Cyclin A Overexpression in Carcinoma of the Renal Pelvis and … · cyclin A to in vito conditions...
Transcript of Cyclin A Overexpression in Carcinoma of the Renal Pelvis and … · cyclin A to in vito conditions...
Vol. 3, 1399-1404, August 1997 Clinical Cancer Research 1399
3 The abbreviation used is: TCC, transitional cell carcinoma.
Cyclin A Overexpression in Carcinoma of the Renal Pelvis and
Ureter including Dysplasia: Immunohistochemical Findings in
Relation to Prognosis’
Mutsuo Furihata,2 Yuji Ohtsuki, Hiroshi Sonobe,
Taro Shuin, Akihiro Yamamoto, Naotami Terao,
and Morimasa KuwaharaDepartments of Pathology II [M. F., Y. 0., H. 5.1 and Urology IT. S.].
Kochi Medical School, Nankoku, Kochi 783: Division of Urology,Kochi Takasu Hospital, Kochi 780 [A. Y., N. TI: and Division of
Urology, Fujisaki Hospital, Karatsu, Saga 847 [M. K.l, Japan
ABSTRACTSeveral in vitro studies have shown that cyclin A gene
alteration in the cell cycle plays an important role in carci-nogenesis. We immunohistochemically examined the expres-
sion of cyclin A protein in 120 patients with transitional cell
carcinoma (TCC) of the renal pelvis and ureter, including
adjacent dysplastic lesions to determine their significancefor the tumor behavior and patient prognosis. Cyclin Aimmunostaining of the nucleus was observed in 29 tumors
(24.2%). Furthermore, 17 cyclin A-positive tumors (58.6%)had dysplastic lesions positive for cyclin A antibody. Theprevalence of cases exhibiting cyclin A staining was higherin the high grade (P < 0.01) and invasive tumors (P < 0.05)
than in the other types of tumors. In the selected 117 cases,patients whose TCCs expressed a high level of cyclin Aprotein had a significantly poorer prognosis than those with-
out cyclin A expression (P < 0.01). These in vivo findings
provide the first evidence for frequent and redundant cyclin
A protein overexpression in TCC and suggest that cyclin Aoverexpression is related to the tumor behavior and patient
prognosis. In addition, our observations indicate that over-
expression of cyclin A may be one of the early events, at least
in some cases, in the carcinogenesis of TCC.
INTRODUCTIONDetermination of the sequences of molecular events lead-
ing to and following deregulation of the cell cycle control is
crucial to our understanding of the development and progression
of malignant tumors. Human cyclins, such as A type, B type,
and 01 (C, D, and E) type cyclins, are prime cell cycle-specific
Received 1 1/5/96; revised 4/22/97; accepted 4/24/97.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported in part by Grants-in Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan.2 To whom requests for reprints should be addressed. Phone: 0888-66-5811: Fax: 0888-80-2336.
regulators ( 1 ), and they play an important role in the control of
major checkpoints of the cell cycle (2, 3). With the discovery of
inappropriate expression of cyclins in some tumors, it has now
been specifically hypothesized that some cyclins are intimately
involved in oncogenesis, acting as proto-oncogenes (2, 3). Di-
verse patterns of redundant expression of particular cyclins,
such as cyclins Dl and E, in different tumor cell lines have
previously been reported in oncogenesis (4-14). A relationship
of cyclin A abnormalities to carcinogenesis has also been no-
ticed. Human cyclin A gene at a site of hepatitis B virus DNA
integration was identified in a hepatocellular carcinoma in as-
sociation with abnormality of the ct-c/in A gene ( I 5, 16). Cyclin
A also associates with adenovirus E1A oncoprotein ( I 7, 18) and
has been identified in complexes containing the E2F transcrip-
tion factor and p107, which is structurally related to the retino-
blastoma protein (1 8 -22). Although little is known concerning
the significant role of cyclin A in tumorigenesis, these studies
suggest that cyclin A may be of value in identifying proliferative
tumor cells, and its alteration may be an important step in the
pathogenesis of certain cell lines and primary tumors.
There is little data available concerning whether premalig-
nant states, including dysplasia, in the transitional epithelium
progress to TCC.3 However, most reports have indicated that
dysplasia acts as a common precursor to TCC because it shows
a high propensity to progress to carcinoma in situ (23, 24). In
addition, the presence of dysplasia adjacent to TCC is associated
with an increased risk of subsequent recurrence and progression.
