Page 306 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Chapter 12
Materials and methods
Patients
Patients were recruited for the Alpe DPD study (clinicaltrial.gov identifier NCT02324452)5 between April 30, 2015 and December 21, 2017, and were newly treated with fluoropyrimidines and genotyped prospectively for four DPYD variants (DPYD*2A, rs3918290, c.1905+1G>A, IVS14+1G>A; c.1679T>G, DPYD*13, rs55886062, I560S; c.1236G>A/HapB3, rs56038477, E412E; and c.2846A>T, rs67376798, D949V). Upon identification of one of these variants, heterozygous variant allele carriers received an initial dose reduction (25 or 50%) based on pharmacogenetic guidelines to prevent severe fluoropyrimidine-induced toxicity. Wild-type patients for these four DPYD variants received standard fluoropyrimidine dosages. After the second cycle the dose could be titrated upwards or downwards according to the occurrence of toxicity. The study was reviewed and approved by the medical ethical committee of the Netherlands Cancer Institute, Amsterdam, the Netherlands, and approval of the board of directors of each individual hospital was obtained for all participating centres. All patients signed informed consent prior to inclusion in the study, which included approval for the use of clinical data and remaining DNA to perform the current GWAS. All patients of whom sufficient DNA was available were genotyped. DPYD variant allele carriers (N=85) received dose reductions based on the four variants mentioned and were therefore excluded in the GWAS analyses.
Clinical data
Baseline characteristics, treatment type and toxicity data were collected for each patient. Ethnicity of the patients was self-reported, merely Caucasian patients participated in the Alpe DPD study. Toxicity was graded according to the National Cancer Institute common terminology criteria for adverse events (CTC-AE; version 4.03) and severe toxicity was defined as CTC-AE grade ≥3.26 Relation to the study drugs 5-FU and capecitabine was recorded for each adverse event and only adverse events classified as possible, probable, or definite were taken into account.
Genotyping and quality control
Patient DNA remaining from the Alpe DPD study was collected. For each patient 200 ng of DNA was required and genotyping was executed at the Human Genotyping Facility of the Erasmus Medical Center, using the Illumina Global Screening Array (GSA).27 The array contains 692,842 SNPs and includes rare variants with allele frequencies <1%. 1000 Genomes reference phase 3 GRCh37.p13 was used to impute the data. Quality control (QC) checks were performed using software R version 3.5.028 and PLINK software, version 1.07.29,30 Patients were excluded from analyses based on an individual genotype call rate <97%, gender mismatch between reported and estimated sex based on genotypes of the X-chromosome (using PLINK), or excess of heterozygous genotypes as measured by the inbreeding coefficient. An inbreeding statistic of F>0.1 was judged to be outlying and patients were removed from the analysis. Genetic markers were excluded based on a SNP call rate <97% and a p-value ≤10-7 for the Hardy-Weinberg equilibrium (HWE) goodness-of-fit test. After exclusion of patients and markers in these marginal QCs, the remaining set was used
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