. RESULTS: The overall median value of plasma uracil was 10.6 ng/mL (range 5.6-23.1 ng/mL). The CVI and CVG were 13.5 and 22.1%, respectively. Plasma uracil remained stable during the day, and there was no day-to-day variation observed. No differences in biological variation components were found between sex and no correlation to age was found. Four samples were calculated to be required to estimate the homeostatic set-point ±15% with 95% confidence. CONCLUSIONS: Plasma uracil is subject to tight homeostatic regulation without semidiurnal and day-to-day variation, however between-subject variation exists. This emphasizes plasma uracil as a well-suited biomarker for evaluation of dihydropyrimidine dehydrogenase activity, but four samples are required to establish the homeostatic set-point in a patient.