(25). Therefore, the features of dysplasia provide ideal systems
to study the proposed multiple-step nature of carcinogenesis.
Molecular and histological studies of preinvasive urothelial be-
sions have frequently demonstrated genomic and pathological
alterations in carcinoma in situ and dysplastic lesions, suggest-
ing that at least some of these abnormalities occur very early in
oncogenesis (26, 27). Recently, increased expression of cyclin
Dl protein was reported in precancerous lesions of coborectal
mucosa (28). These findings prompted us to study the level of
expression of cyclin A protein at different stages along the
multistage process of urothelial carcinogenesis from normal
mucosa to dysplasia and carcinoma.
In the present study, we extended these observations of
cyclin A to in vito conditions in human TCC to elucidate the
potential role of the expression of cyclin A in tumor develop-
ment and to assess their prognostic value, also examining the
dysplastic epithelium adjacent to the tumor. The relationship
with various clinicopathobogical factors was then analyzed.
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1400 Cyclin A in Transitional Cell Carcinoma
MATERIALS AND METHODS
Patients and Tumor Samples. One-hundred twenty
cases of human TCCs of the renal pelvis and ureter, which were
all TCC cases obtained by radical nephroureterectomy between
October 198 1 and January 1997 at the Department of Urology of
Kochi Medical School, the Division of Urology of both Kochi
Takasu Hospital and Fujisaki Hospital, were retrospectively
studied using immunohistochemistry. Tumors were processed in
a similar fashion at all three institutions. Histological or clinical
classification of tumors was performed according to the “Gen-
eral Rules for Clinical and Pathological Studies on Renal Pelvic
and Ureteral Cancer ( 1990)” (29). Tumor specimens were fixed
in 10% buffered formalin, processed routinely, and embedded in
paraffin. In each case, all of the available H&E-stained sections
were reviewed, and a representative block was chosen for fur-
ther studies. There were 67 renal pelvic cancers and 53 ureteral
cancers, including 10 cases with renal pelvic and ureteral can-
cers. Of I 20 cases with TCCs, 2 1 have bladder cancer concur-
rent with renal pelvic or ureteral cancer. The patients included
84 men and 36 women, ages 45-93 years (median age, 71
years).
Immunohistochemistry with Cyclin A Antibody. Five
jim-thick sections from archival formalin-fixed, paraffin-em-
bedded tissue were placed on poly-L-lysine-coated slides (Sigma
Chemical Co., St. Louis, MO) for immunohistochemistry. Cy-
din A protein expression was assessed by immunohistochemi-
cab examination (streptavidin-biotin complex procedure) with a
monocbonal antibody, Cyclin A (NCL-CYCLIN A, 6E6, IgGI;
dilution, 1 :50; Novocastra Laboratory, Inc., Newcastle upon
Tyne, United Kingdom).
Deparaffinized tissue sections were placed in 10 msi citrate
buffer (pH 6.0) and heated to 132 #{176}Cin an autoclave for 20 mm
for antigen retrieval, as shown in our previous study (27). After
blockage of endogenous peroxidase activity with methanol con-
taming 0.3% H,O, for 30 mm, the sections were incubated at 4
#{176}Covernight with the monoclonal antibody to cyclin A protein.
After washing with 0. 1 M PBS (pH 7.4), the streptavidin-biotin
complex (ABC) procedure was performed by using a streptavi-
din biotin complex peroxidase kit (DAKO LSAB kit; Dakopatts,
Kyoto, Japan) and by following the directions in the kit manual.
Finally, slides were counterstained with methyl green. Positive
or negative controls included in each experiment were run in
parallel; these included replacement of the specific or nonspe-
cific mouse IgG 1 antibodies with PBS. The experiment was
repeated, yielding essentially identical patterns of cyclin A
distribution in each instance in each tumor specimen.
Tumor sections exhibiting definite staining of tumor cell
nuclei with cyclin A antibody were scored as cyclin A positive.
A visual assessment was made in such cases of the number of
positive tumor cells as a proportion of the total expression of
cyclin A protein as follows: negative cases (0%, or variable
weak positivity in tumor cells: 0-10%); positive cases consisted
of two types, one with heterogeneous (variable positivity in
tumor cells: 10-60%) and the other with homogeneous (diffuse
strong positivity in tumor cells: >60%) immunostaining.
