Page 272 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Chapter 10
Supplementary material
Calculation of sample size
Within DPYD wild-type patients a variability in DPD enzyme activity exists. We assumed that 95% of the DPYD wild-type patients would be classified as having normal enzyme activity and 5% of the DPYD wild-type patients would be classified with a low DPD enzyme activity (DPD deficient), with an increased risk of toxicity. This results in an unequal sample size, therefore a total sample size of 240 evaluable patients was required to achieve at least 80% power at significance level α=0.05 to detect an increase in the probability of toxicity from an estimated 20% in non-DPD deficient patients to 60% in DPD deficient patients.
Methods assays
DPD enzyme activity1,2
The DPD enzyme activity in PBMCs was determined using a validated radio-assay, which is based on conversion of the radiolabelled probe 4-14C thymine to 4-14C dihydrothymine.1 As this method is considered the gold standard in DPD measurements in the Netherlands, four phenotyping assays were correlated to this assay. Between 8 and 9 am, after overnight fasting, 20 ml blood (EDTA tube) was drawn, combined with a blood draw for determining the endogenous DHU/U ratio. Depending on the hospital of inclusion (N=4), whole blood was either shipped overnight to the Academic Medical Center in Amsterdam for further processing, or was processed at the hospital of blood draw (N=3) as described before, to isolate PBMCs.1 After processing, isolated PMBCs were kept at -80°C before measurement of DPD activity at the Academic Medical Center in Amsterdam.
Endogenous DHU/U ratio and endogenous uracil levels3,4
The uracil and DHU levels were determined in plasma using a validated ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method.4 All samples were measured at the Netherlands Cancer Institute in Amsterdam. In patients who participated in three or four DPD phenotyping assays, a 4 ml blood (heparin tube) was drawn between 8 and 9 am, after overnight fasting, and centrifuged at 4°C at 1500g for 10 minutes. Plasma was kept at -80°C until measurement. In patients from the clinical trial, blood to determine uracil and DHU levels could be drawn throughout the day and in non- fasting state, but information was collected on how long before the blood draw the patient had eaten a meal, as food status could influence the uracil levels in patients.5
Oral uracil loading dose6,7
Previously, a loading dose of 500 mg/m2 uracil was used in this assay. To increase feasibility, a standardized dose of 1,000 mg uracil was administered. Patients had to fast overnight for a minimum of eight hours. Food and drinks had to be abstained for the duration of the assay. Uracil was dissolved in warm water and administered between 8 and 9 am, to minimize effects of circadian rhythm. Four ml blood (EDTA tube) was taken at 60 and 120 minutes after oral intake of uracil. Sample processing consisted of adding 0.15 ml of the DPD inhibitor gimeracil to a 4 ml sample and centrifuging at 4°C at 1,500g for ten minutes. Plasma was kept at -80°C until measurement. Uracil and its metabolite dihydrouracil were determined in
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