Matrix Metalloproteinase-9 Expression in Bladder Washes ...clincancerres.aacrjournals.org/content/clincanres/4/12/3011.full.pdf · Vol. 4, 3011-3016, December 1998 Clinical Cancer

Vol. 4, 3011-3016, December 1998 Clinical Cancer Research 3011

3 The abbreviations used are: CIS, carcinoma in situ: MMP, matrixmetalloproteinase: TIMP, tissue inhibitor of metalloproteinase.

Matrix Metalloproteinase-9 Expression in Bladder Washes from

Bladder Cancer Patients Predicts Pathological Stage

and Grade’

Fernando J. Bianco, Jr., David C. Gervasi,

Rabi Tiguert, David J. Grignon, J. Edson Pontes,

John D. Crissman, Rafael Fridman, and

David P. Wood, Jr.2The Karmanos Cancer Institute and Departments of Urology [F. J. B.,

R. T., J. E. P., D. P. W.] and Pathology [D. C. G., D. J. G., J. D. C.,R. F.], Wayne State UniversityiThe Detroit Medical Center, Detroit,

Michigan 48201

ABSTRACT

The matrix metalloproteinases (MMPs), in particular

the gelatinases (MMP-2 and MMP-9) have been associated

with tumor cell invasion and metastasis in many human

cancers. Here we examined the expression of proMMP-2

(gelatinase A) and proMMP-9 (gelatinase B) proteins in the

cellular component of bladder washes obtained from 65

patients. Twenty-six patients had active bladder cancer, 24had a history of bladder cancer but no evidence of active

disease at the time of cystoscopy (recurrence-free), and 15

patients had lesions other than bladder cancer (controls).

The results were correlated with the cytological findings ofthe bladder wash and the histopathological results of the

tumor resection when performed. In patients with active

transitional cell carcinoma of the bladder, 71 and 38% had

expression and overexpression of the latent form of MMP-9

(proMMP-9), respectively. In contrast, neither latent nor

active MMP-2 could be detected in any of the samples

examined, regardless of tumor status. Overexpression of

proMMP-9 correlated with higher grade (P 0.003) and

pathological stage (P = 0.04) of disease in the active bladder

cancer group. No significant gelatinase expression was de-

tected in the recurrence-free and control cases. Compared

with urine cytology, proMMP-9 expression had an overallhigher sensitivity for bladder cancer identification (71 versus

54%, P = 0.11). Detection of proMMP-9 in bladder washesmay be a novel approach for the identification of patients

with more aggressive forms of bladder cancer.

Received 6/5/98; revised 9/1 1/98; accepted 9/21/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with I 8 U.S.C. Section 1734 solely toindicate this fact.

C Supported by a Virtual Discovery Grant from the Karmanos Cancer

Institute (to R. F., D. J. G., and D. P. W.).2 To whom requests for reprints should be addressed, at Department ofUrology, Wayne State University, 4160 John R., Suite 1017, Detroit, MI

48201. Phone: (313)745-7381; Fax: (313)745-0464.

INTRODUCTION

An estimated 54,400 new cases of bladder cancer will be

reported in the United States, with more than 12,500 deaths

predicted for 1998 (1). It is the fourth most common cancer in

men and the eighth most common in women. Seventy-five

percent of bladder cancers are initially diagnosed as noninvasive

(Ta, TIS) or minimally invasive cancers (T1) (2). Bladder cancer

has a 50% 5-year disease-free survival rate once the tumor

invades the underlying stroma compared to 90% if the tumor is

noninvasive. For patients with superficial cancers (Ta, CIS,3 and

I ) the standard treatment consists of transurethral tumor resec-

tion with or without intravesical therapy. However, 25% of

superficial bladder cancers will eventually invade the bladder

wall and require a radical cystectomy. Currently available prog-

nostic factors are incapable of identifying which superficial

tumors will become invasive. Thus, molecular markers to iden-

tify patients with bladder tumors at high risk for invasion are

critical for the clinician to administer appropriate care.

