Int. J. Med. Sci. 2012, 9
I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f M Me ed di ic ca al l S Sc ci ie en nc ce es s
2012; 9(10):833-837. doi: 10.7150/ijms.4914
VEGF -634C/G Genotype is Predictive of Long-term
Survival after Treatment with a Definitive 5-Fluorou-
racil/cisplatin-based Chemoradiotherapy in Japanese
Patients with Esophageal Squamous Cell Carcinoma
Takao Tamura 1,2, Akiko Kuwahara 2,3, Motohiro Yamamori 2,3, Kohshi Nishiguchi 2,4, Tsutomu Nakamura
2, Tatsuya Okuno 2, Ikuya Miki 2, Yuki Manabe 5 and Toshiyuki Sakaeda 2,5
1. Department of Medical Oncology, Nara Hospital, Kinki University Faculty of Medicine, Nara 630-0293, Japan.
2. Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
3. School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya 663-8179, Japan.
4. Department of Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan.
5. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Corresponding author: Toshiyuki Sakaeda, Ph.D., Center for Integrative Education in Pharmacy and Pharmaceutical Sciences, Graduate
School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan, Tel: +81-75-753-9560, Fax: +81-75-753-9253, e-mail: sa-
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2012.07.23; Accepted: 2012.10.15; Published: 2012.11.01
Background: Reports have been accumulating that genetic properties are predictive of
clinical response after and/or toxicity during cancer chemotherapy, but little information is
available concerning effects on long-term survival. In this study, 49 Japanese patients with
esophageal squamous cell carcinoma (ESCC) were followed up for 5 years after treatment
with a definitive 5-fluorouracil (5-FU)/cisplatin (CDDP)-based chemoradiotherapy (CRT), and
the effects of genotypes of vascular endothelial growth factor (VEGF) were retrospectively
revaluated in terms of prediction of long-term survival.
Methods: A course consisted of the continuous infusion of 5-FU at 400 mg/m2/day for days
1-5 and 8-12, the infusion of CDDP at 40 mg/m2/day on days 1 and 8, and radiation at 2 Gy/day
on days 1 to 5, 8 to 12, and 15 to 19, with a second course repeated after a 2-week interval.
The VEGF genotypes -1498T/C, -1154G/A, -634C/G, -7C/T, 936C/T, and 1612G/A were
Results: The complete response (CR) rate was 46.9% (23/49). The 5-year survival rate was
42.9 % (21/49). There were 7 patients with a CR, but survival of less than 5 years. They died
from myocardial infarction (N=1), sudden cardiac death after suffering from heart failure
(N=1), acute myeloid leukemia that developed from myelodysplastic syndromes (N=1),
factors not specified (N=2), oropharynx cancer (N=1), and tongue cancer (N=1). VEGF
-634C/G had no effect on clinical response, but long-term survival depended on the genotype
(p=0.033, Fisher’s; p=0.038, Cochran-Armitage; p=0.079, Log-rank). The genotype frequency
of 7 patients with a CR, but survival of less than 5 years was different from that for the other
42 patients (p=0.032, Fisher’s). None of the other 5 genotypes evaluated affected either
clinical response or survival.
Conclusions: VEGF -634C/G is possibly predictive of long-term survival after treatment with
a definitive 5-FU/CDDP-based CRT. Further clinical studies with a larger number of cases are
needed to clarify the effects of this genotype.
Key words: esophageal squamous cell carcinoma, chemoradiotherapy, late toxicity, prognosis,
vascular endothelial growth factor.
