A Prospective, multicenter, observational study to identify deleterious novel variants of the DPYD gene in patients of non-Western descent: The DPYD-NOW study

The fluoropyrimidine anticancer drugs 5-fluorouracil (5-FU) and capecitabine
are standard of care in the treatment of early and advanced breast, colorectal
and gastric cancer. There is ample evidence demonstrating that variation in
activity of the fluoropyrimidine-metabolizing enzyme dihydropyrimidine
dehydrogenase (DPD), encoded by the gene DPYD, causes clinically significant
differences in sensitivity to the toxic effects of 5-FU and capecitabine. DPD
deficiency, occurring in up to 5% of the population, is associated with the
risk of severe, potentially lethal, toxicity. It was shown by our group that
the risk of severe fluoropyrimidine related toxicity in patients with a
deleterious Single Nucleotide Polymorphism (SNP) in DPYD (i.e. a SNP that
reduces enzyme activity), (DPYD*2A, c.2846A>T, c.1236G>A and 1679T>G) was
significantly reduced when upfront screening of the DPYD gene and DPYD-guided
dose individualization was applied. Recently, it was shown that these 4 DPYD
variants are not predictive for toxicity in patients of African descent. Twelve
novel non-synonymous variants were identified, seven of which significantly
decreased DPD activity in vitro, in 588 patients of either Somalian or Kenyan
descent. The commonly reported toxicity-associated variants DPYD*2A, c.2846A>T
and c.1679T>G were not detected in any of the Somalian or Kenyan individuals. A
large portion of the population (> 1.5 million) in the Netherlands is of
non-western descent. The five largest groups of non-western descent in the
Netherlands are of Turkish (397,000), Moroccan (386,000), Indonesian (366,800),
Surinamese (349,000) and Antillean (151,000) descent. These groups will most
likely carry different and possibly clinically relevant variants in the DPYD
gene than the four variants (DPYD*2A, c.2846A>T, c.1236G>A and 1679T>G) that
currently routinely screened for in clinical practice. This can result in
unidentified DPD-deficient patients being treated with a full dose of
capecitabine or 5-FU, which can lead to severe toxicity, caused by the lack of
adaptive dosing guided by these variants of DPYD in the non-Western population.
In this study we plan to investigate which variants in the DPYD gene are of
relevance for the population of non-Western descent living in the Netherlands.
We expect to find that approximately 5-8% of this population of patients will
have a partial DPD deficiency, significantly affecting fluoropyrimidine
clearance. Furthermore, we expect to find 5-10 new variants in the DPYD gene,
of which some variants are associated to the development of severe
fluoropyrimidine-related toxicity. Novel deleterious variants in DPYD could be
added to the current routinely used DPYD screening panel. We expect that an
extended screening panel will lead to less severe toxicity, hospital admissions
and reduced risk of death, a better quality of life during and after
fluoropyrimidine treatment and will be cost saving.

Primary objective:
To identify novel variants in the DPYD gene in non-Western patients that are
related to the occurrence of severe (grade *3) toxicity upon treatment with
fluoropyrimidines.
Secondary objective:
* To determine the influence of novel DPYD variants on the variation in DPD
enzyme activity measured in peripheral blood mononuclear cells (PBMCs);
* To determine the ability of the DPYD-varifier to predict if novel variants
are deleterious;
* To determine the frequencies of DPYD variants per ethnic origin;
* To explore the relationship between genetic variants in additional genes
other than DPYD and fluoropyrimidine-related toxicity

The study will last for about 24 months. Approximately 600 patients will be
included. An overview of the study design can be seen in Figure 1. This is a
prospective, multicenter observational study, that will be performed in
multiple centers in the Netherlands. The DPYD gene of patients of non-Western
descent eligible for treatment with capecitabine or 5-FU in the participating
centers will be sequenced. Afterwards, the sequencing data will be compared to
the data of Western patients with sequencing data from the KWF/Alpe study
(n=1103) (NCT02324452). Furthermore, the DPD enzyme activity in PBMCs of all
patients will be determined. Novel DPYD variants that are associated with low
DPD enzyme activity in PBMCs and the development of CTCAE (version 5.0) grade *
3 toxicity will be classified as deleterious. For DPYD variants found to be
associated with low DPD enzyme activity but without leading to severe toxicity,
additional testing will be performed. For these variants functionality will be
evaluated with the use of transfected cells. If these studies also show DPD
deficiency the variant will be classified as deleterious. All novel variants
identified during sequencing will be analyzed using an in silico analysis
(DPYD-varifier) as a secondary analysis to predict if a variant is deleterious.
Results will be correlated with DPD enzyme activity. This is an observational
study, no dose modifications will be applied.

Blood will be drawn from all participating patients for sequencing of the DPYD
gene and DPD enzyme activity measured in PBMCS.