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1131
Expression of Thymidine Phosphorylase as an
Indicator of Poor Prognosis for Patients with
Transitional Cell Carcinoma of the Bladder
Jun-ichirou Arima, M.D.1,2
Yoshiharu Imazono, M.D.1,2
Yuji Takebayashi, M.D.1,3
Kenryu Nishiyama, M.D.2
Tsutomu Shirahama, M.D.2
Suminori Akiba, M.D.4
Tatsuhiko Furukawa, M.D.1
Shin-ichi Akiyama, M.D.1
Yoshitada Ohi, M.D.2
1
Department of Cancer Chemotherapy, Institute
for Cancer Research, Kagoshima University, Kagoshima, Japan.
2
Department of Urology, Kagoshima University,
Kagoshima, Japan.
3
First Department of Surgery, Kagoshima University, Kagoshima, Japan.
4
Department of Public Health, Kagoshima University, Kagoshima, Japan.
BACKGROUND. Thymidine phosphorylase (TP), which is identical to platelet-derived endothelial cell growth factor (PD-ECGF), stimulates chemotaxis of endothelial cells and is involved in the angiogenesis of human solid tumors.
METHODS. The activity and expression of TP were examined in human transitional
cell carcinomas (TCCs) of the bladder, and their association with clinicopathologic
findings was determined. The activity of the enzyme in 37 TCCs and 12 adjacent
nonneoplastic tissues was measured spectrophotometrically. The expression of TP
was also examined by immunoblotting. Immunohistochemical analysis was performed on 108 TCCs.
RESULTS. TP activity in the carcinomas was higher than that in adjacent normal
tissues (P ⫽ 0.002). TP activity in Grade 3 tumors or those classified as pT2– 4 was
higher than in Grade 1 and 2 tumors (P ⫽ 0.017) or those classified as pT1 (P ⫽
0.007). The level of expression of TP detected by immunoblotting correlated well
with TP activity. Immunohistochemical analyses showed that 62 of 108 cases
(57.4%) were TP positive. There was a significant correlation between TP expression and histologic grade, infiltration pattern, local invasion, and lymph node
metastasis. TP expression as a prognostic variable was studied using the Cox
proportional hazards model. TP overexpression was an independent prognostic
factor, as were lymph node metastasis and local invasion.
CONCLUSIONS. These findings suggest that TP activity and its level of expression
influence the progression of TCC and the prognoses of patients with this disease.
Cancer 2000;88:1131– 8. © 2000 American Cancer Society.
KEYWORDS: thymidine phosphorylase, transitional cell carcinoma, prognostic factor, angiogenesis.
Supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, and the
Ministry of Health and Welfare, Japan, and a Research Grant from the Princess Takamatsu Cancer
Research Fund.
The authors thank Ms. Y. Makiyama for technical
assistance, and Drs. K. Shimoinaba (Shimoinaba
Urological Clinic) and M. Ikoma (Ikoma Urological
Clinic) for providing clinical samples.
Address for reprints: Shin-ichi Akiyama, M.D., Department of Cancer Chemotherapy, Institute for
Cancer Research, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
Received April 26, 1999; revision received October
18, 1999; accepted November 22, 1999.
© 2000 American Cancer Society
ngiogenesis is a prerequisite for tumor growth,1 and angiogenic
activity is correlated with a higher incidence of lymph node metastases and poor prognosis for patients with numerous different
types of tumors, including bladder carcinoma.2,3
Thymidine phosphorylase (TP) is involved in pyrimidine nucleoside metabolism, catalyzing the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective bases and
2-deoxyribose-1-phosphate.4 – 6 In mammals, the enzyme consists of 2
identical subunits, each with a molecular weight of about 55kD.7 We
and others previously demonstrated that TP is identical to plateletderived endothelial cell growth factor (PD-ECGF).8 –11 PD-ECGF/TP
has angiogenic activity for which the enzymatic activity of TP is
needed. Among the products of degradation of thymidine by TP,
2-deoxy-D-ribose, a dephosphorylated product derived from 2-deoxy-D-ribose-1-phosphate, has chemotactic activity in vitro and an-
A
1132
CANCER March 1, 2000 / Volume 88 / Number 5
giogenic activity in vivo.12 This suggests that TP phosphorolysis product(s) may stimulate chemotaxis of
endothelial cells and possibly other cells, causing angiogenesis.