Early stages of bladder cancer are characterized by the

presence of a dysplastic and proliferative urothelium that mi-

tially grows into the lumen of the bladder. Cystoscopy and urine

or bladder wash cytology is the traditional method of diagnosing

bladder cancer. Although cystoscopy is highly effective at iden-

tifying papillary tumors, CIS is difficult to identify. Bladder

wash cytology is relatively effective in diagnosing CIS but has

a 20-40% false-negative rate in detecting papillary tumors,

depending on the grade of the tumor (3). Novel methods to

identify bladder cancer and determine the biological aggressive-

ness of the tumor are essential to better select the appropriate

treatment for an individual patient.

Like all carcinomas, bladder cancer invasion into the

neighboring stroma is the hallmark of metastasis formation (4).

The proteolytic degradation of basement membranes has been

considered an essential step for the invasion and metastatic

spread of cancer cells (5). The MMPs are a family of zinc-

dependent endopeptidases that have been associated with the

ability of tumor cells to degrade extracellular matrix cornpo-

nents during tumor cell invasion (5, 6). Considerable evidence

has associated gelatinase A (MMP-2) and B (MMP-9), two

members of the MMP family, with tumor metastasis because of

their ability to degrade basement membrane collagen IV and

their elevated expression in many malignant human tumors (7,

8), including bladder cancer (9-11). Previously, we have shown

enhanced levels of both gelatinases in 42 cases of invasive

bladder cancer by immunohistochemical studies (10). Davies et

al. (9) demonstrated a positive correlation between higher levels

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3012 proMMP-9 in Bladder Washes

Table 1 Cytology and proMMP-9 i n study cohort

Cytology proMMP-9

Samples No

(�z = 65) Positive Negative Expression expression

1” 14 0 15

1 23 1 23

13 13 19 7

Control patients

No active bladder cancer

Active bladder cancer

“ Tissue diagnosis was reported as folicular cystitis: patient had two

subsequent visits with voiding cytology specimens reported as negative

for malignancy.

of proMMP-9 and invasive disease, using gelatin zymography.

Overexpression of proMMP-2 in a rat bladder cancer cell line

increased metastatic potential (12). In other studies, elevated

levels of proMMP-2 ( 1 1 , 1 3) and proMMP-9 ( 13) were detected

in urine samples of patients with transitional cell carcinoma. In

the present study, we analyzed the expression of gelatinases by

gelatin zymography of the bladder washes from patients under-

going cystoscopy. Our goal was to evaluate the ability of this

test to detect the presence of bladder cancer and to determine

whether the presence of malignancy and degree of invasion

correlate with the level of gelatinase expression. The data mdi-

cate that proMMP-9 expression in bladder washes may be a

useful marker for bladder cancer detection and invasion.

MATERIALS AND METHODSStudy Population. Bladder washes were collected from

65 patients undergoing cystoscopy from June 1996 to January

1997 at the Wayne State University/Karmanos Cancer Institute-

affiliated Harper Hospital. Fifty-one men 44-89 years old

(mean, 69.4 years) and 14 women 40-89 years old (mean, 63.6

years) represented the study cohort. Patient characteristics were

collected by chart review. The pathological stages of those with

active bladder cancer were classified according to the 1987

International Union Against Cancer ThM classification of

urothelial cancer (14). Tumors were graded histologically, fol-

lowing the World Health Organization classification (15). The

patients were stratified into three groups: group 1 , transitional

cell carcinoma present on cystoscopic bladder biopsy (active

cancer): group 2, history of transitional cell carcinoma but no

evidence of active disease (recurrence free); and group 3, no

history of transitional cell carcinoma or active disease at the

time of cystoscopy.

Sample Collection. After a patient underwent cystos-

copy and the bladder was emptied and before intravesical ma-

nipulation, 100 ml of a sterile saline solution was pushed back

and forth into the bladder S to 10 times. One-half of the sample

was sent for cytological diagnosis, and the other half was

processed for analysis of gelatinase expression.

