Discovering novel germline genetic variants linked to severe fluoropyrimidine-related toxicity in- and outside DPYD.

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.

A Nomogram to Predict Severe Toxicity in DPYD Wild-Type Patients Treated With Capecitabine-Based Anticancer Regimens.

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.

Survival of Patients With Cancer With DPYD Variant Alleles and Dose-Individualized Fluoropyrimidine Therapy-A Matched-Pair Analysis.

PURPOSE: DPYD-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. METHODS: Data 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. RESULTS: In 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. CONCLUSION: In 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.

Assay performance and stability of uracil and dihydrouracil in clinical practice.

PURPOSE: 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. METHODS: 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. RESULTS: U and DHU levels significantly increased at RT in whole blood and serum after blood sampling with increases of 12.7 and 47.6% after 2 h, respectively. A significant difference (p = 0.0036) in U and DHU levels in serum was found between SSTs and RSTs. U and DHU were stable at - 20 °C at least 2 months in serum and 3 weeks in plasma. Assay performance assessment fulfilled the acceptance criteria for system suitability, calibration standards, and quality controls. CONCLUSION: A maximum of 1 h at RT between sampling and processing is recommended to ensure reliable U and DHU results. Assay performance tests showed that our UPLC-MS/MS method was robust and reliable. Additionally, we provided a guideline for proper sample handling, processing and reliable quantification of U and DHU.

Individualized Dosing of Fluoropyrimidine-Based Chemotherapy to Prevent Severe Fluoropyrimidine-Related Toxicity: What Are the Options?

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.

A review of the bioanalytical methods for the quantitative determination of capecitabine and its metabolites in biological matrices.

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.