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



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-



. 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


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).



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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