Compared with adjacent normal tissues, TP activity has been reported to be increased in a variety of
malignant tumors.13–16 Increased TP expression correlates with microvessel density in several solid turmors,
such as breast carcinomas,17 ovarian carcinomas,15
renal cell carcinomas (RCCs),18 and colorectal carcinomas.19 In RCCs and colorectal carcinomas, TP positivity, but not microvessel density, is an independent
prognostic factor.18,19 These findings suggest that TP is
involved in angiogenesis and the progression of some
solid tumors, and that TP has effects on processes
other than just angiogenesis.
O’Brien et al. reported that tumor cell TP expression in bladder carcinomas correlated with tumor
grade but not with tumor vascularity, relapse free survival, or overall survival.20 On the other hand, Mizutani et al. reported that patients with Ta bladder carcinoma with a low level of PD-ECGF/TP expression
had a longer postoperative tumor free period than
those with a high level of expression during a 3-year
follow-up study.21 Thus, we examined retrospectively
the expression of TP in transitional cell carcinoma
(TCC) and its association with clinicopathologic findings and prognosis.
MATERIALS AND METHODS
Patients
We examined 108 patients with TCC of the bladder (84
males and 24 females) treated by radical cystectomy
from May 1984 to June 1996 at the Department of
Urology, Kagoshima University Hospital, Kagoshima,
Japan. All experiments were performed after obtaining
informed consent from patients according to institutional rules. The average age of patients at the time of
surgery was 64.5 years (range, 35–79). Follow-up of the
patients, including survival analysis, was updated in
March 1997 (the median follow-up was 51 months
[range, 2–151]). The immunohistochemical evaluations were completed in March 1997. At that time, 27
patients had died of TCC, 54 patients were alive, and
27 patients had died of other diseases. None of the
patients had received prior chemotherapy or irradiation. Twelve patients were treated by transurethral
resection before radical cystectomy.
Tumors were graded according to the criteria of
the Japanese Urological Association22 and the TNM
system.23 Histologic grades were classified into two
groups, Grades 1 ⫹ 2 and Grade 3, and a tumor was
given the predominant grade if it had mixed grades.
Infiltration patterns were classified into two groups,
INF ␣ for expanded growth and INF ␤⫹␥ for infiltration with cell clusters and single cells or microclusters.
Venous involvement was classified as pV0 for no venous invasion and pV1 for the presence of venous
invasion. Lymph duct involvement was classified into
two groups, pL0 for no lymph duct invasion and
pL1⫹2 for the presence of lymph duct invasion up to
the superficial muscle layer and beyond it. Local invasion was also classified into two groups, pT1 and
pT2– 4. Lymph node metastases were classified into
two groups, pN0 and pN1–3. The pN category was
determined by intraoperative and preoperative findings from computed tomography scans, ultrasonography, and other radiographic studies. The M category
was based on the results of chest X-rays, skeletal surveys, bone scans, computed tomography, and other
radiographic studies. Patient and tumor profiles are
summarized in Table 1.
Preparation of Monoclonal Antibody against TP
Monoclonal antibodies were raised against a glutathione S-transferase (GST) TP fusion product containing
244 amino acids of TP (residues 7–250), as previously
described.16
Tissue Specimens and Preparation of Homogenates
TCC samples (10 renal pelvic and/or ureter tumors
consisting of 2 pT1G2, 2 pT2G3, 5 pT3G2, and 2
pT3G3, and 27 bladder tumors) were obtained at surgery from 37 patients. Twelve noncancerous urothelia
(8 renal pelvic urothelia from the patients with RCC
and 4 bladder urothelia from the patients with benign
prostatic hypertrophy) were obtained at surgery. Specimens were frozen at ⫺80 °C within 10 minutes and
then homogenized in lysis buffer (50mM Tris-HCl, pH
6.8, containing 1% Triton X-100, 2 mM PMSF, and
0.02% 2-mercaptoethanol). The lysates were centrifuged at 15,000 x g for 30 minutes at 4 °C. The activities
of TP in the supernatants were assayed, and the supernatants were also used for immunoblot analyses.
