Improving the safety of fluoropyrimidine-based chemotherapy by combined DPYD genotype-guided and DPD phenotype-guided dose individualisation: The Alpe2U study

Fluoropyrimidines continue to be the mainstay in the chemotherapeutic treatment
of multiple cancers e.g. colorectal, gastric, and breast cancer. An important
risk factor for fluoropyrimidine-associated toxicity is deficiency of the
metabolic enzyme dihydropyrimidine dehydrogenase (DPD), the rate-limiting
enzyme in the catabolism of fluoropyrimidines. The risk of severe, potentially
lethal, toxicity related to DPD deficiency is potentially avoidable by
identifying DPD-deficient patients and treating them with a reduced
fluoropyrimidine dose. Indeed we have shown in the Alpe-study that DPYD
genotype-based dose reductions improved patient safety of fluoropyrimidine
treatment. A dose reduction of 50% reduced the risk of severe (grade >= 3)
toxicity for DPYD*2A carriers, and proved to be safe in the single c.1679T>G
carrier. A dose reduction of 25% decreased the toxicity risk in c.2846A>T
carriers, although the risk was still higher than for wildtype patients. For
c.1236G>A carriers, a 25% dose reduction was not enough to decrease severe
treatment-related toxicity. A higher initial dose reduction of 50% for
c.2846A>T and c.1236G>A carriers, with subsequent individual dose titrations,
should therefore be considered. Although the abovementioned germ-line
polymorphisms in DPYD are strong predictors for toxicity, they are present in
only ~7% of the patients treated with fluoropyrimidines. These limitations,
which are intrinsic to any genotype-based approach, may be overcome by
combining DPYD screening with a phenotype-directed approach to screen for DPD
deficiency. This approach is based on the conversion of endogenous substrate
uracil (U) into dihydrouracil (DHU) by DPD. Previously, we have shown that high
pretreatment uracil concentrations (> 16 ng/ml) were strongly associated with
overall severe toxicity, as well as severe gastrointestinal toxicity,
toxicity-related hospitalisation and fatal treatment-related toxicity. In order
to further reduce the risk of severe fluoropyrimidine-related toxicity a
combined approach of genotype- and phenotype-guided dose individualization
needs to be studied. This will, therefore be the aim of the Alpe successor
study, the prospective Alpe2U study. In this study the effect of a higher dose
reduction in c.1236G>A and c.2846A>T DPYD variants carriers (50% instead of
25%) will be studied. Additionally, a phenotypic approach will be studied to
determine the additional value of pretreatment uracil level-guided dose
individualization in wildtype patients. Patients with a pretreatment serum
uracil concentration above 16 ng/ml will be treated with a 50% reduced
fluoropyrimidine starting dose. The pretreatment serum uracil levels in DPYD
variant carriers will be assessed retrospectively and non-interventionally.
Based on toxicity (or lack thereof), the dose of patients can be further
individualized (after the first 2 courses), i.e. further decreased or increased
in order to optimize the dose for the individual patient.

To determine whether the incidence of severe toxicity (defined as grade 3 to 5,
grading according to the National Cancer Institute Common Terminology Criteria
for Adverse Events, NCI CTC-AE version 5.0) associated with fluoropyrimidine
treatment can be significantly reduced by individualized dosing of
fluoropyrimidines based on combined genotypic and phenotypic assessment of DPD
deficiency.

Secondary objectives:

• To determine incidence of severe toxicity upon a 50% dose reduction for DPYD
c.1236G>A and c.2846A>T variant carriers treated with fluoropyrimidines;
• To assess the safety and feasibility of dose titration following an initial
dose reduction based on either a genetic or a phenotype result;
• To assess the pharmacokinetic profile of capecitabine, 5-FU and their
metabolites in patients carrying a c.1236G>A or c.2846A>T variant, or carry a
homozygous or compound heterozygous DPYD genotype;
• To assess the pharmacokinetic profile of capecitabine, 5-FU and their
metabolites in DPYD wildtype patients treated with a 50% reduced dose of the
respective drugs based on the phenotypic profile of the patient;
• To assess the safety of treating patients with a homozygous or compound
heterozygous DPYD genotype with fluoropyrimidines based on the DPD enzyme
activity measured in PBMCs;
• To identify predictive geriatric parameters for grade III-V toxicity and/or
treatment discontinuation in patients older than 70 years receiving
fluoropyrimidine-based chemotherapy;
• To retrospectively assess whether composition of wildtype patients receiving
a dose reduction would differ using the DHU/U-ratio compared to the
pre-treatment serum uracil concentration;
• To execute a cost-analysis on individualized dosing based on combined upfront
genotypic and DPD phenotypic assessment of DPD deficiency;
• To retrospectively assess the association between the circadian rhythm and
the pre-treatment serum uracil concentration;
• To retrospectively assess the association between time of administration of
capecitabine and radiotherapy with the development of
fluoropyrimidine-associated toxicity;
• To retrospectively assess the association between time of administration of
capecitabine and the development of fluoropyrimidine-related toxicity.

