(CANCER RESEARCH 58. 1090-1094. March 15. 1998|

Advances in Brief

Elevated and Absent pRb Expression Is Associated with Bladder CancerProgression and Has Cooperative Effects with p53*

Richard J. Cote,2 Matthew D. Dunn,3 Sunanda J. Chatterjee,3 John P. Stein, Shan-Rong Shi, Quoc-Chau Tran,

Shi Xue Hu, Hong Ji Xu, Susan Groshen, Clive R. Taylor, Donald G. Skinner, and William F. BenedictDepartments of Pathology ¡R.J. C.. M. D. D., S. J. C.. J. P. S.. S-R. S.. C. R. T.I, Preventive Medicine IQ-C. T.. S. C./, and Urology [D. C. S.J. University of Southern CaliforniaSchool of Medicine/Norris Comprehensive Cancer Center, Los Angeles, California 90033, and the Departments of Hematology fS. X. H.. W. F. B.I and Molecular Oncology¡H.J. X.I. M. D. Anderson Cancer Center, The Woodlands. Texas 77381

Abstract

Rb protein (pRb) expression was evaluated in 185 cases of transitional cellcarcinoma of the bladder from patients that underwent radical cystectomy.Tumors were stratified into three categories based on the percentage of nucleiexpressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (In \+,1-50% of tumor cells showing nuclear reactivity; and (c)2+, >50% of tumor

cells showing nuclear reactivity. Cases with undetectable (pRb 0) and high(pRb 2+) pRb reactivity had identical rates of recurrence. These cases hadsignificantly higher recurrence (P = 0.0001) and lower survival ratesi/' = 0.0002) compared to cases with moderate (pRb 1+ ) pRb reactivity,

indicating that high levels of pRb expression may reflect a dysfunctional(altered) Rb pathway. The tumors were also examined for alterations in p53expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 0.0001) and survival (P < 0.0001)compared to patients with no alterations in either p53 or pRb; patients withalterations in only one of these proteins had intermediate rates of recurrenceand survival. These results suggest that: (a) bladder cancers with high pRbexpression do not show the tumor suppressor effects of the protein; and (Inalteration in both p53 and pRb may act in cooperative or synergistic ways topromote tumor progression.

Introduction

Cancer is a multistep genetic process involving alterations in onco-

genes and tumor suppressor genes ( 1). Mutations in the tumor suppressorgenes p53 and Rb4 are common events in human cancer, including

bladder cancer (2-6). However, a proportion of tumors showing no

alteration in p53 will progress; this is also true for Rb. Both p53 and Rbexert their control on the cell cycle at the G,-S-phase transition through

independent but interconnected pathways (7). Furthermore, it has recentlybeen shown that germ-line mutations in p53 and Rb may have coopera

tive tumorigenic effects in mice (8). We therefore reasoned that alterations in both p53 and Rb may act in a cooperative or synergistic way topromote bladder cancer progression in humans. Whereas p53 gene mutations often result in the abnormal nuclear accumulation of the alteredprotein (9), mutations of the Rb gene correspond to loss of expression ofthe pRb (6). Because nuclear expression of pRb has been assumed to beindicative of an intact gene and a functional protein, to date, studiesexamining the prognostic significance of pRb determined whether or notthe protein was expressed (4-5). However, it is possible that in some

cases, pRb may have lost functional activity, although it is expressed. Weundertook the present study to determine whether a subset of tumors that

Received 11/25/97; accepted 1/28/98.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by National Cancer Institute Grants CA 70903 (to R. J. C.), ÇA7I921 (to

R. J. C.). and CA 54672 (to W. F. B.).2 To whom requests for reprints should be addressed, at University of Southern

California/Norris Comprehensive Cancer Center. 1441 Eastlake Avenue, Los Angeles,CA 90033. Phone: (213) 764-3270.

