BACKGROUND:Despite the implementation of DPYD genotype-guided dosing, approximately 1 in 3 patients receiving fluoropyrimidine-containing chemotherapy continues to experience severe toxicity. While clinical studies have demonstrated a favorable tolerance among highly selected fit older adults, real-world studies have shown an increased risk of toxicity. OBJECTIVE:To identify predictors of severe toxicity or treatment deintensification in older DPYD wild-type adults receiving fluoropyrimidine-containing chemotherapy. METHOD:Patients wild type for four tested DPYD variants, aged ≥65 years, who participated in a prospective clinical trial investigating genotype-guided individualized fluoropyrimidine dosing, were eligible for the study. The association between tumor-, treatment-, and patient-related characteristics and the occurrence of severe toxicity (grade ≥3, CTCAE v5.0) was analyzed in univariate and multivariate logistic regression analyses. The same analyses were performed for a composite endpoint of severe toxicity or treatment deintensification (including dose reduction, cycle delay, or discontinuation). RESULTS:A total of 311 patients were included. Median age was 71.2 years and 58.8% were male. Grade ≥3 toxicity occurred in 23.2% of patients. In multivariate analysis, none of the characteristics studied were significantly associated with the occurrence of grade ≥3 toxicity. The composite endpoint occurred in 41.2% of patients and was associated with the use of full dose monotherapy in multivariate analysis. CONCLUSION:Despite DPYD genotype-based dosing, grade ≥3 toxicity and treatment deintensification frequently occur in older patients treated with fluoropyrimidine chemotherapy. No patient-related variables were found to be associated with grade ≥3 toxicity, but treatment with dose-reduced monotherapy resulted in fewer treatment deintensification or severe toxicity events.

Abstract 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.

Measurement of endogenous uracil (U) is increasingly being used as a dose-individualization method in the treatment of cancer patients with fluoropyrimidines. However, instability at room temperature (RT) and improper sample handling may cause falsely increased U levels. Therefore we aimed to study the stability of U and dihydrouracil (DHU) to ensure proper handling conditions. Stability of U and DHU in whole blood, serum, and plasma at RT (up to 24 h) and long-term stability (≥ 7 days) at − 20 °C were studied in samples from 6 healthy individuals. U and DHU levels of patients were compared using standard serum tubes (SSTs) and rapid serum tubes (RSTs). The performance of our validated UPLC-MS/MS assay was assessed over a period of 7 months. U and DHU levels significantly increased at RT in whole blood and serum after blood sampling with increases of 12.7 and 47.6

Capecitabine is an anticancer agent and is the oral prodrug of 5-fluorouracil (5-FU). In this study, an ultra-high performance liquid chromatography coupled to turbo ion spray tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify capecitabine and its metabolites including 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5-FU, and fluoro-β-alanine (FBAL) in lithium heparinized human plasma. Analytes were extracted by protein precipitation, chromatographically separated by Acquity UPLC HSS T3 column with gradient elution, and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. Capecitabine and 5'-dFCR were quantified in positive ion mode and 5'-dFUR, 5-FU, and FBAL were quantified in negative ion mode. The total chromatographic run time was 9 min. Stable isotopically labeled internal standards were used for all analytes. The assay was validated over the range from 25.0 to 2,500 ng/mL for capecitabine, 10.0 to 1,000 ng/mL for 5'-dFCR, 5'-dFUR, and 5-FU and 50 to 5,000 ng/ mL for FBAL in human plasma. Validation results have shown the developed assay allows for reliable quantitative analysis of capecitabine, 5'-dFCR, 5'-dFUR, 5-FU, and FBAL in plasma samples. Capecitabine is an anticancer agent and is the oral prodrug of 5-fluorouracil (5-FU). In this study, an ultra-high performance liquid chromatography coupled to turbo ion spray tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify capecitabine and its metabolites including 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5-FU, and fluoro-β-alanine (FBAL) in lithium heparinized human plasma. Analytes were extracted by protein precipitation, chromatographically separated by Acquity UPLC HSS T3 column with gradient elution, and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. Capecitabine and 5'-dFCR were quantified in positive ion mode and 5'-dFUR, 5-FU, and FBAL were quantified in negative ion mode. The total chromatographic run time was 9 min. Stable isotopically labeled internal standards were used for all analytes. The assay was validated over the range from 25.0 to 2,500 ng/mL for capecitabine, 10.0 to 1,000 ng/mL for 5'-dFCR, 5'-dFUR, and 5-FU and 50 to 5,000 ng/ mL for FBAL in human plasma. Validation results have shown the developed assay allows for reliable quantitative analysis of capecitabine, 5'-dFCR, 5'-dFUR, 5-FU, and FBAL in plasma samples.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of < 5%. Therapy options are limited. KRAS is mutated in >85% of PDAC and could be a key target. Preclinical studies have shown that SHP2 and ERK inhibition in KRAS mutant (KRASm) PDAC models results in inhibition of proliferation. The aim of this phase I/Ib trial is to assess safety and efficacy of treatment with SHP2 inhibitor RMC-4630 and ERK inhibitor LY3214996 in KRASm PDAC, non-small cell lung carcinoma (NSCLC) and colorectal carcinoma (CRC) patients. In the expansion phase, only PDAC patients will be included. In preclinical setting, potential resistance markers will be investigated to anticipate patient response. In the dose escalation phase, patients with KRASm PDAC, NSCLC or CRC were treated in a 28-day cycle with RMC-4630 on day 1 and 2 every week and LY3214996 daily. The main objective was to determine the Recommended Phase II Dose (RP2D). Other objectives included safety and tolerability assessed by the incidence of adverse events, efficacy determined by objective response rate (ORR) according to RECIST 1.1, pharmacokinetics (PK), and pharmacodynamics assessed by MAPK downstream protein expression levels. Resistance markers were explored by CRISPR. Eleven patients were enrolled in 2 cohorts: Cohort 1a 140 mg RMC-4630 and 100 mg LY3214996; cohort 1b 100 mg RMC-4630 and 200 mg LY3214996. Eight patients were evaluable for dose limiting toxicity (DLT). In cohort 1a, 2 out of 5 patients developed a DLT: grade 3 thrombocytopenia and renal insufficiency. No DLTs occurred in cohort 1b. PK analysis showed high interpatient variability. The exposure of LY3214966 was dose proportional. The ORR in both cohorts was 0%. In KRASm tumor cell lines, PTEN downregulation, c-JUN hyperactivation and increased PI3K/AKT/mTOR pathways activation were identified as mechanisms of resistance. Two DLTs out of 5 patients limited the tolerability of the combination in cohort 1a, no DLTs were identified in cohort 1b. Patient enrolment is ongoing in the next dose level. The RP2D has not been determined yet. Alterations in the PI3K/AKT/mTOR and the c-JUN pathways could be markers for drug resistance, which data are pending.

