original article
Annals of Oncology 20: 481–485, 2009
doi:10.1093/annonc/mdn667
Published online 12 December 2008
Pharmacogenetic analyses of hematotoxicity in
advanced gastric cancer patients receiving biweekly
fluorouracil, leucovorin, oxaliplatin and docetaxel
(FLOT): a translational study of the Arbeitsgemeinschaft
Internistische Onkologie (AIO)
E. Goekkurt1, S.-E. Al-Batran2, U. Mogck1, C. Pauligk2, J. T. Hartmann3, M. Kramer1, E. Jaeger2,
G. Ehninger1 & J. Stoehlmacher1*
1
Department of Internal Medicine I, University Hospital Carl Gustav Carus, University of Dresden, Dresden; 2Department of Oncology and Hematology, Krankenhaus
Nordwest, Frankfurt; 3Department of Medical Oncology, Hematology, Immunology, Rheumatology, Pneumology, Eberhard-Karls-University, Tuebingen, Germany
Received 8 September 2008; revised 8 September 2008; accepted 9 September 2008
(AGC). However, a high rate of grade 3/4 hematotoxicity was reported with these regimens. Our purpose was to
identify pharmacogenetic markers with potential to detect patients with increased risk to encounter severe
hematotoxicity following treatment with 5-fluorouracil, leucovorin, oxaliplatin and docetaxel (FLOT).
Patients and methods: Polymorphisms of genes involved in DNA repair, drug transport and metabolism were
determined in 50 AGC patients receiving FLOT within a phase II trial. DNA was extracted from peripheral blood.
Genotyping was carried out using PCR-based techniques.
Results: Patients possessing TS-group A genotypes (2R/2R, 2R/3RC, 3RC/3RC) were at increased risk for grade 3/4
hematotoxicity compared with patients harboring a TS-group B genotype (2R/3RG, 3RC/3RG, 3RG/3RG). In all, 59%
(20 of 34) of patients with TS-group A genotypes developed grade 3/4 hematotoxicity compared with 25% (4 of 16) of
those having TS-group B genotypes (P = 0.035). Grade 3/4 neutropenia occurred in 53% (18 of 34) of TS-group A
patients compared with 19% (3 of 16) in TS-group B patients (P = 0.032). Multivariate analyses identified TS-group A
genotypes as significant predictors of grade 3/4 overall hematotoxicity {odds ratio (OR) 4.62 [95% confidence interval
(CI) 1.22; 17.44], P = 0.024} and neutropenia [OR 5.74 (95% CI 1.03; 32.08), P = 0.047].
Conclusion: TS-promoter polymorphisms may be associated with hematotoxicity in AGC patients receiving FLOT.
Key words: advanced gastric cancer, docetaxel, 5-fluorouracil, hematotoxicity, oxaliplatin, polymorphism
introduction
Treatment options for advanced gastric cancer (AGC) are
increasing. Drugs such as oxaliplatin, irinotecan and docetaxel
have been evaluated by phase II and III clinical trials during the
last years and showed substantial efficacy [1–10]. Nevertheless,
prognosis for the majority of patients with AGC remains poor
and 2-year survival rates rarely exceed 20%. Therefore, avoiding
treatment-associated toxicity, which may limit the quality of
live, represents a major objective.
Particularly, the combination of docetaxel, cisplatin and
fluorouracil (DCF) improved several efficacy parameters in
AGC, but was associated with substantial toxicity, including
*Correspondence to: Dr J. Stoehlmacher, University Hospital Carl Gustav Carus,
Department of Internal Medicine I, Fetscherstr. 74, 01307 Dresden, Germany.
Tel: +49-351-458-2311; Fax: +49-351-449-210-630; E-mail: jan.stoehlmacher@
uniklinikum-dresden.de
grade 3/4 neutropenia and complicated neutropenia in 82%
and 29% of patients, respectively [7]. The combination of
docetaxel with the less toxic back bone regimen 5-fluorouracil,
leucovorin and oxaliplatin (FLO) [1, 2] within a recent phase II
study showed improved tolerability as compared with DCF, but
grade 3/4 neutropenia was still observed in 48% of patients
[11]. The purpose of our study was to search for
pharmacogenetic markers that might identify patients with an
increased risk to encounter high-grade hematotoxicity in this
study.