' M. D. D. and S. J. C. contributed equally to this study.4 The abbreviations used are: Rb. retinoblastoma; pRb. Rb protein; wt, wild-type.

express pRb shows evidence of loss of pRb function. We also sought todetermine whether alterations in both pRb and p53 expression exert acooperative or synergistic effect in promoting bladder cancer progression.

Materials and Methods

Patient Information. Tumor specimens were obtained from 185 patientswho underwent en bloc radical cystectomy, pelvic lymphadenectomy, andurinary reconstruction for invasive transitional cell carcinoma of the bladder.Tumor staging was classified according to the tumor-node-metastasis system(10). Median follow-up for this group was 8.6 years.

Antibodies and Immunohistochemistry. The immunohistochemical procedure for the rabbit polyclonal anti-pRb antibody, RB-WL-1. including the

antigen retrieval protocol, has been previously described by us ( 11). The extentof pRb reactivity was classified into three categories based on the percentageof tumor cells showing nuclear reactivity: (a) 0, 0% of tumor cells showingnuclear reactivity; (b) \+, 1-50% of tumor cells showing nuclear reactivity:

and (r) 2+, >50% of tumor cells showing nuclear reactivity (Fig. 1). Thenormal bladder urothelium demonstrated pRb I + nuclear reactivity in allcases. In most cases, tumors showing 2+ pRb nuclear reactivity demonstratedintense staining of more than 80% of tumor cells (Fig. 1). All slides were readindependently at two different institutions (by R. J. C., M. D. D., S. R. S., andJ. P. S. and by W. F. B. and H. J. X.); concordance was reached in nearly allcases. In the discordant cases, the tissue was retested. In all of these cases,concordance was reached.

The immunohistochemical procedure for p53 has been described previouslyby us (2). The level of p53 nuclear reactivity was classified into two categories:(a) p53-, 0-9% of tumor cell nuclei were positive for p53 nuclear reactivity;

and (ft) p53 + , 10-100% of tumor cell nuclei were positive for p53 nuclear

reactivity (2). Based on our previous studies comparing p53 nuclear reactivitywith p53 gene mutations, only tumors showing at least 10% nuclear reactivitywere considered to be p53-altered (p53 + ; Ref. 9).

Statistical Analysis. The clinical outcomes analyzed were time to first recurrence of bladder cancer and overall survival. Time to recurrence was calculated asdays from cystectomy to the date of the first documented clinical recurrence or lastfollow-up visit, if the patient had not recurred; patients who died before recurrence

were censored at the time of death; deaths from any cause were included. Survivalwas calculated as days from cystectomy to death. For the purpose of statisticalanalysis. pRb status was initially classified into two groups, negative (pRb 0) andpositive (pRb 1+ and 2+), and then analyzed in relation to probability ofrecurrence and survival. Classification into three groups (pRb status O, 1+, and2+) was then performed and analyzed in a similar manner. pRb status was then

combined with p53 status and analyzed in relation to recurrence and survival.Contingency tables and Pearson's x2 test ( 12) were used to evaluate the association

of Rb reactivity with pathological stage and lymph node status of the tumor.Kaplan-Meier plots (13) and log-rank tests were produced to evaluate the associ

ation of these prognostic variables with Rb status alone and combined with p53status in regard to survival and recurrence. SEs of these estimates were calculatedusing Greenwood's formula (14).

Results

Rb Status. No pRb nuclear reactivity (pRb 0) was noted in 54tumors (29%), 83 tumors (45%) had 1+ pRb reactivity, and 48 tumors

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Rb AND p53 ALTERATIONS IN BLADDER CANCER

C '» -

t

Fig. 1. Transitional cell carcinomas of Ihe bladder with ¡mmunohislochemical reactivity identifying pRh. using rabbit polyclonal antibody RB-WI.-l. A. no nuclear reactivity (pRh0): the cun-ett arnw shows negative tumor cell nuclei; the strtiif>hl arrow shows pRb expression in surrounding normal lymphiKytes and stromal cells (internal controls). B. low tomoderate (1-50%) nuclear reactivity (pRb \ + ). C, high O509H nuclear reactivity (pRb 2 + ). D. normal bladder transitional cell epithelium lurothelium) showing moderate (pRb 1+ )

nuclear reactivity.

