Page 289 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Compound heterozygous DPYD variant allele carriers
Discussion
Prospective genotyping of DPYD variants followed by individual dose adjustments is increasingly applied as the standard of care for patients starting fluoropyrimidine therapy. Standard dose reductions from CPIC and DPWG guidelines cannot be applied in patients who carry more than one DPYD variant, as the phasing of the variants is unknown. Despite the low population frequency of <0.2%, the absolute number of identified compound heterozygous patients will increase as the number of genotyped patients increases and the panel of tested variants is expanded. To the best of our knowledge, this is the first study that describes a case series of compound heterozygous DPYD variant allele carriers and investigates diagnostic and therapeutic strategies for these patients.
Our study shows the clinical need for further information on the genotype, as four patients were identified as compound heterozygous carriers retrospectively and all of them experienced severe toxicity. These compound heterozygous DPYD variant allele carriers have an increased risk of developing severe fluoropyrimidine-induced toxicity if dosages are not adequately adjusted. Previously, compound heterozygous patients have been described with severe or even lethal side effects after fluoropyrimidine treatment.39,40 Three patients in this study were prospectively identified as compound heterozygous carriers, received initial dose reductions, and developed only mild toxicities.
Out of the four patients for whom we were able to retrieve phasing information, three were in trans and one was in cis orientation. Data from publicly available databases also showed that both in cis and in trans orientations exist. However, the recently updated CPIC guidelines on DPYD assumes in trans phasing for compound heterozygous patients.20 The DPWG guidelines do not mention phasing; however, the dosing recommendations of the DPWG use the GAS, a score based on the activity of individual alleles.19 This implies the need for phasing information. The assumption of in trans phasing could result in the underdosing of patients with variants phased in cis, and thus exemplifies the need for the determination of the phasing of variants.
In this study, we looked at different diagnostic strategies to determine the phasing of DPYD variants in compound heterozygous patients. In four patients, the phasing of DPYD variants could be determined using one of three different molecular methods. These methods are in the early phases of development, not routinely available, quite expensive, and not always conclusive. For two of these techniques, patient RNA is used, which degrades quickly after the blood draw unless specifically designed blood tubes are used. Compound heterozygous patients are rare, yet here we describe seven patients heterozygous for multiple DPYD variants. A limitation of our study is that most patients were identified retrospectively and in different institutions. Because of this, not enough of or not the right material was available for analysis, thus not all genotyping techniques could be executed in each patient. For two samples, tests failed or produced inconclusive results (data not shown). For this reason, a formal comparison of their suitability to identify phasing was not possible. However, of the three explored molecular methods, PacBio sequencing seems most promising. While phasing improved the prediction of DPD enzyme activity, patients with identical combinations of DPYD variants and identical phasing showed considerable differences in DPD enzyme activity, which could potentially limit the added value of the determination of
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