We have investigated the impact of 24 polymorphisms within
15 genes that are involved in transport and metabolism of the
applied drugs within the 5-fluorouracil, leucovorin, oxaliplatin
and docetaxel (FLOT) regimen, in DNA repair or in folate
metabolism on appearance of hematotoxicity in patients with
AGC with FLOT treatment. Selected genes were thymidylate
synthase (TS); methylenetetrahydrofolate reductaxe
ª The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
All rights reserved. For permissions, please email: [email protected]
original
article
Background: Docetaxel-based chemotherapy regimens have demonstrated activity in advanced gastric cancer
original article
(MTHFR); orotate phosphoribosyltransferase (OPRT);
methionine synthase (MTR); glutathione S-tranferases (GSTP1,
GSTT1, GSTM1); xeroderma pigmentosum group D (XPD);
X-ray repair cross complementing group 1 (XRCC1);
xeroderma pigmentosum group A (XPA); excision repair
cross complementing-group 1 (ERCC1); ATP-binding cassette,
subfamily B member 1 (ABCB1); ATP-binding cassette,
subfamily G member 2 (ABCG2) and cytochrome P450 3A4
(Cyp 3A4) and Cyp 3A5.
Annals of Oncology
statistical analyses
Deviation from Hardy–Weinberg Equilibrium (HWE) was analyzed using
R (Version 2.3.1, http://www.r-project.org/). All further statistical analyses
were carried out using SPSS (version 11.0). The Fisher’s exact test (two
sided) and the chi-square test were used to assess univariate associations
between genotypes and toxicity outcome. Multivariate logistic regression
model selection included clinical parameters (performance status, age, sex,
metastasis, number of involved sites and number of administered cycles)
and genotype information and was carried out by stepwise, forward and
backward selection. The significance level for entering an effect into the
model was 0.20. Statistical significance was interpreted as P < 0.05.
patients and methods
study population
In all, 59 patients were enrolled in a phase II study evaluating the safety
and efficacy of a biweekly chemotherapy regimen containing oxaliplatin 85
mg/m2, leucovorin 200 mg/m2 and docetaxel 50 mg/m2, each as a 1- to 2-h
i.v. infusion followed by 5-fluorouracil (FU) 2600 mg/m2 as a 24-h
continuous infusion (FLOT) [11]. Hematotoxicity was monitored prior
each chemotherapy administration and included the evaluation of anemia,
leukopenia, neutropenia and thrombocytopenia according to the National
Cancer Institute—Common Toxicity Criteria (version 2.0). Prophylactic
granulocyte colony-stimulating factor (G-CSF) was not administered.
Blood samples for genotyping were collected prospectively and were
available from 50 patients. All participating patients provided a signed
informed consent for genetic testing and the study including its
translational part was approved by the local ethics committee.
DNA extraction and genotyping
DNA was extracted from whole blood using the QIAamp DNA Mini Kit
(Qiagen, Hilden, Germany) without modifications. Genotypes of TS (TS
28-bp repeat polymorphism, G/C SNP within the second repeat of the
3R variant and TS1394del6), MTHFR (C677T, A1298C), MTR-A2756G,
ERCC1 (C118T, C8092A), XPD (Asp312Asn, Lys751Gln), XRCC1Arg399Gln, XPA-A23G, Cyp3A4*1B, GSTP1-Ile105Val and null-genotypes
of GSTM1 and GSTT1 were determined as described elsewhere [12–16].
We have designed a PCR-based restriction fragment length polymorphism
technique for genotyping the ABCB1-G2677T (G2677A) polymorphism.
Primers used were 5#-TGCAATAGCAGGAGTTGTTGA-3# and 5#AAAAGATTGCTTTGAGGAATGG-3#. PCR was carried out as follows:
95C for 10 min followed by 35 cycles of 95C for 20 s, 58C for 1 min
and 72C for 20 s followed by a final elongation step (72C for 5 s). The
PCR product was then digested by BseY I to discriminate between G>T
(GG = 134, 74 bp; GT = 211, 137, 74 bp; TT = 211 bp) and in a second
assay with Bsr I to discriminate between G>A (GG = 211 bp; GA = 132, 79
bp; AA = 211, 132, 79 bp). Samples were genotyped for OPRT-Gly213Ala,
ABCB11-C1236T, ABCB11-C3435T, ABCG2-C421A, Cyp3A4*3, Cyp3A5*2
and Cyp3A5*3C polymorphisms using a custom-designed Assay-onDemand (OPRT-Gly213Ala: C_1901477_10,rs1801019; ABCB1-C1236T:
C_7586662_10,rs1128503; ABCB11 C3435T: C_7586657_20,rs1045642;
ABCG2-C421A: C_15854163_70,rs2231142; Cyp3A4*3:
C_27535825_20,rs4986910; Cyp3A5*2: C_30633862_10,rs28365083;
Cyp3A5*3C: C_26201809_30,rs776746) from Applied Biosystems
(Darmstadt, Germany). Briefly, 2 ng DNA sample was added to a reaction
volume of 15 ll, containing 7.5 ll TaqMan Universal PCR Master Mix, No
AmpErase UNG and 0.75 ll custom-designed probe. Amplifications were
carried out as follows on an Applied Biosystems 7500 Real Time PCR
System: 95C for 10 min followed by 45 cycles of 93C for 15 s and 60C for
1 min. A post-PCR plate read was used to determine the genotype. A
random sample of 20% of each polymorphism was repeated and showed
100% concordance.