(26%) had 2+ pRb reactivity. Normal bladder urothelium showed

heterogeneous (1 +) pRb nuclear reactivity (Fig. 1). When pRb status

was stratified into two groups based on the presence or absence ofnuclear reactivity [pRb+ (pRb 1+ and 2+) versus pRb- (pRb 0)], a

significant association with stage of disease was noted (P = 0.002);

tumors with pRb+ nuclear reactivity tended to be of lower stage thanpRb— tumors. The pRb 0 group showed increased recurrence

(P = 0.013) and decreased overall survival (P = 0.09) compared with

the pRb 1+/2+ group. The estimated 5-year survival rates were 33

and 54%, respectively.

When stratified into three categories based on the level of pRb

nuclear reactivity (pRb 0 versus 1+ versus 2+), a significant association was seen with stage of disease (P = 0.001; Table 1). However,

this analysis showed that tumors with no (pRb 0) or high (pRb 2+)

expression of pRb were similar with regard to association with disease

stage and were significantly different from tumors that showed mod

erate (pRb 1+) levels of pRb expression. Tumors with moderate (pRb

1+) pRb expression tended to be of lower stage than pRb 0 or 2+

tumors.

Among these three groups (pRb 0 versus 1+ versus 2+), a signif

icant difference in overall rates of recurrence and survival was ob

served (Fig. 2). Patients with tumors demonstrating homogeneous

nuclear reactivity (pRb 2 + ) had nearly identical rates of recurrence

and survival compared to patients with tumors showing no detectable

nuclear reactivity (pRb 0). These patients had significantly higher

rates of recurrence (P < 0.0001 ) and decreased survival (P = 0.0002)

compared to patients with tumors demonstrating moderate nuclearreactivity (pRb 1+ ). The 5-year recurrence rates for patients with pRb

O, 1+, and 2+ bladder tumors were 60. 29, and 61%, respectively(P = 0.0001). The 5-year survival rates for these same groups were

33, 66, and 33%, respectively (P = 0.0002).

When stratified into three categories based on the level of expres

sion, the pRb status was independent of tumor grade, stage, and lymph

node status in predicting recurrence and survival in a multivariableanalysis (P = 0.012 and 0.010 for recurrence and survival, respec

tively).

p53 Status. Of the 185 tumors studied, 109 (61%) demonstrated no

nuclear accumulation of p53 (p53 wt tumors), and 76 (39%) demonstrated nuclear accumulation of p53 protein (p53-altcred tumors),

confirming our previous results (2). Patients with p53 wt tumors had

significantly lower rates of recurrence and better overall survival thanpatients with p53-altered tumors. The 5-year recurrence rates for

patients with p53 wt tumors versus p53-altered tumors were 31 versus

69% (P < 0.0001 ). The 5-year survival rates for patients with p53 wt

tumors versus p53-altered tumors were 63 versus 28% (P < 0.0001 ).

Combined Rb and p53 Status. The combined effects of p53 and

pRb status on tumor progression (relationship to stage, recurrence,

and survival) in patients with invasive bladder cancer are summarized

in Table 1 and Fig. 3. In this analysis, those tumors demonstrating no(pRb 0) or high levels (pRb 2 + ) of nuclear reactivity were con-

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Rb AND p53 ALTERATIONS IN BLADDER CANCER

Table 1 The association of pRb and f>53 status with tumor stage in 1N5 patients \\-ith bladder cancer

Total

No. of cases (%)

LN-"

pRh statuspRhOpRb 1+pRb 2 +Organ-confined14(17)

53 (62)18(21)Extravesical21

(43)14(30)13(27)LN+''20

(38)16(30)17(32)Total54

(29)83 (45)48 (26)P0.001

85(100) 47(100) 53(100) 185(100)

p53/pRb status'

p53 wt/pRb wtp53 wt/pRb alt and p53 alt/pRb wtp53 alt/pRbaltTotal40

(47)34 (40)11(13)85

(100)II

(24)17(36)19(40)47(100)9(17)

21 (40)23(43)53

(100)60(32)

72 (39)53(29)185(100)0.001

" LN-, no evidence of regional lymph node métastases.