Protein content was determined by the method of
Bradford.24
Assay for TP Activity
TP activity was assayed by the spectrophotometric
procedure described by Friedkin and Roberts with
some modifications.4
Supernatants from homogenates, containing 100
␮g protein, were incubated with 0.1 M Tris-arsenate
buffer (pH 6.5) containing 10 mM thymidine in a total
volume of 0.1 mL. After incubation for 1 hour at 37 °C,
the reaction was stopped by adding 1 mL of 0.2 N
NaOH, and the amount of thymine formed was determined by measuring the absorbance at 300 nm. TP
Thymidine Phosphorylase and TCC/Arima et al.
TABLE 1
Patient and Tumor Profiles
1133
Immunohistochemistry
Characteristics
No. of patients (%)
Total no.
Gender
Male
Female
Age (yrs)
Median
Range
Grade
G1
G2
G3
Infiltration pattern
INF␣
INF␤
INF␥
Venous involvement
pV0
pV1
Lymph duct involvement
pL0
pL1⫹2
pT category
pT1
pT2
pT3
pT4
pN category
pN0
ⱖ pN1
108 (100)
84 (77.8)
24 (22.2)
61
35–79
5 (4.6)
46 (42.6)
57 (52.8)
20 (18.5)
67 (62.0)
21 (19.5)
76 (70.4)
32 (29.6)
49 (45.4)
59 (54.6)
49 (45.4)
15 (13.9)
23 (21.3)
21 (19.4)
91 (84.3)
17 (15.7)
INF: infiltration.
activity is expressed as the amount of thymine formed
per ␮g protein per hour.
Immunoblotting
Samples containing 50 ␮g protein were resolved by
11% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins in the gel
were electrophoretically transferred to a sheet of PVDF
as previously described.25 After transfer, the membrane was placed in TTBS (400 mM NaCl, 20 mM
Tris-HCl, pH 8.0, 0.05% Tween-20) containing 5.0%
(weight/vol) skimmed milk for 1 hour. The blots were
incubated overnight with TTBS containing 5%
skimmed milk and a monoclonal antibody against TP
(1:1000 dilution). After washing 4 times in TTBS for 10
minutes each, the sheet was incubated for 1 hour with
horseradish-peroxidase (HRP)– conjugated horse antimouse immunoglobulin (Ig)G (Amersham, Buckinghamshire, UK) diluted 1:1000 in TTBS. After 3 washes
in TTBS for 10 minutes each, the HRP activities were
detected with the ECL Western blot detection system
(Amersham).
TP was investigated immunohistochemically using a
monoclonal antibody against TP and an avidin-biotinperoxidase complex (ABC-PO Vectastain kit, Vector
Laboratories, Inc., Burlingame, CA). Samples were
fixed with 10% formaldehyde in phosphate-buffered
saline (PBS), embedded in paraffin, and cut into
3-␮m-thick sections. The sections were deparaffinized
with xylene and treated with 98% ethanol. Endogenous peroxidase was blocked by immersing the slides
in 0.02% H2O2 in absolute methanol for 20 minutes at
room temperature. The sections were washed 3 times
with PBS for 5 minutes each, incubated with 3%
skimmed milk in PBS for 30 minutes at room temperature, and then incubated at 4 °C overnight with a
monoclonal antibody against TP diluted 400-fold with
3% skimmed milk in PBS. After washing 3 times with
PBS for 5 minutes each, they were then incubated for
30 minutes with biotinylated antimouse IgG at room
temperature. After washing 3 or more times with PBS
for 5 minutes each, the sections were incubated for 30
minutes with avidin-biotinylated horseradish peroxidase complex and then washed 3 times with PBS for 15
minutes each. The immune complexes were visualized
by incubating the sections with 0.5 mg/mL diaminobenzidine and 0.03% (vol/vol) H2O2 in PBS for 7 minutes. The sections were counterstained with hematoxylin and mounted.
