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.

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.

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.

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 cost analysis of upfront DPYD genotype-guided dose individualisation in fluoropyrimidine-based anticancer therapy.

BACKGROUND: Fluoropyrimidine therapy including capecitabine or 5-fluorouracil can result in severe treatment-related toxicity in up to 30% of patients. Toxicity is often related to reduced activity of dihydropyrimidine dehydrogenase, the main metabolic fluoropyrimidine enzyme, primarily caused by genetic DPYD polymorphisms. In a large prospective study, it was concluded that upfront DPYD-guided dose individualisation is able to improve safety of fluoropyrimidine-based therapy. In our current analysis, we evaluated whether this strategy is cost saving. METHODS: A cost-minimisation analysis from a health-care payer perspective was performed as part of the prospective clinical trial (NCT02324452) in which patients prior to start of fluoropyrimidine-based therapy were screened for the DPYD variants DPYD*2A, c.2846A>T, c.1679T>G and c.1236G>A and received an initial dose reduction of 25% (c.2846A>T, c.1236G>A) or 50% (DPYD*2A, c.1679T>G). Data on treatment, toxicity, hospitalisation and other toxicity-related interventions were collected. The model compared prospective screening for these DPYD variants with no DPYD screening. One-way and probabilistic sensitivity analyses were also performed. RESULTS: Expected total costs of the screening strategy were €2599 per patient compared with €2650 for non-screening, resulting in a net cost saving of €51 per patient. Results of the probabilistic sensitivity and one-way sensitivity analysis demonstrated that the screening strategy was very likely to be cost saving or worst case cost-neutral. CONCLUSIONS: Upfront DPYD-guided dose individualisation, improving patient safety, is cost saving or cost-neutral but is not expected to yield additional costs. These results endorse implementing DPYD screening before start of fluoropyrimidine treatment as standard of care.

Effectiveness and safety of reduced-dose fluoropyrimidine therapy in patients carrying the DPYD*2A variant: A matched pair analysis.

Carriers of the genetic DPYD*2A variant, resulting in dihydropyrimidine dehydrogenase deficiency, are at significantly increased risk of developing severe fluoropyrimidine-associated toxicity. Upfront DPYD*2A genotype-based dose reductions improve patient safety, but uncertainty exists whether this has a negative impact on treatment effectiveness. Therefore, our study investigated effectiveness and safety of DPYD*2A genotype-guided dosing. A cohort of 40 prospectively identified heterozygous DPYD*2A carriers, treated with a ~50% reduced fluoropyrimidine dose, was identified. For effectiveness analysis, a matched pair-analysis was performed in which for each DPYD*2A carrier a matched DPYD*2A wild-type patient was identified. Overall survival and progression-free survival were compared between the matched groups. The frequency of severe (grade ≥ 3) treatment-related toxicity was compared to 1] a cohort of 1606 wild-type patients treated with full dose and 2] a cohort of historical controls derived from literature, i.e. 86 DPYD*2A variant carriers who received a full fluoropyrimidine dose. For 37 out of 40 DPYD*2A carriers, a matched control could be identified. Compared to matched controls, reduced doses did not negatively affect overall survival (median 27 months versus 24 months, p = 0.47) nor progression-free survival (median 14 months versus 10 months, p = 0.54). Risk of severe fluoropyrimidine-related toxicity in DPYD*2A carriers treated with reduced dose was 18%, comparable to wild-type patients (23%, p = 0.57) and significantly lower than the risk of 77% in DPYD*2A carriers treated with full dose (p < 0.001). Our study is the first to show that DPYD*2A genotype-guided dosing appears to have no negative effect on effectiveness of fluoropyrimidine-based chemotherapy, while resulting in significantly improved patient safety.

Standard fluoropyrimidine dosages in chemoradiation therapy result in an increased risk of severe toxicity in DPYD variant allele carriers.

