Original Article
5-Fluorouracil degradation rate as a
predictive biomarker of toxicity in breast
cancer patients treated with capecitabine
Andrea Botticelli
1
, Simone Scagnoli
1
, Michela Roberto
2
,
Luana Lionetto
2
, Bruna Cerbelli
2
, Maurizio Simmaco
3
and
Paolo Marchetti
4
Abstract
Capecitabine is an oral prodrug of 5-fluorouracil with a relevant role in the treatment of breast cancer. Severe and
unexpected toxicities related to capecitabine are not rare, and the identification of biomarkers is challenging.
We evaluate the relationship between dihydropyrimidine dehydrogenase, thymidylate synthase enhancer region and
methylenetetrahydrofolate reductase polymorphisms, 5-fluorouracil degradation rate and the onset of G3–4 toxicities in
breast cancer patients. Genetic polymorphisms and the 5-fluorouracil degradation rate of breast cancer patients treated
with capecitabine were retrospectively studied. Genetic markers and the 5-fluorouracil degradation rate were corre-
lated with the reported toxicities. Thirty-seven patients with a median age of 58 years old treated with capecitabine for
stages II–IV breast cancer were included in this study. Overall, 34 (91.9%) patients suffered from at least an episode of
any grade toxicity while nine patients had G3–4 toxicity. Homozygous methylenetetrahydrofolate reductase 677TT was
found to be significantly related to haematological toxicity (OR ¼ 6.5 [95% IC 1.1–37.5], P ¼ 0.04). Three patients had
a degradation rate less than 0.86 ng/mL/106 cells/min and three patients greater than 2.1 ng/mL/106 cells/min. At a
univariate logistic regression analysis, an altered value of 5-fluorouracil degradation rate (values < 0.86 or >2.10 ng/mL/
106 cells/min) increased the risk of G3–4 adverse events (OR ¼ 10.40 [95% IC: 1.48–7.99], P ¼ 0.02). A multivariate logistic
regression analysis, adjusted for age, comorbidity and CAPE-regimen, confirmed the role of 5-fluorouracil degradation rate
as a predictor of G3–4 toxicity occurrence (OR ¼ 10.9 [95% IC 1.2–96.2], P ¼ 0.03). The pre-treatment evaluation of
5-fluorouracil degradation rate allows to identify breast cancer patients at high risk for severe 5-FU toxicity.
Keywords
5-FU degradation rate , breast cancer, capecitabine, chemotherapy toxicity, polymorphism
Date received: 26 May 2019; revised: 13 December 2019; accepted: 15 January 2020
Introduction
Capecitabine is an oral prodrug of 5-fluorouracil
(5-FU), enzymatically activated by timidina phosphor-
ylase in tumour tissue, that was rationally designed
to mimic continuous infusion 5-FU.
1
Capecitabine is
rapidly absorbed from the gastrointestinal (GI) tract
and metabolized by carboxylesterase in liver, and it is
converted to 5
0
deoxy-5-fluorocytidine and then to
5
0
deoxy-5-fluorouridine (5
0
DFUR) by cytidine deam-
inase. Finally, the enzyme thymidine phosphorylase
converts 5
0
DFUR to 5-FU both in normal and
tumour tissues. However, after an oral dose of capeci-
tabine, the concentration of 5-FU in tumor tissue is
higher than in adjacent healthy tissue, as result of an
higher activity of Thymidine phosphorylase (TP).
2
Oral
administration seems to be preferred by patients and
allows to avoid complications and costs linked to 5-FU
IV infusion.
3
1
Azienda Policlinico Umberto I Roma, Roma, Italy
2
Azienda Ospedaliera Sant’Andrea, Roma, Lazio, Italy
3
Department of Neurosciences, Mental Health and Sensory Organs
(NESMOS), Sapienza University of Rome, Roma, Italy
4
Department of Medical Oncology, St Andrea University Hospital, Rome,
Italy
Corresponding author:
Simone Scagnoli, Azienda Policlinico, Umberto I Viale del Policlinico, 155
Roma, Roma 00161, Italy.
Email: simone.scagnoli@hotmail.it
J Oncol Pharm Practice
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DOI: 10.1177/1078155220904999
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In advanced breast cancer (ABC), the efficacy and
safety of capecitabine have been demonstrated both as
monotherapy and in combination with other drugs.