PURPOSEDPYD-guided fluoropyrimidine dosing improves patient safety in carriers of DPYD variant alleles. However, the impact on treatment outcome in these patients is largely unknown. Therefore, progression-free survival (PFS) and overall survival (OS) were compared between DPYD variant carriers treated with a reduced dose and DPYD wild-type controls receiving a full fluoropyrimidine dose in a retrospective matched-pair survival analysis.METHODSData from a prospective multicenter study (ClinicalTrials.gov identifier: NCT02324452) in which DPYD variant carriers received a 25% (c.1236G>A and c.2846A>T) or 50% (DPYD*2A and c.1679T>G) reduced dose and data from DPYD variant carriers treated with a similarly reduced dose of fluoropyrimidines identified during routine clinical care were obtained. Each DPYD variant carrier was matched to three DPYD wild-type controls treated with a standard dose. Survival analyses were performed using Kaplan-Meier estimates and Cox regression.RESULTSIn total, 156 DPYD variant carriers and 775 DPYD wild-type controls were available for analysis. Sixty-one c.1236G>A, 25 DPYD*2A, 13 c.2846A>T, and-when pooled-93 DPYD variant carriers could each be matched to three unique DPYD wild-type controls. For pooled DPYD variant carriers, PFS (hazard ratio [HR], 1.23; 95% CI, 1.00 to 1.51; P = .053) and OS (HR, 0.95; 95% CI, 0.75 to 1.51; P = .698) were not negatively affected by DPYD-guided dose individualization. In the subgroup analyses, a shorter PFS (HR, 1.43; 95% CI, 1.10 to 1.86; P = .007) was found in c.1236G>A variant carriers, whereas no differences were found for DPYD*2A and c.2846A>T carriers.CONCLUSIONIn this exploratory analysis, DPYD-guided fluoropyrimidine dosing does not negatively affect PFS and OS in pooled DPYD variant carriers. Close monitoring with early dose modifications based on toxicity is recommended, especially for c.1236G>A carriers receiving a reduced starting dose.

DPYD-guided dosing has improved the safety of fluoropyrimidine-based chemotherapy in recent years. However, severe toxicity remains in ~ 23% of patients not carrying DPYD variant alleles treated with capecitabine. Therefore, we developed a predictive model based on patient-related and treatment-related factors aimed at estimating the risk of developing severe capecitabine-related toxicity. The nomogram was developed using data from two large clinical trials (NCT00838370 and NCT02324452). Patients with cancer carrying a DPYD variant allele (DPYD*2A, c.1236G>A, c.2846A>T, and c.1679T>G) were excluded. Univariable and multivariable logistic regression using predetermined predictors based on previous findings, including age, sex, body surface area, type of treatment regimen, and creatinine levels were used to develop the nomogram. The developed model was internally validated using bootstrap resampling and cross-validation. This model was not externally or clinically validated. A total of 2,147 DPYD wild-type patients with cancer treated with capecitabine-based chemotherapy regimens were included of which complete data of 1,745 patients were available and used for the development of the nomogram. Univariable and multivariable logistic regression showed that age, sex, and type of treatment regimen were strong predictors of severe capecitabine-related toxicity in DPYD wild-type patients. Internal validation demonstrated a concordance index of 0.68 which indicates a good discriminative ability for prediction of severe capecitabine-related toxicity. The developed nomogram includes readily available parameters and may be a helpful tool for clinicians to assess the risk of developing severe capecitabine-related toxicity in patients without known risk DPYD variant alleles treated with capecitabine-based anticancer regimens.