Cytology. Samples of bladder washes were centrifuged

at 2,000 rpm for 10 mm, and two cytospmn slides were prepared

from the sediment of each sample and fixed in 95% ethanol.

Fixed smears were stained by the modified Papanicolaou tech-

nique (16). Diagnostic interpretation of the cellular material was

performed by a cytopathologist on the basis of criteria described

previously (17). The cases were classified as positive or nega-

tive for malignant cells. All suspicious cases were included in

the positive group. The bladder wash cytology results analyzed

were from the original reports, carried out as a routine labora-

tory procedure, without subsequent revision.

Sample Preparation for Gelatin Zymography. Bladder

washes (40-50 ml) were centrifuged (2,000 rpm, 10 mm, 4#{176}C)

to obtain the cellular fraction. The cell pellets were washed

twice with cold PBS (pH 7.2), centrifuged, and resuspended in

200-400 p.1 of lysis buffer [25 mrvi Tris buffer (pH 7.5), 100 mr�i

NaC1, and 1% NP4O] containing 1 mr�i phenylmethylsulfonyl

fluoride, 2 mM EDTA, 10 �i.g/ml aprotinin, 1 p�g/ml leupeptin,

and 2 mM benzamidine. The lysates were rocked at 4#{176}Cfor 2 h,

centrifuged (13,000 x g, 30 mm, 4#{176}C),and the supernatants

were collected. The protein content in each supernatant was

determined using the BCA Protein Kit (Pierce, IL). The samples

were stored at - 80#{176}Cuntil use.

Gelatin Zymography. Gelatin zymography was per-

formed using 10% SDS-polyacrylamide gels containing 0.1%

gelatin, as described previously (18). Equal amounts of protein

(2 p.g) from each bladder wash were mixed with Laemmli

sample buffer without reducing agents and subsequent boiling.

Recombinant human proMMP-9 and proMMP-2 produced in a

vaccinia expression system ( 1 9) were used as controls. At the

end of the electrophoretic run, the gels were incubated (20 mm,

22#{176}C)in a solution of 2.5% Tniton-XlOO in distilled H2O, and

then washed in distilled H20 for another 20 mm. The gels were

then incubated (16 h, 37#{176}C)in 50 m�i Tris-HC1, 5 msi CaC12

(pH 8), and then stained with 0.25% Coomassie Blue in a

solution of 10% methanol and 5% acetic acid. Bands with

gelatinolytic activity, detected as clear bands against the blue-

stained gelatin background, were visualized after the gels were

destained with 10% methanol-5% acetic acid. To analyze the

expression of gelatinases in the cellular precipitates of the

bladder washes, samples were subjected to gelatin zymography.

It is known that some members of the MMP family, and in

particular the gelatinases, can be detected in complex mixtures

using SDS-polyacrylamide gels impregnated with gelatin be-

cause of their ability to degrade denatured collagen. After elec-

trophoresis under nonreducing conditions, the proteinases in the

sample are allowed to renature by removal of the SDS, which

results in the in situ degradation of substrate, indicated by a

gelatinolytic band at the corresponding size of the proteinase.

The intensity of the bands was analyzed by densitometry using

an Ambis Radioanalytic Imaging System. This system software

allowed the quantification of the gelatinases expression in the

samples, measuring the intensity rate of the bands against the

baseline control. An intensity rate of 25-89% of the control was

defined as expression, and 90% or more was defined as over-

expression.

Statistical Analysis. The Fisher exact test was used to

determine the significance between gelatinase expression and

overexpression and stage and grade of disease. For statistical

comparison between levels of gelatinases, we used the Student

t test.