Statistical Analysis. The correlations between the cx-
pression of cyclin A protein and the various clinicopathological
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Fig. I Positive staining patterns of anti-cyclin A antibody in human
TCC showing invasive growth (TCC, grade 3). Note the strong staining
confined to the nuclei of cancer cells, some revealing negative staining(arrotv/ieads). Arrows, mitotic figures were frequently observed. X250.
factors considered were determined using the �2 test at the 5%
level.
Association between Cyclin A Expression and Progno-
sis. Survival was calculated from operation to the date of death
or the date of the last follow-up (either a clinical visit or a
discussion with the patient’s referring physician). Of all 120
patients selected, excluded from the follow-up studies were 3
patients (2.5%), who did not have at least 6 months of follow-up
or who did not survive at least 6 months after operation. In the
remaining I 17 patients, all were clinically followed up more
than 6 months, and there were no patients with inadequate
follow-up. Median follow-up was 3.4 years (range, 0.5-10.5).
At last follow-up, 66.2% of the patients were alive.
Analyses of survival data were performed using survival
curves and the Kaplan-Meier method and bog rank test. In
addition, the Cox proportional hazards model was used to cal-
culate and estimate the postoperative survival rate and to deter-
mine the significance of each prognostic factor used in histo-
logical or clinical classification. For multivariate analysis,
variables were selected on condition that they were statistically
significant and were only poorly correlated with each other.
RESULTS
Immunohistochemistry with Cyclin A Antibody. Cy-
din A antibody immunoreactivity was homogeneous in 16.7%
(20 of 120) of the TCCs. A certain degree of heterogeneous
staining in the neoplastic cell population of immunoreactive
cases was observed in 7.5% (9 of 1 20) tumors. Staining was
predominantly observed in the nucleus (Fig. I ). Mitotic figures
were found in cyclin A-positive tumors (Fig. I , arrows). Of the
tumors regarded as negative, 14 showed very weak traces of
nuclear staining. In the present study, dysplastic lesions adjacent
to carcinomas were observed in 101 cases (101 of 120; 84.7%),
including cyclin A-positive 29 tumors. Furthermore, cyclin A
expression was frequently detected in dysplastic lesions adja-
cent to 17 cyclin A-positive TCCs (17 of 29; 58.6%; Fig. 2,
A-C). In the remaining 72 cyclin A-negative tumors with dys-
plasia, negative immunoreaction for cyclin A was also found in
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Clinical Cancer Research 1401
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Fig. 2 A. positive nuclear staining with anti-cyclin A antibody was
detected in cancer cells (TCC. grade 2 > 1 : arrow) as well as dysplastic
cells (arrowhead: X 100). B and C, high magnification of both cancer
(B) and dysplasia (C). X250. D, cyclin A immunoreactivity was re-
stricted to the cells of the basal layer in the transitional epithelium of theurinary tract. X250.
dysplastic lesions. In the normal tissues of the urinary tract,
cyclin A immunorcactivity was restricted to the subsets of cells
of the basal layer in the transitional epithelium (Fig. 2D) and
some invading lymphocytes and histiocytes. These observations
of reasonable expression of cyclin A provided additional sup-
porting evidence for the specific detection of cyclin A with its
antibody.
Statistical Analysis. Table 1 shows the relationships be-
tween the rates of detection of overexpressed cyclin A in pri-
mary tumors and clinical or pathological features. The relation-
ships of the cyclin A-positive cases with high nuclear grade
(P < 0.01) and also with an invasive tumor growth pattern (P <
0.05) were both statistically significant. In contrast, no signifi-
cant correlation was detected between cyclin A overexpression
and the other clinical and pathological parameters, such as sex,
Table I Summary of the relationship between the cyclin A
immunoreactivity and clinicopathobogical factors
Cyclin Cyclin
A (91 A� (29cases) cases)
Age
<70 46 12
70-80 34 13
>80 II 4
SexMale 66 18
Female 25 1 1
Grade”1:1>2 14 2
2:2>3orl 48 9
3:3>2 29 18 P<0.0l
pT”
is,a 21 2
1 22 2
2 12 8
3 29 10
4 6 7
by or v”
(-) 66 18(+) 25 11
m on n”
(-) 85 20(+) 10 9
Growth”Non-invasive 21 4
NNT 4 2
PNT 17 2Invasive 70 25
NIT 21 12 P<0.05
PIT 49 13
(-) 39 10
(+) 52 19
“ Subjects followed by the method of the Japanese Unobogicab
Association and the Society of Pathology (29). Grade, tumor grade: pT,
depth of penetration: ly or v, lymphatic or venous invasion: m or n,
distant organ or lymph node metastasis: growth: tumor growth pattern:
NNT, nonpapillary noninvasive type: PNT, papillary noninvasive type:
NIT, nonpapilbary invasive type: PIT, papillary invasive type.