Several studies have demonstrated the efficacy of cape-
citabine at the dose of 1250 or 1000 mg/m
2
twice
daily for 14 over 21 days in metastatic breast cancer
patients.
4,5
The oral prodrug has also been tested in
an older population of patients (>65 years old) result-
ing effective and well tolerated at the dose of
1000 mg/m
2
.
6
Capecitabine is a landmark treatment
also in HER2þ metastatic disease in association
with lapatinib, after initial progression to first or
second line.
7
Conversely, the role of capecitabine in the adjuvant
setting is still uncertain. Several trials of adjuvant cape-
citabine administered in combination with other effec-
tive drugs did not show an advantage over regimens
without capecitabine.
8,9
Recently, in the CRATE
study, about 900 patients with HER2 negative breast
cancer with residual disease after standard neoadjuvant
chemotherapy have been randomized to receive adju-
vant capecitabine or no treatment (control group).
The results showed improvement in disease-free surviv-
al (74.1% vs. 67.6%r at 5 years, respectively, HR 0.70,
p: 0.01) and overall survival (OS) (89.2% vs. 83.6%
respectively, HR 0.59; P ¼ 0. 01) in treatment group
and even more significant in TNBC subgroup.
10
As
expected, the addition of capecitabine caused
treatment-related toxicities.
11,12
Regardless cancer primary site and setting of treat-
ment, however, capecitabine is related with several high
grade toxicities, some of them with quick onset and
unexpected severity.
13
Diarrhea is the most common,
adverse event that occurs in 55–60% of patients, with a
severe grade 3 or 4 of toxicity in about 12–15%. Other
common GI adverse events are nausea and vomiting.
Moreover, haematological adverse events like neutro-
penia, anaemia and thrombocytopenia are very
common during the treatment. Finally, skin reactions
are common: rash and hand&foot syndrome are fre-
quent but also severe skin toxicities such as Stevens–
Johnson syndrome and toxic epidermal necrolysis are
reported.
14–16
In view of the wide use of capecitabine in breast
cancer, the identification of predictive factors for
capecitabine-related toxicities is a pressing need. Pre-
emptive identification of patients that will develop
severe toxicities is still an open issue. Several tests
have been developed to identify those patients with
alterations in 5-FU metabolism that can lead to
undue and unexpected toxicities. We previously dem-
onstrated that the evaluation of dihydropyrimidine
dehydrogenase (DPYD) polymorphisms and the
5-FU degradation rate (5FUDR) can help to distin-
guish patients prone to develop severe side effects in
colorectal and gastric patients treated with 5-FU in
both adjuvant and metastatic setting.
17
The aim of our study is to explore the association
between 5FUDR, DPYD, thymidylate synthase
enhancer region (TSER) and methylenetet rahydrofo-
late reductase (MTHFR) polymorphisms and toxicities
in breast cancer patients treated with capecitabine.
Patients and methods
Patients
Clinical data of 37 patients treated for locally advanced
and metastatic breast cancer at our institutions were
retrospectively collected. Inclusion criteria were: age
>18 years old, diagnosis of metastatic breast cancer,
previous treatment with capecitabine for metastatic
breast cancer, PS 0-1 at baseline, absence of liver or
kidney impairment. Exclusion criteria were: PS 2 or
more at baseline, less than one month of treatment
with capecitabine and patient lost at follow up during
treatment with capecitabine. As a clinical practice,
CAPE was used at 1000–1250 mg/mq twice daily for
14 days followed by a seven-day rest period. We per-
formed a monthly assessment of treatment toxicity
according to the National Cancer Institute-Common
Terminology Criteria for Adverse Events version 5
(CTCAE v.5, 2017). Patien ts were instructed to
report and were usually asked for common toxicities
during follow-up visits. Total toxicity was defined as
the percentage of patients who suffered from at least
one adverse event irrespective of type and grade. The
study was conducted in accordance with the Declaration
of Helsinki and the protocol approved by the institutional
(Sapienza University) ethical committee.
