DPYD c.85 T > C, DPYD c.496 A>G, and DPYD c.2194 G>A and severe toxicity to fluoropyrimidines in PREPARE and Alpe-DPD trials. An original study and meta-analysis.

Current DPYD genotyping panels used to prevent fluoropyrimidine (FL)-related toxicity explain only a limited proportion of severe adverse events. The clinical relevance, beyond current testing, of additional DPYD variants, including DPYD*6 (c.2194 G>A, rs1801160), DPYD c.496 A>G (rs2297595), and DPYD*9 A (c.85 T > C, rs1801265) remains debated. This study evaluated each polymorphism individually, irrespective of the allelic configuration at the other investigated loci, and as part of three-locus haplotypes in relation to severe FL-related toxicity. For single-variant effects, data from patients enrolled in two trials (PREPARE, Italian cohort; Alpe-DPD) were combined with 31 eligible published studies in a meta-analysis comprising 33 studies and 17,485 patients, with subgroup analyses by ethnicity. Haplotype analyses were performed in the 1296 trial participants. LC-MS was applied to evaluate plasma uracil and dihydrouracil levels. In the meta-analysis, DPYD c.2194 G>A (OR=1.70; 95%CI: 1.48-1.95) and c.496 A>G (OR=1.55; 95%CI: 1.19-2.02) were associated with FL toxicity. No overall association was observed for c.85 T > C; however, it improved c.496 A>G associated risk prediction in a haplotype analysis. Haplotype #3 (minor c.496 G with major c.85 T) was consistently associated with increased severe overall toxicity (PREPARE: OR=2.62, 95%CI: 1.06-6.50; Alpe-DPD: OR=2.09, 95%CI: 1.01-4.37) and gastrointestinal toxicity (PREPARE: OR=5.65, 95%CI: 2.11-15.15; Alpe-DPD: OR=2.71, 95%CI: 1.23-5.97). An exploratory Receiver Operating Characteristic (ROC) curve analysis demonstrated a significant incremental gain in risk discrimination achieved by adding DPYD c.85 T > C to a model that already included c.496 A>G. Haplotype #4 (minor alleles at both loci) was not associated with toxicity, suggesting a functional interaction between the variants. DPYD c.2194 G>A did not add to haplotype-based stratification. In PREPARE, pre-treatment plasma uracil and dihydrouracil confirmed reduced DPD function in haplotype #3 carriers. Overall, DPYD c.2194 G>A and c.496 A>G increased severe FL-toxicity risk when evaluated individually, whereas the integration of c.85 T > C refined risk prediction in a haplotype-based approach, supporting further refinement of pharmacogenetic risk stratification.