RESULTS

Of the 65 patients examined, 26 had active cancer, 24 were

recurrence-free, and 15 were control patients who underwent

cystoscopy for a variety of noncancerous reasons, including



Pathological diagnosis

No. ofpatients

Cytological diagnosis proMMP-9 proMMP-2

Expression No expressionPositive Negative Expression No expression

Dysplasia 4 1” 3

Squamous metaplasia 2 0 2

Cystitis follicularis 7 1 6

Cystitis glandularis 2 0 2

Cystitis cystica 2 0 2

Nephrogenic adenoma 2 0 2

Total 19 2 17a This positive result came from the same patient, who 6 months later on follow-up cytoscopy with biopsy was diagnosed with recurrent bladder

carcinoma.

3

27

0

0

0

0

0

0

0

0I

1

1

18

0

0

0

0

41

71

1

I

19

Clinical Cancer Research 3013

Table 2 Patients without transitional cell carcinoma confirmed by tissue biopsy

Table 3 Stage and grade of bladde r tumors

Tumor grade

Pathological stage I II III Total

Ta 3 5 1 9

TIS 0 0 3 3T 0 2 6 8T,-T4 0 0 6 6Total 3 7 16 26

evaluation of irritative or obstructive voiding symptoms or for

treatment of renal calculi. All 65 patients had cytological diag-

nosis and zymographic studies from the bladder wash samples

as shown in Table 1 . Histopathological diagnosis was estab-

lished in 45 patients, 19 with noncancerous conditions and 26

with transitional cell carcinoma of the bladder, as shown in

Tables 2 and 3, respectively. The other 20 patients had no

suspicious lesions on cystoscopy, and therefore, biopsy was not

performed. Nine of the 15 control patients had bladder biopsies

with the following histopathological diagnosis: cystitis follicu-

laris (3), cystitis glandularis (2), squamous metaplasia ( 1 ), neph-

rogenic adenoma (1), and cystitis cystica (1). A biopsy was

performed in 10 of the recurrence-free patients with the follow-

ing histology: cystitis follicularis (4), nephrogenic adenoma (1),

cystitis cystica ( I ), and mild dysplasia (4). Transitional cell

carcinomas were present in 26 patients: 3 were grade I, 7 were

grade II, and 16 were grade III. The pathological stage was TIS

in 3 patients, Ta in 9 patients, T1 in 8 patients, and T,-T4 in 6

patients.

As shown in Fig. 1, purified human recombinant

proMMP-9 produces a gelatinolytic band of Mr 92,000, consist-

ent with the enzyme being in the latent form (18). The recom-

binant proMMP-2 produces an Mr 72,000 band, representing

latent enzyme, and an M1 62,000 band, representing the active

form as a result of partial autoactivation (20). In patients with no

history of bladder cancer or no suspicious lesions on cystoscopy,

the zymogram of the bladder wash revealed little or no expres-

sion of gelatinases in the samples, with the exception of a very

faint band of Mr 92,000 (Fig. 1). Similar results were observed

with the bladder washes of patients with a history of bladder

cancer but who were recurrence-free at the time of the study, as

shown in the zymogram of Fig. 2.

Representative expression of gelatinases in bladder washes

of patients with active bladder cancer is shown in Fig. 3. Most

of the samples derived from patients with bladder cancer

showed significantly higher levels of proMMP-9 expression

when compared with the control patients. In contrast, expression

of proMMP-2 or MMP-2 could not be detected in any of the

samples examined regardless of pathological stage and grade.

When an arbitrary known amount of recombinant proMMP-9

was used as a standard, samples were classified as expressing

and overexpressing proMMP-9 as described in “Materials and

Methods.” Table 4 describes the expression and overexpression

of proMMP-9 according to pathological stage in patients with

active bladder cancer. Although there are only a few cases,

patients with TIS were more likely to have a positive cytology

(67%) or proMMP-9 expression (100%) compared with patients

with Ta disease (22% had positive cytology and 44% had

proMMP-9 expression). In patients with invasive bladder cancer

(T1-T4), cytology studies were positive in 9 of 14 cases,

whereas 12 of 14 cases had proMMP-9 expression. In this study,

proMMP-9 expression had 71% sensitivity, 95% specificity, and

91% positive predictive value in detecting bladder cancer. Con-

versely, cytology had 50% sensitivity, 95% specificity, and 81%

positive predictive value. However, because of small sample

size these numbers were not statistically significantly (P =

0. 1 1). A positive cytology and proMMP-9 expression were

generally more common in patients with grade III bladder

cancer. Interestingly, 30 and 40% of the patients with grade I or

II bladder cancer had a positive cytology or proMMP-9 expres-

sion, compared with 62 and 94% of patients (P = 0.02) with

grade III disease, respectively.