482 | Goekkurt et al.
results
patients’ characteristics
Patients’ demographic and tumor-related information are
provided in Table 1. Hematotoxicity grade 3/4 including
Table 1. Patients’ characteristics
Patient characteristics
All enrolled
patients (n)
Total no. of patients
59
Ineligible disease
2
Age
Median
60
Range
29–76
Sex
Female
18
Male
41
Primary tumor site
Esophagogastric junction
26
Gastric
31
Not applicable
1
Prior surgery
25
ECOG status
Median
1
0–1
49
2–3
9
Unknown
1
Disease status
Newly diagnosed
39
Recurrent
20
Locally advanced
4
Metastatic
55
No. of organs involved
Median
3
1
12
2
15
3
17
4
11
‡5
4
Organs involved (primary tumor excluded)
Liver
22
Lymph nodes
38
Peritoneum
20
Lung
12
Other
23
%
Genotyped
patients (n)
%
50
1
60
29–76
30.5
69.5
14
36
28
72
44.1
52.5
3.4
42.4
25
24
1
21
48
50
2
42
83.1
15.3
1.7
1
44
6
0
88
12
0
66.1
33.9
6.8
93.2
34
16
2
48
68
34
4
96
20.3
25.4
28.8
18.6
6.8
3
10
13
15
10
2
20
26
30
20
4
37.3
64.4
33.9
20.3
39.0
18
33
15
10
22
36
66
30
20
44
ECOG, Eastern Cooperative Oncology Group.
Volume 20 | No. 3 | March 2009
original article
Annals of Oncology
anemia, leukopenia, neutropenia and thrombocytopenia was
observed in 24 of 50 eligible patients (48%). Neutropenia grade
3/4 was the most observed grade 3/4 hematotoxicity with
a frequency of 42% (n = 21) followed by grade 3/4 leucopenia
with 24% (n = 12). Grade 3/4 anemia occurred only in two
patients (4%) and grade 3/4 thrombocytopenia in only one
patient (2%). Detection of genetic polymorphisms was
successful in all patients. All analyzed single-nucleotide
polymorphisms (SNP) showed no deviation from the HWE.
and increased risk for grade 3/4 neutropenia could be
confirmed by stepwise multivariate logistic regression analyses
(Table 3). In addition, increasing numbers of administered
treatment cycles could be determined as an independent risk
p* = .035
100
25
59
90
80
% of pts
TS polymorphisms and hematotoxicity
We observed a significant association between TS promoter
polymorphisms and hematotoxicity. TS promoter
polymorphisms were divided into a low-expressing group A
(2R/2R, 2R/3RC, 3RC/3RC) and a high-expressing group B
(2R/3RG, 3RC/3RG, 3RG/3RG) according to the literature
[17, 18]. Patients possessing one of the group A genotypes were
at significantly increased risk for grade 3/4 hematotoxicity
compared with patients harboring a TS-group B genotype
(Table 2). In all, 59% (20 of 34) of patients with TS-group A
genotypes developed grade 3/4 hematotoxicity compared with
only 25% (4 of 16) of patients with TS-group B genotypes
(P = 0.035, Fisher’s exact test, two sided) (Figure 1). In fact,
83% of patients with grade 3 or 4 toxicity harbored a TS-group
A genotype. Furthermore, TS-group A genotypes were
identified as the sole independent risk factor for grade 3/4
overall hematotoxicity [hazard ratio 4.62 (95% confidence
interval 1.22; 17.44), P = 0.024] using a stepwise multivariate
logistic regression model.