We examined at least 200 carcinoma cells by light
microscopy to determine whether or not the cells were
positive for TP. The evaluation of TP expression was
done by two investigators (J.A. and Y.I.) who had no
knowledge of the clinicopathologic characteristics, using a “two-headed” light microscope.
We separated the patients into two groups, each
with about the same number of cases. As a result, a
cutoff point of 5% was selected; samples were considered to be TP positive when more than 5% of the
parenchyma cells were stained and TP negative when
less than 5% of the cells were stained.
Statistical Methods
Demographic and clinicopathologic characteristics
were compared using the chi-square test or the Student t test.26 We used the Kaplan–Meier method to
estimate survival rates. The Cox proportional hazards
models were used in survival analysis. All P values
presented are two-sided.
RESULTS
TP Activity in Tumors and Normal Urothelium
We measured the TP activity in 37 TCCs and 12 adjacent nonneoplastic tissues, including those from 4
1134
CANCER March 1, 2000 / Volume 88 / Number 5
FIGURE 1. The correlations between thymidine phosphorylase (TP) activity and (A) nonneoplastic or cancer tissues, (B) tumor grade, and (C) tumor stage, analyzed
by the unpaired Student t test, are shown. Boxes correspond to interquartile ranges. Lines in boxes represent mean values. The open circles represent the outliers.
benign urologic diseases. The average TP activity in
TCC (8.54 nmol/100 ␮g protein/hour) was 10.2-fold
higher than in normal urothelia (0.84 nmol/100 ␮g
protein/hour) (P ⫽ 0.002). The average TP activity in
G3 tumors (15.0 nmol/100 ␮g protein/hour) was 3.3fold higher than in G1–2 tumors (4.6 nmol/100 ␮g
protein/hour) (P ⫽ 0.017), and the average in pT2– 4
tumors (11.7 nmol/100 ␮g protein/hour) was 6.0-fold
higher than in T1 (2.0 nmol/100 ␮g protein/hour) (P ⫽
0.007) (Fig. 1).
Immunoblotting of TP from Tumors and Normal
Urothelium
Homogenates from 7 bladder TCCs and one noncancerous urothelium were subjected to immunoblot
analysis. A monoclonal antibody against TP was used
to examine the levels of TP expression in tissues. We
detected TP with a molecular weight of 55,000 in carcinomas and nonneoplastic tissues. The level of TP
expression in cancer tissues was higher than in nonneoplastic tissues. TP expression as well as TP activity
seemed to be correlated with tumor grade and stage.
In high grade tumors (G2, pT3 or G3, pT3), it tended to
be higher than in low grade tumors (G1, pT1 or G2,
pT1) (Fig. 2). More experiments and statistical analyses are needed to confirm these differences.
Correlations among Pathologic Findings, Clinical
Outcome, and TP Expression
The expression of TP in carcinomas was examined by
immunohistochemical staining with a monoclonal antibody against TP. The cytoplasms of TP positive carcinoma cells were as intensely stained as those of
FIGURE 2.
Expression of thymidine phosphorylase (TP) in transitional cell
carcinomas (TCCs), detected by immunoblotting with a monoclonal antibody
against TP, is shown. Lane 1, nonneoplastic tissue; Lanes 2– 8, TCCs ( 2,
G1pT1; 3, G2pT1; 4, G3pT1; 5, G2pT2; 6, G3pT2; 7, G2pT3; 8, G3pT3).
stromal cells (Fig. 3). The correlations between clinical
or pathologic features and TP expression in human
TCC are summarized in Table 2. Sixty-two (57.4%) of
108 patients were TP positive (Fig. 4). There was a
significant correlation between TP expression and histologic grade, infiltration pattern, local invasion, and
lymph node metastasis or extent of disease. No significant association was found between TP expression
and gender, venous involvement, or lymph duct invasion.