Int. J. Med. Sci. 2012, 9
A clinical report published in 1999, the RTOG
(Radiation Therapy Oncology Group) 85-01 trial in-
volving 134 patients with T1-3, N0-1, and M0 esoph-
ageal cancer, was of great interest in terms of clinical
outcome because it demonstrated a 5-year survival
rate of 26 % [1-4]. This treatment consists of a
96-hr-infusion of 5-fluorouracil (5-FU) at a daily dose
of 1,000 mg/m2/day in weeks 1, 5, 8, and 11, infusion
of cisplatin (CDDP) at 75 mg/m2/day on the first day
of weeks 1, 5, 8, and 11, and concurrent radiation at 50
Gy in 25 fractions over 5 weeks. Immediately there-
after, another version of chemoradiotherapy (CRT)
was proposed for advanced esophageal squamous cell
carcinoma (ESCC), which consists of a 120-hr-infusion
of 5-FU at 400 mg/m2/day in weeks 1, 2, 6, and 7,
infusion of CDDP at 40 mg/m2/day on the first day of
weeks 1, 2, 6, and 7, and concurrent radiation at 60 Gy
in 30 fractions over 8 weeks [5, 6]. Two independent
clinical investigations have shown curative potential
using this regimen for unresectable ESCC with T4 or
M1a [5, 6], and a long-term evaluation of efficacy and
toxicity with 139 patients resulted in a complete re-
sponse (CR) rate of 56%, along with a 5-year survival
rate of 29 % [7-9]. Of note, however, treatment-related
late toxicities were severe, and life-threatening car-
diopulmonary toxicities might have occurred, in-
cluding pericarditis, heart failure, myocardial infarc-
tion/cardiac ischemia, pleural effusion, and radiation
pneumonitis [8-10]. The incidence of mortality due to
cardiopulmonary late toxicities was relatively high for
patients with a CR [8, 9]; however, it was low in two
large randomized trials, in which the benefits of com-
bining CRT with surgical treatment were evaluated
[11, 12]. Currently, a definitive 5-FU/CDDP-based
CRT is recognized as one of the most promising
treatments for esophageal cancer, but given the ex-
tensive inter-individual variation in clinical outcome,
future improvements will
dose-modification of these regimens, incorporation of
a novel anticancer drug, pharmacokinetically guided
administration of 5-FU or CDDP, and identification of
responders via patient genetic profiling .
A series of studies has been performed to find a
marker predictive of clinical response 1 month after or
severe acute toxicities during treatment with a defini-
tive 5-FU/CDDP-based CRT in Japanese patients with
ESCC [14-19]. The genotypes of vascular endothelial
growth factor (VEGF), an endothelial cell-specific
mitogen, were evaluated , since clinical reports
suggested that their genetic variations have the po-
tential to influence the expression of VEGF, and
thereby the growth of tumors, metastatic spread, and
likely require the
response to treatment [20-23]; however, they failed to
predict the response . Obviously, the final goal of
cancer chemotherapy is an improvement in long-term
survival, not a short-term clinical response, so pa-
rameters predicting prognosis have been absolutely
imperative. In this study, patients with ESCC were
followed up for 5 years after treatment with a defini-
tive 5-FU/CDDP-based CRT, and the effects of VEGF
genotypes were retrospectively re-evaluated in terms
of prediction of long-term survival.
Patients and study protocol
Forty-nine ESCC patients treated with a defini-
tive 5-FU/CDDP-based CRT at Kobe University
Hospital, Japan, from October, 2003 to June, 2006 were
followed up for 5 years. Survival time was defined as
the time from treatment initiation to death from any
cause or to the last date of confirmation of survival.
Survival data were updated on June 25, 2011. The
inclusion criteria for treatment basically included; 1)
clinical stage T1 to T4, N0 or N1, and M0 or M1 ac-
cording to the International Union Against Cancer
tumor-node-metastasis (TNM) classification; 2) age
less than 85 years; 3) an Eastern Cooperative Oncol-
ogy Group performance status of 0 to 2; 4) adequate
bone marrow, renal, and hepatic function; 5) no prior
chemotherapy; 6) no severe medical complications;
and 7) no other active malignancies (except early
cancer). The tumors were histologically confirmed to
be primary, and no patients with recurrence were
included in this study.