The association between TS promoter polymorphisms and
hematotoxicity was most significant for neutropenia with 53%
(18 of 34) of TS-group A patients showing grade 3/4
neutropenia compared with only 19% (3 of 16) of TS-group B
patients (P = 0.032, Fisher’s exact test) (Figure 2). In fact, 86%
of patients with grade 3 or 4 neutropenia harbored a TS-group
A genotype. The association between TS-group A genotypes
75
70
60
50
40
41
30
20
Hematotoxicity
CTC grade 3/4
10
CTC grade 0/1/2
0
Group A
Group B
TS5' 28 bp-repeat + G/C SNP
Figure 1. Distribution of overall hematotoxicity according to TS5
promoter genotype. Group A: 2R/2R, 2R/3RC, 3RC/3RC; Group B: 2R/
3RG, 3RC/3RG; 3RG/3RG. TS, thymidylate synthase; bp, base pair; SNP,
single-nucleotide polymorphism. CTC, common toxicity criteria. *Fisher’s
exact test (two sided).
p* = .032
100
53
Table 2. Distribution of hematotoxicity by TS promoter genotype
19
90
Anemia
Grades 0–2
Grades 3–4
Leucopenia
Grades 0–2
Grades 3–4
Neutropenia
Grades 0–2
Grades 3–4
Thrombocytopenia
Grades 0–2
Grades 3–4
Overall hematotoxicity
Grades 0–2
Grades 3–4
TS promoter genotype
Group A (n)
Group B (n)
P value*
33
1
15
1
24
10
14
2
0.292
16
18
13
3
0.032
0.542
80
% of pts
Toxicity
81
70
60
50
40
47
30
20
Neutropenia
CTC grade 3/4
10
33
1
16
0
CTC grade 0/1/2
0
1
Group A
Group B
TS5' 28 bp-repeat + G/C SNP
14
12
*Fisher’s exact test (two sided).
TS, thymidylate synthase.
Volume 20 | No. 3 | March 2009
20
4
0.035
Figure 2. Distribution of neutropenia according to TS promoter
genotypes. Group A: 2R/2R, 2R/3RC, 3RC/3RC; Group B: 2R/3RG, 3RC/
3RG; 3RG/3RG. TS, thymidylate synthase; bp, base pair; SNP, singlenucleotide polymorphism. *Fisher’s exact test (two sided).
doi:10.1093/annonc/mdn667 | 483
original article
Annals of Oncology
Table 3. Factors predicting grade 3/4 neutropenia in AGC
Factor
OR
95% CI
Pa
Age
Administered cycles
TS5#-28 bp repeat + G/C SNP
Group B genotypes
Group A genotypes
GSTT1
Positive
Negative
ABCB1-C1236T
C/C
CT
T/T
1.07
1.36
0.988; 1.155
1.032; 1.783
0.098
0.029
1
5.74
1.025; 32.081
0.047
1
4.77
0.699; 32.500
0.111
1
0.26
1.76
0.046; 1.442
0.153; 20.325
0.123
0.650
a
Multivariate logistic regression model.
AGC, advanced gastric cancer, OR, odds ratio; CI, confidence interval; TS,
thymidylate synthase; SNP, single-nucleotide polymorphisms; GSTT1,
glutathione S-transferase T1; ABCB1, ATP-binding cassette, subfamily B
member 1.
factor for developing grade 3/4 neutropenia (Table 3). None of
the other analyzed polymorphisms was found to have
significant impact on hematotoxicity in this study population.
discussion
Here, we demonstrated for the first time an association between
TS promoter polymorphisms and hematotoxicity in AGC
patients treated with a docetaxel-based triplet chemotherapy
regimen. Patients with a TS-group A genotype were likely to
develop grade 3/4 neutropenia. Nearly 90% of observed grade
3/4 neutropenia in this study could be attributed to patients
harboring group A genotypes. Reduced TS expression has been
shown to be associated with enhanced 5-fluorouracil (5-FU)
activity [19, 20] and these group A genotypes have been shown
to be associated with decreased TS expression [17, 18].
However, in vivo data that are linking TS promoter
polymorphisms to 5-FU-induced toxicity are rare [21, 22] and
in case of AGC even lacking. The TS promoter contains
a double polymorphism that is characterized by a 28-bp repeat
with mainly two or three repeat (2R, 3R) with a G>C SNP
within the second repeat of the 3R variant [17, 18]. In general,
the 2R variant could be linked to decreased TS expression
and the 3R variant to increased TS expression [17, 18, 22, 23].