BACKGROUND: Prospective DPYD genotyping prevents severe fluoropyrimidine (FP)-induced toxicity by decreasing dosages in DPYD variant allele carriers. FP dosages in chemoradiation therapy (CRT) are lower than those in other FP-containing regimens. Pharmacogenetic guidelines do not distinguish between regimens, leaving physicians in doubt to apply dose reductions. Our aim was to investigate severe toxicity in DPYD variant allele carriers receiving CRT. METHODS: Medical records of 828 patients who received FP-based CRT were reviewed from three centres. Severe (grade ≥III) toxicity in DPYD variant allele carriers receiving upfront FP dose reductions according to pharmacogenetic dosing guidelines and DPYD variant allele carriers not receiving FP dose reductions was compared with DPYD wild-type patients receiving standard dose of FPs in CRT. RESULTS: DPYD variant allele carriers treated with standard dosages (N = 34) showed an increased risk of severe gastrointestinal (adjusted OR = 2.58, confidence interval [CI] = 1.02-6.53, P = 0.045) or severe haematological (adjusted OR = 4.19, CI = 1.32-13.25, P = 0.015) toxicity compared with wild-type patients (N = 771). DPYD variant allele carriers who received dose reductions (N = 22) showed a comparable frequency of severe gastrointestinal toxicity compared with wild-type patients, but more (not statistically significant) severe haematological toxicity. Hospitalisations for all DPYD variant allele carriers were comparable, independent of dose adjustments; however, the mean duration of hospitalisation was significantly shorter in the dose reduction group (P = 0.010). CONCLUSIONS: Standard FP dosages in CRT resulted in an increased risk of severe toxicity in DPYD variant allele carriers. We advise to apply FP dose reductions according to current guidelines in DPYD variant allele carriers starting CRT.

DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis.

BACKGROUND: Fluoropyrimidine treatment can result in severe toxicity in up to 30% of patients and is often the result of reduced activity of the key metabolic enzyme dihydropyrimidine dehydrogenase (DPD), mostly caused by genetic variants in the gene encoding DPD (DPYD). We assessed the effect of prospective screening for the four most relevant DPYD variants (DPYD*2A [rs3918290, c.1905+1G>A, IVS14+1G>A], c.2846A>T [rs67376798, D949V], c.1679T>G [rs55886062, DPYD*13, I560S], and c.1236G>A [rs56038477, E412E, in haplotype B3]) on patient safety and subsequent DPYD genotype-guided dose individualisation in daily clinical care. METHODS: In this prospective, multicentre, safety analysis in 17 hospitals in the Netherlands, the study population consisted of adult patients (≥18 years) with cancer who were intended to start on a fluoropyrimidine-based anticancer therapy (capecitabine or fluorouracil as single agent or in combination with other chemotherapeutic agents or radiotherapy). Patients with all tumour types for which fluoropyrimidine-based therapy was considered in their best interest were eligible. We did prospective genotyping for DPYD*2A, c.2846A>T, c.1679T>G, and c.1236G>A. Heterozygous DPYD variant allele carriers received an initial dose reduction of 25% (c.2846A>T and c.1236G>A) or 50% (DPYD*2A and c.1679T>G), and DPYD wild-type patients were treated according to the current standard of care. The primary endpoint of the study was the frequency of severe (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 grade ≥3) overall fluoropyrimidine-related toxicity across the entire treatment duration. We compared toxicity incidence between DPYD variant allele carriers and DPYD wild-type patients on an intention-to-treat basis, and relative risks (RRs) for severe toxicity were compared between the current study and a historical cohort of DPYD variant allele carriers treated with full dose fluoropyrimidine-based therapy (derived from a previously published meta-analysis). This trial is registered with ClinicalTrials.gov, number NCT02324452, and is complete. FINDINGS: Between April 30, 2015, and Dec 21, 2017, we enrolled 1181 patients. 78 patients were considered non-evaluable, because they were retrospectively identified as not meeting inclusion criteria, did not start fluoropyrimidine-based treatment, or were homozygous or compound heterozygous DPYD variant allele carriers. Of 1103 evaluable patients, 85 (8%) were heterozygous DPYD variant allele carriers, and 1018 (92%) were DPYD wild-type patients. Overall, fluoropyrimidine-related severe toxicity was higher in DPYD variant carriers (33 [39%] of 85 patients) than in wild-type patients (231 [23%] of 1018 patients; p=0·0013). The RR for severe fluoropyrimidine-related toxicity was 1·31 (95% CI 0·63-2·73) for genotype-guided dosing compared with 2·87 (2·14-3·86) in the historical cohort for DPYD*2A carriers, no toxicity compared with 4·30 (2·10-8·80) in c.1679T>G carriers, 2·00 (1·19-3·34) compared with 3·11 (2·25-4·28) for c.2846A>T carriers, and 1·69 (1·18-2·42) compared with 1·72 (1·22-2·42) for c.1236G>A carriers. INTERPRETATION: Prospective DPYD genotyping was feasible in routine clinical practice, and DPYD genotype-based dose reductions improved patient safety of fluoropyrimidine treatment. For DPYD*2A and c.1679T>G carriers, a 50% initial dose reduction was adequate. For c.1236G>A and c.2846A>T carriers, a larger dose reduction of 50% (instead of 25%) requires investigation. Since fluoropyrimidines are among the most commonly used anticancer agents, these findings suggest that implementation of DPYD genotype-guided individualised dosing should be a new standard of care. FUNDING: Dutch Cancer Society.

Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers.

AIMS: This study aimed to determine the effect of food intake on uracil and dihydrouracil plasma levels. These levels are a promising marker for dihydropyrimidine dehydrogenase activity and for individualizing fluoropyrimidine anticancer therapy. METHODS: A randomized, cross-over study in 16 healthy volunteers was performed, in which subjects were examined in fasted and fed state on two separate days. In fed condition, a high-fat, high-caloric breakfast was consumed between 8:00 h and 8:30 h. Whole blood for determination of uracil, dihydrouracil and uridine plasma levels was drawn on both test days at predefined time points between 8:00 h and 13:00 h. RESULTS: Uracil levels were statistically significantly different between fasting and fed state. At 13:00 h, the mean uracil level in fasting state was 12.6 ± 3.7 ng ml-1 and after a test meal 9.4 ± 2.6 ng ml-1 (P < 0.001). Dihydrouracil levels were influenced by food intake as well (mean dihydrouracil level at 13:00 h in fasting state 147.0 ± 36.4 ng ml-1 and in fed state 85.7 ± 22.1 ng ml-1 , P < 0.001). Uridine plasma levels showed curves with similar patterns as for uracil. CONCLUSIONS: It was shown that both uracil and dihydrouracil levels were higher in fasting state than in fed state. This is hypothesized to be an direct effect of uridine plasma levels, which were previously shown to be elevated in fasting state and reduced after intake of food. These findings show that, when assessing plasma uracil and dihydrouracil levels for adaptive fluoropyrimidine dosing in clinical practice, sampling should be done between 8:00 h and 9:00 h after overnight fasting to avoid bias caused by circadian rhythm and food effects.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing: 2017 Update.

The purpose of this guideline is to provide information for the interpretation of clinical dihydropyrimidine dehydrogenase (DPYD) genotype tests so that the results can be used to guide dosing of fluoropyrimidines (5-fluorouracil and capecitabine). Detailed guidelines for the use of fluoropyrimidines, their clinical pharmacology, as well as analyses of cost-effectiveness are beyond the scope of this document. The Clinical Pharmacogenetics Implementation Consortium (CPIC® ) guidelines consider the situation of patients for which genotype data are already available (updates available at https://cpicpgx.org/guidelines/guideline-for-fluoropyrimidines-and-dpyd/).