Prognostic Relevance of TP Expression
Kaplan–Meier product limit estimates of overall survival are plotted in Figure 5. Patients with TP positive
carcinomas had significantly (P ⬍ 0.001) poorer survival than those with negative tumors. Thus, we investigated whether TP positivity is an independent prognostic variable. A Cox proportional hazards model was
constructed using established prognostic factors and
TP expression. Univariate analysis showed that histologic grade (P ⬍ 0.001), infiltration pattern (P ⫽ 0.001),
local invasion (P ⬍ 0.001), venous involvement (P ⫽
Thymidine Phosphorylase and TCC/Arima et al.
1135
FIGURE 3. Immunoreactivity to a monoclonal
antibody against thymidine phosphorylase (TP)
of transitional cell carcinoma (TCC) tissue is
shown. (A) TP negative TCC, (B) TP positive TCC.
0.001), lymph duct invasion (P ⬍ 0.001), N category
(P ⬍ 0.001), and TP expression (P ⫽ 0.001) were significant prognostic factors (Table 3). Multivariate survival
analysis showed that lymph node metastasis (P ⫽ 0.019),
local invasion (P ⫽ 0.046), and TP expression (P ⫽ 0.047)
were significant prognostic factors (Table 4).
DISCUSSION
Angiogenic activity appears to be an important determinant of the behavior of bladder carcinomas because
increased angiogenesis, as assessed by vessel counts
using immunohistochemistry, is associated with
shorter overall survival for patients with invasive
TCC.2 The expression of several angiogenic factors has
been examined in bladder carcinoma. Basic fibroblast
growth factor (bFGF) was found in the urine of patients with bladder carcinoma, particularly in those
with metastatic disease.27 Immunohistochemical
studies demonstrated increased expression of acidic
FGF in bladder carcinomas compared with normal
bladders, and the strongest expression of acidic FGF
was found in high grade tumors.28
Expression of vascular endothelial growth factor
(VEGF) in superficial bladder tumors was significantly
higher than in invasive tumors and in normal bladder.29 TP expression in invasive bladder tumors was
reported to be higher than in superficial tumors and
normal bladder.29 Our study also shows that TP expression is higher in human transitional cell carcinoma than in normal urothelium, and that TP activity
TABLE 2
Relations between TP Expression in TCCs and Clinicopathologic
Characteristics
No. of patients
Gender
Male
Female
Histologic grade
G1⫹2
G3
Infiltration pattern
INF␣
INF␤ ⫹ ␥
Local invasion
pT1
pT2–4
Venous involvement
pV0
pV1
Lymph duct involvement
pL0
pL1⫹2
Lymph node metastasis
pN0
pN1–3
Disease extention
pT1–3 and pN0
pT4 or pN1–3
No. of patients
Positive
Negative
P valuea
84
24
51 (60.7%)
11 (45.8%)
33 (39.3%)
13 (54.2%)
NS
51
57
23 (45.1%)
39 (68.4%)
28 (54.9%)
18 (31.6%)
P ⫽ 0.014
20
88
7 (35.0%)
55 (62.5%)
13 (65.0%)
33 (37.5%)
P ⫽ 0.025
49
59
20 (40.8%)
42 (71.2%)
29 (59.2%)
17 (28.8%)
P ⫽ 0.002
76
32
40 (52.6%)
22 (68.8%)
36 (47.4%)
10 (31.2%)
NS
49
59
25 (51.0%)
37 (62.7%)
24 (49.0%)
22 (37.3%)
NS
91
17
47 (51.6%)
15 (88.2%)
44 (48.4%)
2 (11.8%)
P ⫽ 0.003
87
21
44 (50.6%)
18 (85.7%)
43 (49.4%)
3 (14.3%)
P ⫽ 0.002
INF: infiltration; NS: not significant.
a
P values were obtained by the chi-square test.
1136
CANCER March 1, 2000 / Volume 88 / Number 5
TABLE 3
Results of Univariate Survival Analysis
FIGURE 4. Percentages of carcinoma cells obtained from 108 patients with
transitional cell carcinoma (TCC) expressing thymidine phosphorylase (TP) are
shown.