The described G>C SNP is supposed to reduce the expression
level of the 3R variant to that of the 2R variant [17, 18].
The reports by Pullarkat et al. and Lecomte et al. both
describe an association between the 2R/2R genotype and
enhanced 5-FU-derived high-grade toxicity [21, 22]. However,
these reports did not include the G>C SNP in their analyses.
In our present study, there was also a trend for more grade 3/4
hematotoxicity in patients with the 2R/2R or 2R/3R genotype
compared with patients harboring the 3R/3R genotype
(data not shown). Neutropenia grade 3/4 occurred in 66.7%,
38.5% and 25% of 2R/2R, 2R/3R and 3R/3R carriers,
respectively (P = 0.10, chi-square test). We also carried out
484 | Goekkurt et al.
haplotype-based analyses including the TS promoter
polymorphisms and the TS1394del6 within the 3#-untranslated
region (UTR) of the gene. Thus, haplotype analyses did not reveal
additional significant information (data not shown). In addition,
we could not identify a significant association between TS
promoter genotypes or occurrence of high grade hematotoxicity
and treatment efficacy in terms of overall response, progressionfree and overall survival in this study (data not shown).
There are three possible explanations for the observed
association between TS promoter genotypes and the occurrence
of grade 3/4 hematotoxicity. The first and obvious one is the
mentioned association between reduced TS expression and
enhanced 5-FU activity. TS, as the sole de novo source of
thymidylate, is the target of 5-FU. The lower the TS expression
level the more 5-FU activity can be expected. Second, TS
plays an important role in various folate-dependent pathways
such as purine synthesis, methionine regeneration or DNA
methylation [24]. In conclusion, TS represents an important
enzyme in proliferating or regenerating cells, including those of
the hematopoietic system. An impaired TS function due to
reduced TS expression, therefore, may lead to impaired
regeneration after chemotherapy-induced bone marrow
depression caused by highly immunosuppressive agents such as
docetaxel. This suggestion provides a possible explanation of
the observed association of TS-group A genotypes with grade
3/4 neutropenia in this study since neutropenia is
a consequence of toxic injury of proliferating myeloid
precursor cells in the bone marrow. To date, in vitro or in vivo
data proving this hypothesis are lacking. In addition,
a highly synergistic effect of docetaxel and 5-FU has been
shown [25]. Within this synergy between taxanes and
fluoropyrimidines, TS expression seems to play an important
role [26]. In preclinical studies, exposure of gastric and colon
cancer cell lines to taxanes have led to changes in TS
expression [27, 28] suggesting a direct role of TS also in taxane
activity. These observations may provide an additional
mechanism that might underlie the observed predictive value of
TS promoter polymorphisms on hematotoxicity in our study.
In this study, a triple combination regimen consisting of
FLOT has been used. FLOT results from the addition of
docetaxel at 50 mg/m2 every 2 weeks to the doublet regimen
FLO [1, 2]. Within the limitations of a cross-study comparison,
the addition of docetaxel to the FLO regimen has raised the
observed hematotoxicity from 11.8% [2] to 48% [11]. Due to
the increased rate of grade 3/4 neutropenia with the FLOT
regimen compared with the FLO regimen, special focus was set
on pharmacogenetics of docetaxel. Therefore, polymorphisms
of Cyp 3A4, Cyp 3A5, ABCB1 and ABCG2 have been analyzed.
However, none of these polymorphisms turned out to have an
impact on hematotoxicity in this translational study. These
findings are in agreement with a study by Marsh et al. [29]
investigating these and other polymorphisms in a large cohort
of ovarian cancer patients with taxane plus platinum-based
chemotherapy regimens. The authors did not identify any clear
pharmacogenetic marker for toxicity prediction in their study.
We have also analyzed polymorphisms of genes being involved
in metabolism of platinum compounds and in DNA repair
(GSTP1, GSTT1, GSTM1, XPD, XPA, ERCC1, XRCC1). Again
we did not find an association between these polymorphisms
Volume 20 | No. 3 | March 2009
Annals of Oncology
and hematotoxicity in our study which also is in line with the
study by Marsh et al. [29].
Grade 3/4 neutropenia is the main source of infectious or
febrile complication during chemotherapy treatment of
patients. Two patients suffered from complicated neutropenia
(both with TS-group A promoter genotypes) in this study.
Therefore, if confirmed, AGC patients with a TS-group A
promoter genotype should receive an intensified safety
monitoring during FLOT therapy and may qualify for
prophylactic G-CSF administration.
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