Overexpression of proMMP-9 was common in invasive or

grade III tumors (Tables 4 and 5). Eight of the 14 patients with

T1-T4 tumors overexpressed proMMP-9 compared to 2 of 12

patients with CIS or Ta disease (P = 0.04). Similarly, 63% (10

of 16) of the patients with grade III tumors and 0% (0 of 10) of

the patients with grade I or II tumors overexpressed proMMP-9,

respectively (P = 0.003).

DISCUSSION

Bladder carcinoma is an important public health problem.

It is the fifth most common human cancer, with more than

54,000 new cases estimated in 1998 accounting for more than

12,000 deaths (1). Transitional cell carcinoma, the most com-

mon histopathological type (94%), usually presents as a super-

ficial lesion in 70-80% of the cases, recurs in 50-70% of

patients, and progresses to muscle invasive disease or presents



Ii

123

I�S. .1456 7 8 9 10


proMMP-9 #{149}‘�

proMM P-2

MMP-2

Fig. I Zymography of gelatinase expression in bladder washes from patients with no history of bladder cancer. The cellular fraction (2 p.gllane) of

bladder washes was subjected to gelatin zymography as described in “Materials and Methods.” Lane I, squamous metaplasia: Lane 2, cystitis

glandulanis: Lane 3, no bladder lesion identified by cystoscopy: Lane 4. nephrogenic adenoma: Lanes 5 and 6, dysplasia: Lane 7. cystitis folliculanis:

Lanes 8 and 9. cystitis cystica: and Lane 10, no bladder lesion identified by cystoscopy. Purified human recombinant proMMP-9 (10 ngllane) and

proMMP-2 (containing a fraction of active MMP-2, 10 ng/lane) were used as standards.

proMMP-9 #{149}proMMP-2

MMP-2

12345678

Fig. 2 Zymography of gelatinase expression in bladder washes from

patients with history of bladder cancer but no active disease at time of

cystoscopy. Samples (2 jig/lane) of bladder washes prepared as de-

scribed in Fig. 1 were subjected to gelatin zymography. Lanes 1, 2. 4.

and 8. cystitis follicularis: Lane 3. cystitis glandularis: Lane 5. nephro-

genic adenoma: Lane 6, squamous metaplasia: and L�ine 7, dysplasia.Recombinant proMMP-9 and proMMP-2 were used as standards as

described in Fig. 1.

de ,im’o at an invasive stage in 15-30% of cases. Stage and

grade represent the most reliable prognostic indicators of this

disease. Fewer than 2% of patients with grade I tumors, approx-

imately I 1 % of patients with grade II tumors, and 45% of

patients with grade III:pT�, tumors are likely to progress within

2 years of diagnosis. Patients with bladder cancer that infiltrates

the muscle wall have a worse outcome, with 50% developing

metastasis within 2 years and a 5-year survival rate between 20

and 40%. (2 1 ) However. recent data in PT2�PT3a, N0, M0

patients have revealed increased 5-year survival rates (60-70%)

after radical treatment (2). This highlights the importance of

distinguishing aggressive superficial tumors that will eventually

invade and muscle-invasive tumors that will metastasize.

Here we have examined the expression of gelatinases in the

cellular component of bladder washes from three patient cate-

gonies: those with active histologically proven transitional cell

carcinoma; those with a history of bladder cancer but are cur-

rently recurrence-free: and those undergoing cystoscopy for

benign conditions that have no history of bladder cancer. We

wished to test whether expression of these enzymes in gelatin

zymography correlates with the presence of disease and/or the

invasiveness of the tumor. Bladder washes are known to contain

cellular elements, derived from the bladder wall, that after

isolation aid in the cytological diagnosis of bladder tumors.