A course consisted of the continuous infusion of
5-FU at 400 mg/m2/day for days 1-5 and 8-12, the
infusion of CDDP at 40 mg/m2/day on days 1 and 8,
and radiation at 2 Gy/day on days 1 to 5, 8 to 12, and
15 to 19, with a second course repeated after a 2-week
interval [5, 6]. If disease progression/recurrence was
observed, either salvage surgery, endoscopic treat-
ment, or another regimen of chemotherapy was
scheduled. The VEGF genotypes -1498T/C (known as
-460T/C, rs833061) and -1154G/A (rs1570360) in the
promoter region, -634C/G (known as 405C/G,
rs2010963) and -7C/T (rs25648) in the 5’ untranslated
region (UTR), and 936C/T (rs3025039) and 1612G/A
(rs10434) in the 3’UTR, were evaluated using periph-
eral blood and the TaqManR MGB probe-based pol-
ymerase chain reaction and confirmed by direct se-
quencing. This study was conducted with the au-
thorization of the institutional review board and fol-
lowed the medical research council guidelines of Ko-
be University. Written informed consent was obtained
from all participants prior to genotyping. The associ-
Int. J. Med. Sci. 2012, 9
ations with clinical response and severe acute toxici-
ties were already reported , and their effects on
long-term survival were evaluated in this study. It
should be noted that the patients in this study were
almost the same as those in the previous report .
The clinical response was evaluated as reported
previously [5-9]. Briefly, a CR was defined as the
complete disappearance of all measurable and as-
sessable disease at the first evaluation, which was
performed 1 month after the completion of CRT to
determine whether the disease had progressed. The
clinical response was evaluated by endoscopy and
chest and abdominal computed tomography (CT)
scans in each course. A CR at the primary site was
given by endoscopic examination when all of the fol-
lowing criteria were satisfied on observation of the
entire esophagus: 1) disappearance of the tumor le-
sion; 2) disappearance of ulceration (slough); and 3)
absence of cancer cells in biopsy specimens. If small
nodes of 1 cm or less were detected on CT scans, the
recovery was defined as an “uncertain CR” after con-
firmation of no progression for at least 3 months. An
“uncertain CR” was included as a CR when calculat-
ing the CR rate. When these criteria were not satisfied,
a non-CR was assigned. The existence of erosion, a
granular protruded lesion, an ulcer scar, and 1.2
w/v% iodine/glycerin-voiding lesions did not pre-
vent an evaluation of CR. The evaluations were per-
formed every month for the first 3 months, and when
the criteria for CR were not satisfied at 3 months, the
result was changed to non-CR. Follow-up evaluations
were performed thereafter every 3 months for 3 years
by endoscopy and CT scan. After 3 years, patients
were seen every 6 months. During the follow-up pe-
riod, a routine course of physical examinations and
clinical laboratory tests was performed to check the
Data analysis and statistics
All values reported are the mean±standard de-
viation (SD) without any explanatory notes. The
Fisher’s exact test, Cochran-Armitage test, Recessive
and Dominant test and Log-rank test were used for
evaluation of the effects of genotypes on long-term
survival. P values of less than 0.05 (two tailed) were
considered to be significant.
Demographic and clinicopathologic characteris-
tics of 49 Japanese ESCC patients are summarized in
Table 1. The average age was 64.5±7.4 years (range:
48-78) and the average weight was 56.1±9.6 kg (range:
33-79). The ratio of T1/T2/T3/T4 was 16/6/15/12,
that of N0/N1 was 22/27, and that of M0/M1 was
41/8, resulting in a stage I/II/III/IV ratio of
12/10/19/8. The CR rate and 5-year survival rate
were 46.9% (23/49) and 42.9 % (21/49), respectively.
The 5-year survival rate was 69.6 % (16/23) for the
patients with a CR, but only 19.2 % (5/26) for those
with a non-CR, and survival depended on clinical
response, i.e., CR or non-CR (P=0.0005, Fisher’s;
The frequencies of VEGF genotypes in the 49
patients are listed in Table 2. Among the 6 genotypes
evaluated, only -634C/G affected survival (p=0.033,
Fisher’s; p=0.038, Cochran-Armitage). Figure 1 shows
the association of VEGF -634C/G with survival. The
median overall survival time (±SE) was 29.5±22.3
months for all patients, and more than 60 months,
more than 60 months, and 13.0±9.0 months for the
patients with CC-634, CG-634, and GG-634, respectively,
but the genotype did not have enough power to pre-
dict survival (P=0.079, Log-rank). There were 7 pa-
tients with a CR, but a survival of less than 5 years.
