New advances in DPYD genotype and risk of severe toxicity under capecitabine

Abstract

Background: Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine.

Methods: Two-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. DPYD exome, along with flanking intronic regions 3'UTR and 5'UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement.

Results: Among the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient (among which, F100L and A26T, both pathogenic in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3-4 toxicity (sensitivity 16.7%, positive predictive value (PPV) 71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3-4 toxicity (PPV 72.7%, RR 7.6, p<0.001), and was significantly associated with grade 4 toxicity (sensitivity 60%, PPV 27.3%, RR 31.4, p = 0.001), suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR.

Conclusions: Exploring an extended set of deleterious DPYD variants improves the performance of DPYD genotyping for predicting both grade 3-4 and grade 4 toxicities (digestive and/or neurologic and/or hematotoxicities) related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.

Fig 1. CONSORT diagram.

Fig 2. Location of F100L variant within the N-terminal Fe-S cluster containing the alpha helical domain I of the DPD protein.

Protein modeling was performed using UCSF Chimera version 1.8. The pig crystal structure (PDB ID 1gTH) was used as a template. The F100L variant could impair enzyme function by disrupting a conserved residue F100 important for electron transfer via the [4Fe-4S] cluster.

Fig 3

Distribution of pre-treatment plasma UH2/U ratio (A) and Uracil concentrations (B) for the 205 patients with validated phenotypic data, according to DPYD variants of interest: variant *2A (3 heterozygous patients), D949V (3 heterozygous patients), R592W (1 heterozygous patient), D342G (1 heterozygous patient), HapB3 (4 heterozygous patients), 166VV (3 homozygous patients) vs any other variations (185 patients) vs no variation (5 patients). DPD deficiency is reflected by plasma UH2/U decrease or plasma uracil increase. All indicated genotypes were mutually exclusive. Horizontal solid lines indicate median values (10.6 for UH2/U and 9.6 ng/ml for Uracil concentration). Horizontal dotted line on Uracil plot indicates the 91st percentile (16 ng/ml) associated with enhanced grade 3–4 toxicity. Open diamonds indicate patients with toxicity grade 0-1-2 and solid bow ties indicate patients with grade 3–4 toxicity. For variants carried by at least 3 patients, distribution of phenotype was compared between carriers and non-carriers using the non-parametric Mann-Whitney test (* indicates 0.01≤p<0.05 and ** indicates p<0.01).