Gender
Male
Female
Histologic grade
G1⫹2
G3
Infiltration pattern
INF␣
INF␤ ⫹ ␥
Local invasion
pT1
pT2–4
Venous involvement
pV0
pV1
Lymph duct invasion
pL0
pL1–2
Lymph node metastasis
pN0
pN1–3
TP expression
Negative
Positive
No. of patients
HRa
95% CIa
P valuea
84
24
0.91
Reference
0.39–2.15
0.845
51
57
Reference
7.46
2.25–24.72
20
88
Reference
9.76
1.32–71.77
49
59
Reference
7.54
2.63–21.68
76
32
Reference
3.46
1.68–7.14
49
59
Reference
6.65
2.53–17.44
91
17
Reference
7.82
3.59–17.02
46
62
Reference
4.95
1.89–12.98
⬍ 0.001
0.001
⬍ 0.001
0.001
⬍ 0.001
⬍ 0.001
0.001
INF: infiltration; HR: hazard ratio; CI: confidence interval.
a
Hazard ratios and 95% confidence intervals were obtained from the Cox proportional hazards model.
b
P values were obtained by the chi-square test.
TABLE 4
Results of Multivariate Survival Analysis
FIGURE 5. Kaplan–Meier survival curves of patients with transitional cell
carcinoma are shown. TP: thymidine phosphorylase.
in invasive bladder carcinoma classified as pT2– 4 is
significantly higher than in superficial bladder carcinoma classified as pT1. TP expression has also been
associated with the extent of invasion in colorectal
carcinoma,19 squamous cell carcinoma of the esophagus,30,31 and extrapancreatic neural plexus invasion
in ductal adenocarcinoma of the pancreas.32 These
findings suggest that TP plays an important role in
invasion of various kinds of tumor cells as well as TCC
cells. TP expression was also correlated with histologic
grade, infiltration pattern, local invasion, and lymph
node metastasis in this study, suggesting that TP expression has an important role in tumor growth and
metastasis in bladder carcinoma.
Tumor vascularity is an important indicator of a
poor prognosis in patients with invasive TCC,2 and TP
positivity was a predictor of poor prognosis in patients
with TCC in our study. Mizutani et al. reported that
Histologic grade
Local invasion
Lymph node metastasis
TP expression
HRa
95% CIa
P valueb
2.33
3.28
2.76
2.68
0.54–9.86
0.92–11.67
1.20–6.33
0.94–7.59
0.230
0.046
0.019
0.047
HR: hazard ratio; CI: confidence interval; TP: thymidine phosphorylase.
a
Hazard ratios and 95% confidence intervals were obtained from the Cox proportional hazards model,
adjusted for variables shown.
b
P values were obtained by the chi-square test.
elevated PD-ECGF/TP expression predicted early recurrence of Ta bladder carcinoma.21 On the other
hand, TP expression in bladder carcinomas treated by
transurethral resection with or without radiotherapy
was reported to have no prognostic value.20 The difference between these findings may be attributed to
the different antibodies used, differences in the tumors and the treatments (i.e., radical cystectomy vs.
transurethral resection with or without radiotherapy),
and/or differences in the cutoff values used to deter-
Thymidine Phosphorylase and TCC/Arima et al.
mine TP positivity. PD-ECGF/TP expression in bladder carcinomas was previously reported not to be
correlated with tumor vascularity.20 However, this
does not necessarily mean that PD-ECGF/TP is not
correlated with prognosis. In RCC and colorectal carcinoma, TP positivity, but not microvessel density, is
an independent prognostic factor.18,19 Recently, we
proposed that TP has some roles, other than in angiogenesis, in the progression of solid tumors.19 In an
experimental in vitro system, TP conferred resistance
to apoptosis induced by hypoxia, and the degradation
products of thymidine were involved in the resistance.33 This effect of TP may confer invasive and
metastatic properties to tumor cells. Expression of
PD-ECGF/TP may play an important role in the progression of invasive TCC; and inhibitors of PD-ECGF/
TP, such as 5-chloro-6-[1-C2-iminopyrrolidinyl) methyl]
uracil hydrochloride (TPI),34,35 may suppress the
growth and metastasis of the tumors.
13.
14.
15.
16.
17.
18.
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