We thank Thomas et al. for the comments on our publication and continued discussion regarding the feasibility and clinical validity of plasma uracil concentration as a marker for DPD deficiency. The authors request the number of patients per hospital included in the analysis, which we provided in Figure 1a. Thomas et al. propose that the reliability of our conclusion is undermined, because we used uracil data irrespective of sample origin. We tend not to agree with this, as our conclusion focusses on the feasibility of using uracil for DPD phenotyping in routine clinical practice, across a range of treatment centers. Additional analyses excluding the six centers with significantly higher uracil concentrations are suggested. However, this would not be a methodologically justified approach, being a nonpreplanned analysis and without further justification of why to exclude certain sites. Nevertheless, we performed this analysis and this showed similar results, with no association with severe toxicity (Figure 1b). In addition, an analysis using the reference center alone showed similar negative results (Figure 1c). As Thomas et al. mentioned, French authorities recommend a maximum blood storage of 1.5 hours at room temperature. However, there is no consensus for the maximum time of blood storage and several studies have demonstrated consistent increases in uracil concentrations when whole blood is stored at room temperature before centrifugation.2– 4 More specifically, average uracil concentrations were found to be increased by 27% after 1 hour, 21% after 1.5 hours, and ~25% after 2 hours. Our unpublished data shows an increase of 12.7% after 2 hours in whole blood and at room temperature. Therefore, delayed processing could potentially result in misclassification of patients. The instability of uracil makes it a highly complex marker for predicting DPD deficiency accurately. In addition, Thomas et al. provided data from three academic laboratories. We acknowledge that these data look more reassuring. Nevertheless, for two of these laboratories, they also show significant differences in uracil concentrations between centers, despite the relatively large sample sizes. Aside from remaining questions around preanalytical processing, the clinical validation of uracil as a biomarker to guide fluoropyrimidine dosing is also incomplete. In our recent Alpe2U study, we aimed to validate this method. We gave a 50% dose reduction advice for DPYD wild type patients with uracil levels > 16 ng/mL. Despite this, these patients had a 56% lower AUC of 5FU than expected, indicating underdosing. In conclusion, we deem that there are outstanding concerns around the feasibility, clinical validation, and usefulness of uracil testing in clinical practice.

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.

The bioanalysis of the oral anticancer drug capecitabine and its metabolites has been investigated extensively over the past years. This paper reviews methods for the bioanalysis of capecitabine and its metabolites. The focus of this review will be on sample pre-treatment, chromatography and detection. Furthermore, the choice of standards and analytical problems encountered during analysis of capecitabine and its metabolites in biological matrices will be discussed. The major challenges in the bioanalysis of capecitabine and its metabolites are the simultaneous extraction and analysis due to the differences in polarity of the analytes. Furthermore we evaluate currently described methods for the quantification of capecitabine and its metabolites. Future wishes and perspectives are stated that could serve as an inspiration for further development of assays for the quantification of capecitabine and its metabolites.

Fluoropyrimidines are widely used in the treatment of several types of solid tumors. Although most often well tolerated, severe toxicity is encountered in ~ 20–30% of the patients. Individualized dosing for these patients can reduce the incidence of severe fluoropyrimidine‐related toxicity. However, no consensus has been achieved on which dosing strategy is preferred. The most established strategy for individualized dosing of fluoropyrimidines is upfront genotyping of the DPYD gene. Prospective research has shown that DPYD ‐guided dose‐individualization significantly reduces the incidence of severe toxicity and can be easily applied in routine daily practice. Furthermore, the measurement of the dihydropyrimidine dehydrogenase (DPD) enzyme activity has shown to accurately detect patients with a DPD deficiency. Yet, because this assay is time‐consuming and expensive, it is not widely implemented in routine clinical care. Other methods include the measurement of pretreatment endogenous serum uracil concentrations, the uracil/dihydrouracil‐ratio, and the 5‐fluorouracil (5‐FU) degradation rate. These methods have shown mixed results. Next to these methods to detect DPD deficiency, pharmacokinetically guided follow‐up of 5‐FU could potentially be used as an addition to dosing strategies to further improve the safety of fluoropyrimidines. Furthermore, baseline characteristics, such as sex, age, body composition, and renal function have shown to have a relationship with the development of severe toxicity. Therefore, these baseline characteristics should be considered as a dose‐individualization strategy. We present an overview of the current dose‐individualization strategies and provide perspectives for a future multiparametric approach.