Capecitabine-based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidines are frequently used anti-cancer drugs. It is known that patients with reduced activity of dihydropyrimidine dehydrogenase (DPD), the key metabolic enzyme in fluoropyrimidine inactivation, are at increased risk of developing severe fluoropyrimidine-related toxicity. Upfront screening for DPD deficiency and dose reduction in patients with partial DPD deficiency is recommended and improves patient safety. For patients with complete DPD deficiency, fluoropyrimidine-treatment has generally been discouraged. During routine pretreatment screening, we identified a 59-year-old patient with a sigmoid adenocarcinoma who proved to have a complete DPD deficiency. Genetic analyses showed that this complete absence of DPD activity was likely to be caused by a novel DPYD genotype, consisting of a combination of amplification of exons 17 and 18 of DPYD and heterozygosity for DPYD*2A. Despite absence of DPD activity, the patient was treated with capecitabine-based chemotherapy, but capecitabine dose was drastically reduced to 150 mg once every 5 days (0.8% of original dose). Pharmacokinetic analyses showed that the area under the concentration-time curve (AUC) and half-life of 5-fluorouracil were respectively tenfold and fourfold higher than control values of patients receiving capecitabine 850 mg/m2 . When extrapolating from the dosing schedule of once every 5 days to twice daily, the AUC of 5-fluorouracil was comparable to controls. Treatment was tolerated well for eight cycles by the patient without occurrence of capecitabine-related toxicity. This case report demonstrates that a more comprehensive genotyping and phenotyping approach, combined with pharmacokinetically-guided dose administration, enables save fluoropyrimidine-treatment with adequate drug exposure in completely DPD deficient patients.

Letter regarding Zhao et al. entitled " DPYD gene polymorphisms are associated with risk and chemotherapy prognosis in pediatric patients with acute lymphoblastic leukemia".

Zhao et al. investigated the association between germline genetic polymorphisms in DPYD, the gene encoding dihydropyrimidine dehydrogenase, and (1) the risk of developing pediatric acute lymphoblastic leukemia and (2) outcome of acute lymphoblastic leukemia following the treatment with 5-fluorouracil plus oxaliplatin (FOLFOX). The authors found that the common DPYD variant c.85T>C (rs1801265, DPYD*9A) was significantly associated with (1) risk of developing pediatric acute lymphoblastic leukemia, (2) complete response rate, (3) event-free survival, and (4) treatment-related toxicity. The authors conclude that patients carrying the c.85T>C C allele have an increased risk of developing acute lymphoblastic leukemia and have inferior outcome, and that DPYD c.85T>C can be used as a guide for individualized treatment and the decision to utilize 5-fluorouracil in acute lymphoblastic leukemia patients. In our view, the published article gives rise to multiple critical issues regarding the study's rationale and the methodology used, which strongly question the validity of the authors' conclusions.

Pretreatment serum uracil concentration as a predictor of severe and fatal fluoropyrimidine-associated toxicity.

BACKGROUND: We investigated the predictive value of dihydropyrimidine dehydrogenase (DPD) phenotype, measured as pretreatment serum uracil and dihydrouracil concentrations, for severe as well as fatal fluoropyrimidine-associated toxicity in 550 patients treated previously with fluoropyrimidines during a prospective multicenter study. METHODS: Pretreatment serum concentrations of uracil and dihydrouracil were measured using a validated LC-MS/MS method. The primary endpoint of this analysis was global (any) severe fluoropyrimidine-associated toxicity, that is, grade ⩾3 toxicity according to the NCI CTC-AE v3.0, occurring during the first cycle of treatment. The predictive value of uracil and the uracil/dihydrouracil ratio for early severe fluoropyrimidine-associated toxicity were compared. Pharmacogenetic variants in DPYD (c.2846A>T, c.1679T>G, c.1129-5923C>G, and c.1601G>A) and TYMS (TYMS 5'-UTR VNTR and TYMS 3'-UTR 6-bp ins/del) were measured and tested for associations with severe fluoropyrimidine-associated toxicity to compare predictive value with DPD phenotype. The Benjamini-Hochberg false discovery rate method was used to control for type I errors at level q<0.050 (corresponding to P<0.010). RESULTS: Uracil was superior to the dihydrouracil/uracil ratio as a predictor of severe toxicity. High pretreatment uracil concentrations (>16 ng ml-1) were strongly associated with global severe toxicity (OR 5.3, P=0.009), severe gastrointestinal toxicity (OR 33.7, P<0.0001), toxicity-related hospitalisation (OR 16.9, P<0.0001), as well as fatal treatment-related toxicity (OR 44.8, P=0.001). None of the DPYD variants alone, or TYMS variants alone, were associated with severe toxicity. CONCLUSIONS: High pretreatment uracil concentration was strongly predictive of severe, including fatal, fluoropyrimidine-associated toxicity, and is a highly promising phenotypic marker to identify patients at risk of severe fluoropyrimidine-associated toxicity.

