Genetic Variants Associated with Fluoropyrimidine-Induced Toxicity in Real-World Patients After Pre-Emptive DPYD Pharmacogenetic Testing

<b>Background/Objectives</b>: As a treatment, fluoropyrimidines are often associated with early moderate-to-severe toxicity. Although pre-emptive <i>DPYD</i> genotyping enables genotype-guided dosing, significant adverse events still occur in patients classified as <i>DPYD</i> wild-type (WT). The aim of this study was to identify <i>DPYD</i> variants and evaluate the contributions of <i>TYMS</i>, <i>ENOSF1</i>, <i>CDH4,</i> and <i>CDA</i> variants to fluoropyrimidine toxicity. <b>Methods</b>: A total of 256 European ancestry patients (aged ≥ 18 years, had completed ≥6 cycles of chemotherapy, and were WT for the <i>DPYD</i> variants routinely tested for) underwent genotyping for <i>TYMS</i>, <i>ENOSF1</i>, <i>CDH4,</i> and <i>CDA</i> variants, and full <i>DPYD</i> exon sequencing was performed in 56 of these patients. Toxicity was defined as fluoropyrimidine-related adverse events requiring a dose reduction. Multivariable models were adjusted for sex, fluoropyrimidine type, and the Charlson Comorbidity Index, and time-to-event was assessed using the Kaplan-Meier/Cox proportional hazards models. <b>Results</b>: A subgroup of 117 patients experienced toxicity requiring a dose reduction. The most frequent events were asthenia, gastrointestinal toxicity, hand-foot syndrome, and haematological toxicity. The <i>ENOSF1</i> rs2612091 C allele was associated with fluoropyrimidine withdrawal and a shorter time to dose reduction. In the patients treated with 5-fluorouracil, <i>TYMS</i> rs11280056 was associated with toxicity. <i>DPYD</i> exon sequencing identified thirteen variants, nine of which were more prevalent in the toxicity group. These included a canonical splice site (c.150+1G>A) and a stop-gained (c.1863G>A) variant, which is predicted to result in loss of function. <b>Conclusions</b>: In real-world practice, despite undergoing standard <i>DPYD</i> genotyping, <i>DPYD</i> WT patients receiving a full dose of fluoropyrimidines develop clinically relevant toxicity. The presence of rare <i>DPYD</i> variants and associated genes (<i>ENOSF1</i> and <i>TYMS</i>) suggests that broader, prospectively validated pharmacogenetic strategies may improve toxicity prevention.