Page 234 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Chapter 9
fatal, as is explained in the following example of DPYD genotyping for fluoropyrimidines (5-fluorouracil/5-FU, and capecitabine).15 There is compelling evidence on the reduction of severe fluoropyrimidine-induced toxicity when using prospective PGx for four DPYD variants, and dosing recommendations for these four DPYD variants have been published by the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG).16-19 Despite this, clinical implementation is not yet part of routine clinical care in many hospitals.20 When exposed to standard dosages of fluoropyrimidines, carriers of a DPYD variant are at high risk for severe, or even fatal, toxicity. Despite the low frequency of DPYD variants, prospective genotyping of DPYD variants in all patients prior to initiating fluoropyrimidine treatment was shown to be cost-saving.21 Thus, it is safer, but not more expensive to genotype patients. Misclassification of the DPYD genotype can result in suboptimal therapy (false positive) or even have lethal consequences from fluoropyrimidine treatment in standard dosages (false negative). In addition, therapeutic drug monitoring (TDM) could be used to monitor the 5-FU dose during treatment, but is rarely executed. For capecitabine, the oral pro-drug of 5-FU, TDM protocols need to be developed. This particular example shows the clinical importance and substantial consequences of PGx testing and illustrates why it is of utmost importance to report the correct result.
The dilemma
Laboratories apply different genotyping techniques to generate PGx results. Sanger sequencing remains the gold standard for DNA sequencing,22 even though this can be prone to errors.23 In general, PCR-based assays (including Sanger sequencing) are considered a robust methodology with reliable results. Each assay is subjected to extensive validation by the company or laboratory to reduce the risk of a priori errors. However, after the implementation of a test in clinical practice, it is still possible to have false positive or false negative results, e.g. due to allele dropout.24 Allele dropout can be caused by a newly acquired variant located at the site of a primer, causing the binding of this primer to fail. A genetic variant located on that DNA strand will not be genotyped, and the patient is misclassified as homozygous carrier of the variant on the other strand.
To mitigate the risk of allele dropout a laboratory can use a second, independent method that uses different primers to confirm results. However, this results in increased costs, labour and turn-around-time. Should laboratories execute a second method to confirm results, or not? The dilemma of the quality control aspect of PGx testing is based on the probability of a genotyping error to occur, the level of increased effort and costs to detect the error and the consequence of not detecting the error. A genotyping error, e.g. due to allele dropout, can be detected by a second, independent genotyping assay, which is the most adequate, but comprehensive, available method. Abolishing a second method or repetition can thus save both time and costs, possibly increasing the likeliness of use of PGx testing since cost-effectiveness is often reported as a barrier for implementing PGx testing.15 The consequence of an error in PGx can be substantial, yet it is unrealistic to aim to never have an incorrect result. This dilemma is why differences in confirmation practices in laboratories could exist and why guidelines are required to align laboratory practices. These differences could be overcome by clear guidelines from regulatory authorities, however,
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