Patients homozygous for DPYD c.1129-5923C>G/haplotype B3 have partial DPD deficiency and require a dose reduction when treated with fluoropyrimidines.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) is a critical determinant of 5-fluorouracil pharmacology, and reduced activity of DPD as a result of deleterious polymorphisms in the gene encoding DPD (DPYD) can result in severe treatment-related toxicity. Dosing recommendations to individualize treatment have been provided for three DPYD variants (DPYD*2A, c.2846A>T, and c.1679T>G). A fourth variant, c.1129-5923C>G/HapB3, has been shown to increase the risk of fluoropyrimidine-associated toxicity, but little is known about the functional effects of this variant. METHODS: By performing a large retrospective screen for DPYD variants, we identified three patients who were homozygous for c.1129-5923C>G/HapB3. We describe their clinical course of treatment and analyzed DPD activity and DPYD gene expression, to provide insight into the phenotypic effects of c.1129-5923C>G/HapB3. RESULTS: DPD activity could be measured in two patients and was 4.1 and 5.4 nmol/mg/h (DPD activity 41 and 55 % compared to controls, respectively). The fluoropyrimidine dose had to be reduced during treatment in both patients. In line with partial DPD deficiency in both patients, sequence analysis of DPD cDNA demonstrated a normal-sized (wild type) cDNA fragment of 486 bp, as well as a larger-sized (mutant) 530-bp fragment containing an aberrant 44-bp insertion in intron 10. Patient three tolerated treatment well, but DPD activity measurement was not possible as the patient had deceased at the time of performing the study. CONCLUSIONS: The presented functional and clinical data indicate that the c.1129-5923C>G variant is both functionally and clinically relevant, and support an upfront dose reduction of the fluoropyrimidine starting dose in patients carrying c.1129-5923C>G homozygously.

Rs895819 in MIR27A improves the predictive value of DPYD variants to identify patients at risk of severe fluoropyrimidine-associated toxicity.

The objective of this study was to determine whether genotyping of MIR27A polymorphisms rs895819A>G and rs11671784C>T can be used to improve the predictive value of DPYD variants to identify patients at risk of severe fluoropyrimidine-associated toxicity (FP-toxicity). Patients treated previously in a prospective study with fluoropyrimidine-based chemotherapy were genotyped for rs895819 and rs11671784, and DPYD c.2846A>T, c.1679T>G, c.1129-5923C>G and c.1601G>A. The predictive value of MIR27A variants for early-onset grade ≥3 FP-toxicity, alone or in combination with DPYD variants, was tested in multivariable logistic regression models. Random-effects meta-analysis was performed, including previously published data. A total of 1,592 patients were included. Allele frequencies of rs895819 and rs11671784 were 0.331 and 0.020, respectively. In DPYD wild-type patients, MIR27A variants did not affect risk of FP-toxicity (OR 1.3 for ≥1 variant MIR27A allele vs. none, 95% CI: 0.87-1.82, p = 0.228). In contrast, in patients carrying DPYD variants, the presence of ≥1 rs895819 variant allele was associated with increased risk of FP-toxicity (OR 4.9, 95% CI: 1.24-19.7, p = 0.023). Rs11671784 was not associated with FP-toxicity (OR 2.9, 95% CI: 0.47-18.0, p = 0.253). Patients carrying a DPYD variant and rs895819 were at increased risk of FP-toxicity compared to patients wild type for rs895819 and DPYD (OR 2.4, 95% CI: 1.27-4.37, p = 0.007), while patients with a DPYD variant but without a MIR27A variant were not (OR 0.3 95% CI: 0.06-1.17, p = 0.081). In meta-analysis, rs895819 remained significantly associated with FP-toxicity in DPYD variant allele carriers, OR 5.4 (95% CI: 1.83-15.7, p = 0.002). This study demonstrates the clinical validity of combined MIR27A/DPYD screening to identify patients at risk of severe FP-toxicity.

Prospective DPYD genotyping to reduce the risk of fluoropyrimidine-induced severe toxicity: Ready for prime time.