Methods
Peripheral blood samples from all patients enrolled
were collected at the baseline as clinical practice in
our Institution to perform genotype analysis and eval-
uate 5FUDR on peripheral blood cells. Genotyping of
DPYD GIVS14A (rs3918290), MTHFR C677T
(rs1801133) and A1298C (rs1801131) SNPs was per-
formed by pyro-sequencing technology. PCR analysis
was used for genotyping TSER polymorphism
(rs34743033). The individual 5FUDR was assessed by
a liquid chromatography–tandem mass spectrometry
on peripheral blood mononuclear cells (PBMC).
5FUDR is determined in vitro by measuring the
decrease of a fixed amount of 5-FU (10 mg/mL) added
to a solution of PBMC, after 2 h incubation, expressed
as nanogram per millilitre of 5-FU degraded per
minute 10 cells. The assay is composed of three
steps: (1) PBMC isolation from peripheral blood, (2)
2 Journal of Oncology Pharmacy Practice 0(0)
PBMC incubation with 5-FU in vitro and (3) determi-
nation of 5-FU amount to calculate the degradation
rate.
18
5FUDR is the result of the whole intracellular
metabolizing process, regardless the presence or not of
a single enzyme alterations. As previously reported,
patients were categorized in three groups according
to their 5FUDR value: below the fifth centile (poor
metabolizers–PMs), above the 95th centile (ultra-rapid
metabolizers–UMs) and within the 5–95th centile (0.85–
2.2 ng/mL/106 cells/min) (extensive metabolizers–EM).
Statistical analysis
SPSS statistical software, Version 24 (SPSS Inc.
Chicago, Illinois, USA), was used. Each MTHFR gen-
otypes C677T (CC, CT and TT) and A1298C (AA, AC,
CC), TSER genotypes (2 R/2R, 2 R/3R, 3 R/3R) and
DPYD (GG/GA) were tested. The v
2
test and t test
for unpaired data were applied to compare the frequen-
cies and mean, respectively. Genotype variant associa-
tion with toxicity events was first analysed using
univariate logistic regression and further by a multi var-
iate logistic regression including patient age (60 vs.
<60 years old), comorbidity (2 vs. <2) and type of
chemotherapy regimen (CAPE alone vs. CAPE plus
navelbine/lapatinib). A P value < 0.05 was considered
as statistically significant.
Results
Overall, 37 Caucasian patients with a median age of
58 years old (range 34–79) treated with CAPE -based
chemotherapy for stage II–IV breast cancer were
included in this study. Their baseline and demographic
characteristics are shown in Table 1. A 25% dose
reduction was done in nine cases (24.3%), and the
treatment was prematurely stopped in six (16.2%)
patients due to G1–4 GI (40%) and haematological
(60%) adverse events. The most common treatment-
related adverse events are reported in Figure 1.
CAPE was administered with an adjuvant intent in
16 (43.2%) patients and in 21 (56.8%) patients affected
by ABC, and it was administered alone or in combina-
tion with other drugs in 15 (40.5%) and 22 (59.5%)
cases, respectively. Overall, 34 (91.9%) patients suf-
fered from at least an episode of any grade toxicity
while 9 (24.3%) patients had G3–4 toxicity. No toxic
death was reported. The frequency of toxicity (73.7 vs.
77.3% and 26.7 vs. 22.7% for any grade and G3–4
toxicity, respectively) did not differ between patients
who received CAPE alone or in combination with
other drugs. Besides, capecitabine plus navelbine regi-
men showed a higher incidence of any grade haemato-
logical toxicity and G1–2 GI toxicity than capecitabine
alone or in combination with lapatinib (Figure 2).
Pharmacogenetic variant analyses
The distribution of the analysed genotypes did not
deviate from Hardy–Weinberg equilibrium (DPYD,
P ¼ 0.9, MTHFR 677, P ¼ 0.86; MTHFR 1298,
P ¼ 0.50; TSER, P ¼ 0.87). Homozygous DPYD
Table 1. Clinicopathological parameters of patients.