Trott and Edwards (22) reported that bladder wash cytologies

were more accurate in detecting the presence of bladder cancer

in comparison with voided urine cytology. It has been shown

that the barbotage action enhances cell shedding and provides

better preserved cells for diagnostic examination (22). Our data

indicate that the cellular pellets of bladder washes can be used

to detect gelatinases in patients with bladder cancer, using a

simple zymographic assay. Analysis of 65 cases revealed that

proMMP-9 is the major gelatinase expressed in the bladder

washes, whereas proMMP-2 could not be detected. The expres-

sion of proMMP-9 correlated strongly with the presence of

bladder cancer because 73% of patients with bladder cancer had

proMMP-9 expression in their bladder wash, whereas only 5%

of patients with benign bladder conditions had proMMP-9 ex-

pression (P = 0.001). Expression of proMMP-9 correlated with

higher tumor grade (P < 0.02). but interestingly, overexpression

correlated with tumor grade (P = 0.003) and pathological stage

(P < 0.04), suggesting a potential prognostic value for

proMMP-9 in patients with active bladder cancer. In contrast,

our data indicate that cytological analyses of the same samples

failed to correlate with either tumor grade (P = 0. 12) or path-

ological stage (P = 0. 1 1).

Moses et a!. ( 13) found MMP expression in the urine of

cancer patients that correlated with disease status. In addition,

they demonstrated that expression of proMMP-9 and

pnoMMP-2 in urine were comparable to or better than other

tumor markers in predicting metastatic cancer of any tumor type

(13). These investigators measured proMMP-9 expression in

urine and hypothesized that metastatic tumors secrete

proMMP-9 in the urine. We, however, measured proMMP-9

expression in exfoliated bladder cells. Therefore, our data are

specifically indicative of the role of proMMP-9 in bladder

cancer. In addition, our data are in agreement with previous

studies indicating the importance of proMMP-9 in cancer pro-

gression (23-25). In our previous irnmunohistochemical study

( 1 0), we could not find a positive correlation between gelati-

nases expression and pathological stage, tumor grade, and/or

outcome in 42 cases of invasive bladder cancer. However, we

found that the expression of TIMP-2 in the tumor stroma

strongly correlated with poor survival. Attempts to measure

TIMP-l and TIMP-2 by immunoblot analysis in the bladder

washes were unsuccessful and probably require a more sensitive

detection assay. Thus, the levels of TIMPs in the bladder washes

remain to be determined. However, a previous study of 69

patients with bladder cancer demonstrated high serum levels of

TIMP-l and a positive correlation between TIMP-l levels and

invasion (26). These results and our results in the present and

previous studies further demonstrate a complex relationship

between levels of protemnases and their inhibitors in cancer

progression (10, 27).



1 2 3 4 5678 9 10 11 12 13 14 15 16 17

Clinical Cancer Research 3015

proMMP-9

proMMP-2

MMP-2

Fig. 3 Zymography of gelatinase expression and overexpression in bladder washes from patients with active bladder cancer. Samples (2 p.gllane)

of bladder washes prepared as described in Fig. 1 were subjected to gelatin zymography. Expression of proMMP-9: Lanes I and 8, TC grade III: Lane

3, TC grade II: Lanes 5 and 16, Ta grade II: and Lane 13, CIS grade III. Overexpression of proMMP-9: Lane 2. Tia grade III: Lanes 4 and 10, T, grade

III; Lane 9, T3,, grade III: Lane 14, Ta grade III: and Lane 17, CIS grade III. Lesions with no expression of proMMP-9: Lane 6, TC grade II: Lane

7, Ta grade I; Lane ii, Ta grade I: Lane 12. Ta grade I: and Lane 15, Ta grade II. Recombinant proMMP-9 and proMMP-2 were used as standards

as described in Fig. 1.