5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.

Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data.

BACKGROUND: The best-known cause of intolerance to fluoropyrimidines is dihydropyrimidine dehydrogenase (DPD) deficiency, which can result from deleterious polymorphisms in the gene encoding DPD (DPYD), including DPYD*2A and c.2846A>T. Three other variants-DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A-have been associated with DPD deficiency, but no definitive evidence for the clinical validity of these variants is available. The primary objective of this systematic review and meta-analysis was to assess the clinical validity of c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity. METHODS: We did a systematic review of the literature published before Dec 17, 2014, to identify cohort studies investigating associations between DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A and severe (grade ≥3) fluoropyrimidine-associated toxicity in patients treated with fluoropyrimidines (fluorouracil, capecitabine, or tegafur-uracil as single agents, in combination with other anticancer drugs, or with radiotherapy). Individual patient data were retrieved and analysed in a multivariable analysis to obtain an adjusted relative risk (RR). Effect estimates were pooled by use of a random-effects meta-analysis. The threshold for significance was set at a p value of less than 0·0167 (Bonferroni correction). FINDINGS: 7365 patients from eight studies were included in the meta-analysis. DPYD c.1679T>G was significantly associated with fluoropyrimidine-associated toxicity (adjusted RR 4·40, 95% CI 2·08-9·30, p<0·0001), as was c.1236G>A/HapB3 (1·59, 1·29-1·97, p<0·0001). The association between c.1601G>A and fluoropyrimidine-associated toxicity was not significant (adjusted RR 1·52, 95% CI 0·86-2·70, p=0·15). Analysis of individual types of toxicity showed consistent associations of c.1679T>G and c.1236G>A/HapB3 with gastrointestinal toxicity (adjusted RR 5·72, 95% CI 1·40-23·33, p=0·015; and 2·04, 1·49-2·78, p<0·0001, respectively) and haematological toxicity (adjusted RR 9·76, 95% CI 3·03-31·48, p=0·00014; and 2·07, 1·17-3·68, p=0·013, respectively), but not with hand-foot syndrome. DPYD*2A and c.2846A>T were also significantly associated with severe fluoropyrimidine-associated toxicity (adjusted RR 2·85, 95% CI 1·75-4·62, p<0·0001; and 3·02, 2·22-4·10, p<0·0001, respectively). INTERPRETATION: DPYD variants c.1679T>G and c.1236G>A/HapB3 are clinically relevant predictors of fluoropyrimidine-associated toxicity. Upfront screening for these variants, in addition to the established variants DPYD*2A and c.2846A>T, is recommended to improve the safety of patients with cancer treated with fluoropyrimidines. FUNDING: None.

The use of combinations of monoclonal antibodies in clinical oncology.

Treatment with monoclonal antibodies is becoming increasingly important in clinical oncology. These antibodies specifically inhibit signaling pathways in tumor growth and/or induce immunological responses against tumor cells. By combining monoclonal antibodies several pathways may be targeted simultaneously, potentially leading to additive or synergistic effects. Theoretically, antibodies are very suitable for use in combination therapy, because of limited overlapping toxicity and lack of pharmacokinetic interactions. In this article an overview is given of preclinical and clinical data on twenty-five different combinations of antibodies in oncology. Some of these combinations have proven clinical benefit, for example the combination of trastuzumab and pertuzumab in HER2-positive breast cancer, which exemplifies an additive or synergistic effect on antitumor activity in clinical studies and the combination of nivolumab and ipilimumab, which results in significant increases in progression-free and overall survival in patients with advanced melanoma. However, other combinations may lead to unfavorable results, such as bevacizumab with cetuximab or panitumumab in advanced colorectal cancer. These combinations result in shorter progression-free survival and increased toxicity compared to therapy with a single antibody. In summary, the different published studies showed widely varying results, depending on the combination of antibodies, indication and patient population. More preclinical and clinical studies are necessary to unravel the mechanisms behind synergistic or antagonistic effects of combining monoclonal antibodies. Most research on combination therapies is still in an early stage, but it is expected that for several tumor types the use of combination therapy of antibodies will become standard of care in the near future.