The study will probably have an inclusion period of about 18 months. An
overview of the study design can be seen in Figure 1. We will study the added
value of dose-individualization based on the pre-treatment serum uracil
concentration as a DPD phenotypic marker in wildtype patients. Wildtype
patients with a pre-treatment serum uracil concentration above 16 ng/ml will
receive a 50% dose-reduction. Pre-treatment serum uracil concentrations will be
measured in all patients fasted and between 8 and 10 am to minimize the effect
of food and circadian rhythm. The inclusion ends when 36 wild-type patients are
included in the group with a serum uracil concentration above 16 ng/ml.
Furthermore, the DPD enzyme activity measured in peripheral blood mononuclear
cells (PBMCs) will be measured in patients with a pretreatment uracil
concentration above 16 ng/ml. Additionally, prospective screening for four
single nucleotide polymorphisms (SNPs) in DPYD (DPYD*2A, c.2846A>T,
c.1236G>A/HapB3 and c.1679T>G) is executed. Patients with a SNP in DPYD will be
treated with a 50% reduced starting dose. Furthermore, patients with a
homozygous or compound heterozygous genotype will receive a dose-reduction
based on the DPD enzyme activity measured in PBMCs. The dose can be titrated in
all patients after 2 completed cycles, according to tolerability of the patient
to achieve maximal safe exposure, as shown in Figure 2 and 3. Additional
pharmacokinetic assessment will be performed in the wildtype patients receiving
a 50% dose-reduction, patients carrying a c.2846A>T or c.1236G>A variant and
patients with homozygous or compound heterozygous DPYD-genotype. Furthermore,
the DHU concentration will be measured in all patients to retrospectively
assess whether the group of wildtype patients receiving a dose reduction will
differ using the DHU/U-ratio compared to the pre-treatment serum concentration.
All patients above the age of 70 will undergo a short battery of geriatric
tests which will take around 10 minutes per patient. Patients treated with a
standard dose will be followed until the second cycle. Patients who received a
dose reduction will be followed until end of treatment. The cost-analysis of
combined DPYD genotype- and DPD phenotype-guided dosing will be calculated. An
interim analysis will be executed when 50% of the required 36 patients with a
pretreatment serum uracil concentration above 16 ng/ml is included.
Pharmacokinetic data and DPD enzyme activity measured in PBMCs will be assessed
to determine if patients are not being treated sub optimally.

Patients with a pretreatment serum uracil concentration above 16 ng/ml and
patients with c.1236G>A or c.2846A>T variant wil receive a 50% dose reduction.
Patients with a homozygous or compound heterozygous DPYD variant will be dosed
according to the DPD enzyme activity measured in PBMCs.

Blood will be drawn from all participating patients for determining the
genotype and the DHU/U ratio, prior to start of the fluoropyrimidine therapy.
Extra bloodsampling will be performed in patients carrying a homozygous or
compound heterzygous DPYD genotype to determine the DPD enzyme activity in
PBMCs. Pharmacokinetic analyses will be performed on a subgroup of the
participating patients (all patients carrying a c.1236G>A, c2846A>T, homozygous
or compound heterozygous DPYD genotype and wildtype patients with a
pretreatment serum uracil concentration above 16 ng/ml),
for which hospitalization for the duration of 9 hours is necessary and blood
samples at 9 (for capecitabine) different time points will be collected. Since
dosages will be reduced for patients carrying a c.1236G>A, c2846A>T,
homozygous or compound heterozygous DPYD genotype and wildtype patients with a
pretreatment serum uracil concentration above 16 ng/ml there will be a small
risk of underdosing for these patients. This risk will be very small, since
dose escalation according to tolerance after the first two courses will be
performed. Furthermore, all patients above the age of 70 in participating
hospitals will undergo a short battery of geriatric tests which will take
around 10 minutes per patient.