Table 4 Cytopathological diagnosis and proMMP-9 expression in bladder washes according to pathological stage

Cytological diagnosis proMMP-9 expression proMMP-9 overexpression

Pathological stage No. of patients Positive Negative Positive Negative Positive Negative

Ta 9 2 7 4 5 1 8

TIS 3 2 1 3 0 1 2

T-T4 14 9 5 12 2 8” 6

Total 26 13 13 19 7 10 16

“ P = 0.04 (T1-T4 vs. TIS + Ta).

Table 5 Cytopathological diagnosis and proMMP-9 expression in bladder washes according to tumor grade

Cytological diagnosis proMMP-9 expression proMMP-9 overexpressionTumor No. ofgrade patients Positive Negative Positive Negative Positive Negative

I 3 1 2 0 3 0 3

II 7 2 5 4 3 0 7

III 16 10 6 15” 1 10” 6

“ P = 0.02 (grade III vs. I + II).I, p = 0.003 (grade Ill vs. I + II).

Expression of gelatinases has been correlated with tumor

invasion and metastasis in many human tumors, including blad-

den cancer. Davies et a!. (9) correlated proMMP-2 activation

with tumor grade, using zymography of bladder tumor extracts

that detected the presence of active MMP-2 forms in the more

malignant tumors compared to the low-grade tumors. In the case

of proMMP-9, the studies by Davies et a!. (9) showed that levels

of the latent form of the enzyme are enhanced in invasive

bladder cancer, in agreement with our results. Interestingly, the

expression of gelatinase mRNAs by in situ hybridization is

mostly in stroma cells, with occasional expression in the tumor

cells (9). Although we have not determined the precise nature of

the cells in the bladder washes, they are most likely to contain

epithelial and some inflammatory cells as reported previously

(22). However, it is unlikely that the major source of proMMP-9

in the bladder washes are inflammatory cells because bladder

washes obtained from patients with inflammatory conditions

showed no presence of proMMP-9, suggesting that proMMP-9

expression is associated with the epithelial cells. Indeed, immu-

nohistochemical studies indicated that both proMMP-9 and

proMMP-2 are present in bladder cancer cells (10). In other

studies, human bladder cancer cell lines cultured in vitro were

shown to express both gelatinases after exposure to basic fibro-

blast growth factor (28). Thus, bladder cancer cells under certain

conditions can potentially express both proMMP-9 and

proMMP-2. Alternatively, expression of gelatinases in bladder

cancer cells may be the result of paracrine secretion of enzymes

by stromal cells and subsequent binding of the enzymes to the

tumor cells (18).

It was interesting to observe the lack of proMMP-2 detec-

tion in the bladder washes in spite of previous reports of

proMMP-2 presence in bladder tumors ( 10) and in the urine of

patients with transitional carcinoma (1 1). The reason for this

discrepancy is unclear but may be due to a preferential entrap-

rnent of proMMP-9 in the bladder wall, whereas proMMP-2 is

readily secreted and detectable in the urine. Alternatively, the

samples of the bladder washes are too small to allow for the

detection of low levels of proMMP-2. Moses et a!. (13) detected

proMMP-2 in only 30% of the urine specimens from patients

with bladder cancer, whereas proMMP-9 was detected in 70%

of the cases in the urine samples. Although the precise role of

these enzymes in bladder cancer progression remains unclear,

our data suggest that expression of proMMP-9 in bladder

washes can be useful for identifying patients with bladder can-




14. International Union Against Cancer. ThM classification of malig-

nant tumors. Geneva, Switzerland: Springer-Verlag, 1987.

cer and differentiating high-grade cancers from superficial dis-

ease.

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1998;4:3011-3016. Clin Cancer Res F J Bianco, Jr, D C Gervasi, R Tiguert, et al. bladder cancer patients predicts pathological stage and grade.Matrix metalloproteinase-9 expression in bladder washes from

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