Parameter Number %
Total 37 100
Median age years (range) 58 (34–79)
Comorbidity
<2 32 86.5
2 5 13.5
Stage
II–III 16 43.2
IV 21 56.8
Estrogen receptor
Median (range) 80 (0–100)
Negative 11 30.6
Progesteron receptor
Median (range) 49.5 (0–90)
Negative 10 27.8
Her2/neu
Positive 10 27.0
Negative 27 73.0
Grading
1 2 5.4
2 6 16.2
3 22 59.5
Missing 7 18.9
Ki 67 expression
Median (range) 32 (7–89)
Capecitabine-based therapy
Mono-chemotherapy 15 40.5
Plus navelbine 17 46.0
Plus lapatinib 5 13.5
TSER
2R/2R 7 18.9
3R/3R 14 37.8
2R/3R 14 37.8
Missing 2 5.4
MTHFR 677
CC 11 29.7
CT 19 51.4
TT 6 16.2
Missing 1 2.7
MTHFR 1298
AA 17 45.9
AC 15 40.5
CC 5 13.5
5FU degradation value
EM 31 83.8
PM 3 8.1
UP 3 8.1
5FU: 5-fluorouracil; EM: extensive metabolizer; PM: poor metabolizer,
UM: ultra-rapid metabolizer.
Botticelli et al. 3
IVS14 þ 1G > A SNP nor heterozygous DPYD was
not observed in the cohort. Homozygous MTHFR
677TT was found to be significantly related to haema-
tological toxicity (OR ¼ 6.5 [95% IC 1.1–37.5],
P ¼ 0.04). However, no association was detected
between each other SNPs and toxicity (Table 2).
Overall, the mean value SD of 5FUDR was 1.45
0.45 (range: 0.49–2.50) ng/mL/106 cells/min. Three
patients had a degradation rate less than 0.86 ng/mL/
106 cells/min (PMs) and three patients greater than
2.1 ng/mL/106 cells/min (UMs). No association was
found between 5FUDR and either TSER or MTHFR
genotypes (Table 3). At a univariate logistic regression
analysis, an altered value of 5FUDR (values < 0.86 or
>2.10 ng/mL/106 cells/min) increased the risk of G3–4
adverse events (OR ¼ 10.40 [95% IC: 1.48–7.99],
P ¼ 0.02) (Table 2). A multivariate logistic regression
analysis, adjusted for age, comorbidity and CAPE reg-
imen, confirmed the role of 5FUDR as a predictor
of G3–4 toxicity occurrence (OR ¼ 10.9 [95% IC 1.2–
96.2], P ¼ 0.03.
Discussion
Several enzymes are involved in the capecitabine
metabolism.
19
The dihydropyrimidine dehydrogenase
enzyme (DPD) metabolizes about 80% of the admin-
istered 5-FU into the inactive metabolite 5,6-dihydro-5-
fluorouracil. The remaining 20% is converted into
active metabolites that cause the inhibition of thymidy-
late synthase (TYMS) and RNA/DNA damage.
20
Several genotypes of the DPD have been associated
Leucopenia
Neutropenia
Anaemia Nausea Vomit Constipation Diarrhoea Mucositis
Hand-foot
syndrome
G1–G2
2661772944
G3–G4
042110402
0
2
4
6
8
10
12
14
16
18
N. of patients
Figure 1. Most common toxicities in study population.
G1–G2 G3–G4 G1–G2 G3–G4 G1–G2 G3–G4
Haematological toxicity Gastrointestinal toxicity Hand-foot syndrome
CAPE
408422
CAPE plus Nvb
7410210
CAPE plus lapatinib
003110
0
2
4
6
8
10
12
N. of patients
Figure 2. Type and severity of toxicity according to the therapeutic scheme administered.
4 Journal of Oncology Pharmacy Practice 0(0)
with reduced enzyme activity that could lead to severe
toxic adverse events of capecitabine or fluoropyrimi-
dine.
21
The most used pharmacogenetic test to predict
DPD activity is base d on the detection of IVS14 þ
1G > A polymorphism in the DPYD gene, which
leads to the production of an inactive protein and
severe toxicity in about one-half of carrier patients.
22
Moreover, a decreased value of 5FUDR is linked to
DPYD haplotype, and it could be related to adverse
events development;
23
however, this polymorphism has
a low frequency. Other enzymes are involved in 5-FU
metabolism, and their polymorphisms could result in
increased and unexpected toxicities such as MTHFR,
one of the most relevant enzyme that regulates intra-
cellular folate levels that affect DNA synthesis and
methylation and TYMS.
24,25
The single-nucleotide polymorphisms (SNPs) of
MTHFR 677 C > T and 1298 A > C are clinically
relevant and have been associated with the toxicity
of 5-FU.