They died from myocardial infarction (N=1), sudden
cardiac death after suffering from heart failure (N=1),
acute myeloid leukemia that developed from myelo-
dysplastic syndromes (N=1), factors not specified
(N=2), oropharynx cancer (N=1), and tongue cancer
(N=1). Their genotype frequency is listed in Table 3.
Their genotype frequency was different from that for
the other 42 patients (p=0.032, Fisher’s).
Table 1. Demographic and clinicopathologic characteris-
tics of 49 Japanese patients with esophageal squamous cell
Performance status, 0/1/2/unknown
64.5±7.4 (48 -78)
The values are the mean±SD, with the range in parentheses.
Int. J. Med. Sci. 2012, 9
Table 2. VEGF genotypes and survival after treatment with
a definitive 5-fluorouracil/cisplatin-based chemoradiother-
apy in 49 Japanese patients with esophageal squamous cell
total 5 years
less than 5
a) more than 5 years vs. less than 5 years; P values by Fisher’s exact test for
genotype frequencies with allele frequencies in parentheses.
Figure 1. Association of VEGF -634C/G genotype with long-term
survival in Japanese patients with esophageal squamous cell car-
cinoma. Lines: patients with CC-634 (N=9), CG-634 (N=26), and
GG-634 (N=14). The survival tended to depend on the genotype
(p=0.079, Log-rank test).
Table 3. VEGF -634C/G genotype in the patients with a
complete response, but survival of less than 5 years.
5 years or more
Less than 5 years
The VEGF-634C/G genotype frequency in the patients with a complete
response (CR), but survival of less than 5 years, was different from others
(Fisher’s exact test, p=0.032 for genotype, p=0.018 for allele).
VEGF, an endothelial cell-specific mitogen, was
discovered in the 1980s [24-29], and more than 20
years of investigation has clarified that VEGF inhibits
the apoptosis of endothelial cells, increases vascular
permeability, and promotes angiogenesis [30-32].
VEGF is a member of a structurally related VEGF
family; VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D,
and placenta growth factor (PlGF) [30-32]. VEGF and
related proteins are critical for development, growth
and metastasis of tumors, and their expression levels
have been demonstrated to be predictive of resistance
to treatment [30-32]. Genetic polymorphisms in the
promoter region, 5’UTR, and 3’UTR of the VEGF gene
are associated with VEGF levels in plasma or tumors,
and therefore can be diagnostic, prognostic, and pre-
dictive biomarkers for patients with tumors [20-23].
In this study, it was suggested that the VEGF
-634C/G genotype is possibly predictive of long-term
survival after treatment
5-FU/CDDP-based CRT (Table 2, Figure 1). For this
protocol, the cardiopulmonary late toxicities are a
matter of debate, and the incidence of mortality was
reported to be 3-12% for the patients with a CR [8, 9].
Recently, it has been suggested that the cardiopul-
monary toxicities and subsequent malignancy ob-
served in cancer survivors are presumably due to
radiation and/or a certain class of anticancer drugs
[33, 34]; however, the incidence of mortality due to
cardiopulmonary late toxicities was 2% or less in two
large randomized trials, in which CRT alone was
compared with CRT with surgical treatment [11, 12].
Here, there were 7 patients with a CR, but survival of
less than 5 years. Their VEGF -634C/G genotype was
different from others, and 2 of 7 patients died from
cardiopulmonary late toxicities. If we can obtain
compelling evidence concerning the VEGF -634C/G
genotype, we can optimize therapeutic treatment
based on VEGF genetic profiling.
Angiogenesis plays a protective role in ischemic
heart disease and myocardial infarction, and VEGF
with a definitive
Int. J. Med. Sci. 2012, 9 Download full-text
expression in the myocardium increases tentatively
after hypoxia . Acute and chronic hypoxias result
in an increase in the secretion of VEGF into the lung
. We do not have enough data to clarify the role of
VEGF in cardiopulmonary diseases, but VEGF genetic
variations might be biomarkers for patients with car-
diopulmonary diseases. In conclusion, VEGF -634C/G
is possibly predictive of long-term survival after
treatment with a definitive 5-FU/CDDP-based CRT.
Further clinical studies with a larger number of cases
are needed to clarify the effects of this genotype.
The authors have declared that no competing
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