I, Chemo., chemotherapy.
age, depth of penetration, lymphatic or venous invasion, distant
organ or lymph node metastasis, and treatment with chemother-
apy.
Association between Cyclin A Expression and Progno-
sis. Fig. 3 shows the Kaplan-Meier survival curves based on a
comparison of the two groups; one group consists of the patients
with positive immunostaining with cyclin A and the other with
negative immunostaining with cyclin A. In all I 17 patients
tested, the postoperative 10-year survival rate of the cyclin
A-positive group (n = 28) was 26.5%, whereas that of the cyclin
A-negative group (n = 89) was 62.4%. There was a significant
difference between these two groups (P < 0.01 ; Fig. 3A). Of the
72 patients with low grade TCC (grade 3>, 3 < 2, or 1), the
postoperative 9-year survival rate of the cyclin A-positive group
(n I I) was 35.4%, whereas that of the cyclin A-negative
group (n = 61) was 70.2%; the difference between these rates
was statistically significant (P < 0.05: Fig. 3B). Of the 45
patients with high grade TCC (grade 3 > 2, or I), the postop-
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. cyckn A-negative (n -89)
100
90
80
70
�4oQ. 30
YEARS AFTER NEPHROURETERECT0My
(n -17)
I cyclin A-n.g.t.v. (n - 21)
0 cychn A-pos.t.v. (n -4)
(� . Psi0i�nts with hich cir�de TCC (n = 451
i i i 4 5 a � 8 9 10
YEAJ� AFTER NEPHRouau-rEI�cToMY
El � Patianta with non- invasiva tvna nf TC(� (n =
1 2 3 4 5 6 iYEARS AFTER NEPHROURETERECTOMV
9 10
> a cyckn A-negative (n -68)
YEARS AFTER NEPHROURETERECTOMV
10
1402 Cyclin A in Transitional Cell Carcinoma
100
90
80
70
�60
� so
�4o
20
10
0
20
10
A-positiv.(n-
B : Patients with low arade TCC (n = 721
i 2 3 4 5 6 7 8 9 i�o
YEA� AFTER NEPHROURETERECTOMy
100 1 �
90
80
70
�6o�5o
20
10
Fig. 3 The cumulative Kaplan-Meier survival curves of patients with carcinoma of renal pelvis and ureter. There are statistically significant
differences between the cyclin A-positive and cyclin A-negative group in all 117 patients tested (A, P < 0.01) and in patients with low grade TCC(B, P < 0.05) and high grade TCC (C, P < 0.05). Although no significant difference in the survival rate for the patients with noninvasive type ofTCC is detected (D), there is a statistically significant difference between the cyclin A-positive and cyclin A-negative group for the patients with the
invasive type of ICC (E. P < 0.05).
erative 9-year survival rate of the cyclin A-positive group (n =
17) was 13.8%, whereas that of the cyclin A-negative group
(n 28) was 48.2%; the difference between these rates was also
statistically significant (P < 0.05; Fig. 3C). In the tumor growth
pattern, there was no significant difference in the survival rate
for the 25 patients with the noninvasive type of TCC between
cyclin A-positive (n = 4) and cyclin A-negative groups (n = 21;
Fig. 3D). In contrast, the postoperative 10-year survival rates for
the 92 patients with the invasive type of TCC was 18.6% for the
cyclin A-positive group (n = 24) and 50.3% for the cyclin
A-negative group (n 68), respectively. There was a significant
difference between these groups (P < 0.05; Fig. 3E).