26
Moreover, variations of the TSER in the
promoter of TYMS gene have been related to both
survival/response outcomes and toxicities in patients
affected by colorectal cancer treated with 5-FU-based
chemotherapy.
27–30
Finally, we previously described
a non-genomic assay that seems to be able to predict
5-FU toxicity by the assessment of the 5FUDR in the
PBMC.
31
This parameter indicates the amount of drug
consumed by cells in a time unit and reflexes the result
of the entire 5-FU degradation metabolism, not only a
single enzyme activity.
32
Applying the assay on colo-
rectal patients, we previously described two different
classes of patients with a higher risk to develop 5-FU
unexpected toxicities: poor metabolizers and ultrarapid
metabolizers.
33,34
Besides, we showed that 5FUDR is
associated with progression-free survival in metastatic
colorectal patients with an advantage for ultra/poor
metabolizers versus normal metabolizers.
35
Up to now, no data were available on the correla-
tion between 5FUDR and DPD/MTHFR/TSER poly-
morphism and cape citabine-related toxicity specifically
in breast cancer patients. Hence, we carried out a ret-
rospective study aimed to evaluate the impact of each
of the fol lowing gene polymorphisms MTHFR C667T,
MTHFR A1298C, DPYD IVS14 þ 1G > A, TSER and
the 5-fluorouracil degradation rate (5-FUDR) on
toxicities in breast cancer patients treated with capeci-
tabine. Our hypothesis was that ultra/poor metabolizer
patients have a higher percentage of total adverse
events.
Our results suggest that 5FUDR is a possible
predictor of G3–4 toxicity in both metastatic and
non-metastatic breast cancer patients treated with
capecitabine. UMs and PMs patients developed
higher rate of severe toxicities compared with NM,
and these results are similar to our previous findings
on colorectal cancer patients treated with capecitabine,
Table 3. Incidence of toxicity by patients genotype and 5FUDR.
Biomarker Genotype N
Haematoxicity
(G1–4)
(%)
OR
(95% CI) P
GI toxicity
(G1–4)
(%)
OR
(95% CI) P
HFS
(G1–4)
(%)
OR
(95% CI) P
G3–4
Toxicity
(%)
OR
(95% CI) P
MTHFR
C677T
CC 11 18 91 35 36
CT 16 56 75 6 21
TT 6 66 6.5 (1.1–37.5) 0.04 83 0.3 (0.1–3.3) 0.35 20 0.2 (0.1–1.2) 0.08 17 0.4 (0.1–2.1) 0.30
MTHFR
A1298C
AA 15 53 73 21 29
AC 14 43 85 7 13
CC 5 20 0.5 (0.1–2.0) 0.33 100 3.1 (0.5–19.8) 0.23 40 0.7 (0.1–4.0) 0.67 40 0.6 (0.1–2.7) 0.50
TSER 3R/3R 7 43 86 0 29
2R/3R 12 42 83 25 29
2R/2R 13 54 1.2 (0.2–6.6) 0.81 77 0.6 (0.1–6.8) 0.73 23 1.5 (0.2–8.9) 0.65 14 0.7 (0.1–4.4) 0.68
5-FU
dRate
NM 28 43 1.3 (0.2–7.8) 86 22 16 10.4 (1.5–72.9)
PM 3 33 67 0.3 (0.1–2.4) 0 67
UM 3 67 0.75 67 0.28 0 0.0 0.99 67 0.02
Bold- significant difference in G3-4 toxicities between normal metabolizers (NM) and ultra/poor metabolizers (PM/UM) (p ¼ 0.02).
Table 2. 5FUDR descriptive statistics by demographic and
genetic characteristics.
N
5FUDR
(mean value SD) P
Age
<60 years 19 1.39 0.51 0.26
70 years 18 1.56 0.37
MTHFR C677T
CC 11 1.56 0.63 0.72
CT 19 1.42 0.39
TT 6 1.48 0.28
MTHFR A1298C
AA 17 1.57 0.45 0.14
AC 15 1.47 0.36
CC 5 1.12 0.57
TSER
3R3R 7 1.30 0.47 0.60
2R3R 14 1.51 0.41
2R2R 14 1.46 0.43
Botticelli et al. 5