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Clinical Cancer Research 1403
To determine the most informative combination of mdc-
pendent factors for prognosis, the variables identified as statis-
tically significant in predicting survival (tumor grading, depth of
penetration, lymphatic or venous invasion, distant organ or
lymph node metastasis, tumor growth pattern, and cyclin A
overexpression) were subjected to a multivariate analysis using
Cox’s stepwise proportional hazard model. Each of these factors
had a statistically significant effect on the survival rate. A
stepwise selection of these factors was made, based on the
relative magnitude of their contribution to survival. This anal-
ysis demonstrated that the most important factor affecting sun-
vival was the distant organ and/or lymph node metastasis (P <
0.001). Moreover, cyclin A overexpression was also an impor-
tant factor affecting survival (P < 0.0 1) as well as tumor
grading (P < 0.05) and the tumor growth pattern (P < 0.05).
DISCUSSION
In this study, we directly examined the relevance of cyclin
derangement to in vivo conditions by immunohistochemically
measuring and comparing the expression of cyclin A protein in
tumor samples from patients with TCC. Immunohistochemistry
using antibodies to cyclin A is optimally designed for precise
measurement of the expression rates of this protein and its
pattern of expression in an individual tumor cell, and this
technique may be suitable for screening. In the present study,
24.2% (29 of 120) of the tumor samples had increased levels of
cyclin A protein predominantly detected in the nuclei of cancer
cells. Recently, Keyomarsi and Pardec (6) also showed the
unscheduled patterns and timing of overexpression of cyclin A
through G1 to G2-M phase in human breast cancer cell lines (6).
Our present data and their findings suggest that, in the synchro-
nized populations of cyclin A-positive tumor cells observed,
cyclin A protein is no longer a cell cycle regulator; instead, it
appears to be constitutively regulated in the cell cycle. However,
in these cyclin A-positive tumors, mitotic figures were com-
monly observed, and the intensity of positive nuclear staining
varied between tumor cells within the identical tumor. These
observations support the hypothesis that cyclin A protein is not
constantly expressed and may be degraded or expelled from the
nucleus during progression of the cell cycle.
Urothelial ICC usually exhibits significantly different be-
havior, depending on the nuclear grade of the cancer cells and
the patterns of growth. The present study showed that tumors
with cyclin A protein overexpression became progressively
more abundant with an increase in the degree of tumor grade
(P < 0.01) and tumor invasion (P < 0.05). Thus, our results
suggest that cyclin A overexpression may be associated with the
clinical behavior of high grade and progressive TCCs. In the
present study, our findings also indicated that the groups with a
high level of cyclin A expression was associated with a signif-
icantly poorer prognosis than the groups without its expression.
These in vivo findings suggest that cyclin A overexpression is
also related to the poor prognosis for the patients with renal
pelvic and ureteral TCCs.
We also investigated the expression of cyclin A in
samples containing normal and dysplastic lesions adjacent to
the primary tumors. One important finding in this study was
that approximately 58% (17 of 29) of the dysplastic lesions
adjacent to cyclin A-positive tumors eventually featured cy-
din A overexpression. It is also of interest to note that
physiological levels of cyclin A protein was detected in the
subsets of cells of the basal layer in the nonneoplastic tnan-
sitional epithelium. This observation supports the importance
of abnormal levels of expression of this protein in urothebiab
dysplasia. In the present study, these findings suggest that
unscheduled overexpression ofcycbin A may be due to loss of
the cell cycle regulation at an early stage of the pathogenesis
of some cases of urinary neoplasms. In addition, detection of
cyclin A overexpression in premalignant lesions of the un-
nary tract may potentially be useful in diagnosis and deter-
mination of their roles in the natural history of ICC.
Although the molecular basis for the positive immuno-
staining is still indefinite, this is the first report of frequent
cyclin A protein overexpression in a large series of primary
human TCCs including dysplasia. In addition, the present study
also demonstrated that immunohistochemical detection of cyclin
A protein is useful in the search for novel and potentially useful
prognostic markers in ICC also. Recent reports have further
demonstrated the presence of a link between oncogenesis and
the cell cycle by correlating the deranged expression of cyclins
to the loss of growth control (4, 6, 14). Theretbre, the frequent
alterations of cyclin A protein in tumor cells are also suggestive
of its role as an oncogene. Up to the present time, most inves-
tigations into the relationship between the expression of cyclin
and carcinogenesis were mainly carried out in vitro using certain
cell lines. Further comprehensive studies using excised human
carcinoma tissue samples from greater numbers of human tu-
mors and including measurement of DNA and/or RNA levels
will be needed to elucidate the function and clinical significance
of cyclin A overexpression.
ACKNOWLEDGMENTS
We are grateful to Dr. T. Moniki for providing clinical materials.
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