LN + , métastasesto regional lymph nodes.' p53 wt. <IO% nuclear reactivity; p53 alt. a 10% nuclear reactivity; pRb wl. 1+ nuclear reactivity; pRb alt. 0 or 2+ nuclear reactivity.

siderei! pRb-altered. because their recurrence and survival rateswere nearly identical. Tumors with moderate levels of pRb expression (pRb 1+ ) were considered to have a pRb wt phenotype. Thecombined p53/pRb status was significantly associated with tumorstage (Table 1); low-stage tumors tended to have fewer alterationsthan did high-stage tumors. Patients with p53 wt/pRb wt bladdertumors had significantly decreased recurrence and increased survival rates when compared to patients with tumors showing alterations in either p53 or pRb (p53 wt/pRb-altered tumors or p53-altered/pRb wt tumors; P < 0.0001 and 0.0001, respectively).Patients with alterations in either p53 or pRb expression demonstrated decreased recurrence and increased survival rates comparedto patients with tumors showing altered expression of both p53 andpRb (p53-altered/pRb-altered tumors; Fig. 3). The 5-year recurrence rates for patients with tumors demonstrating p53 wt/pRb wtversus p53 wt/pRb-altered or p53-altered/pRb wt versus p53-al-tered/pRb-altered expression were 22 versus 42 versus 79%, respectively (P < 0.0001); the 5-year survival rates for these groupswere 71 versus 51 versus 16%, respectively (P < 0.0001). Inpatients with organ-confined (Pl-P3a) lymph node-negative disease, the estimated 5-year recurrence rates for patients with tumorsdemonstrating p53 wt/pRb wt versus p53 wt/pRb-altered or p53-altered/pRb wt versus p53-altered/pRb-altered expression were 21versus 27 versus 78%, respectively (P < 0.0001); the 5-yearsurvival rates for these groups were 82 versus 75 versus 27%,respectively (P < 0.0001).

Discussion

Our results strongly suggest that at least in the case of bladdercancer, high levels of pRb expression may reflect an altered Rbpathway. In our study, patients with high levels of pRb nuclearreactivity in their tumors had essentially identical recurrence andsurvival rates as did patients with tumors showing no detectable pRbexpression. Both of these groups of patients had significantly increased recurrence and decreased survival rates compared to patientswith tumors demonstrating heterogeneous patterns of pRb reactivity.These findings suggest that both loss of pRb reactivity and overex-pression of pRb involve perturbation of normal Rb function. It isknown that absent pRb reactivity is indicative of loss of gene expression (generally through mutation; Ref. 6). We hypothesize that pRboverexpression reflects an alteration in the Rb pathway resulting inloss of tumor suppressor function.

Although the mechanism for increased pRb expression and possiblealterations is not clear, a number of possibilities exist. The over-

expressed pRb may represent a hyperphosphorylated form of theprotein. Rb acts to control cell proliferation through regulation ofthe cell cycle at the G,-S-phase transition. In its underphosphory-

lated state. pRb inhibits this transition by sequestering the transcription factor E2F (7). However, when pRb is phosphorylated bythe cyclin-dependent kinase complexes (15), E2F is released, and

the cell can initiate DNA synthesis (16). Hyperphosphorylation ofpRb has been demonstrated in acute myeloid leukemias, in whichit is associated with poor prognosis (17). Phosphorylation of pRbis accomplished by cyclin-dependent kinase complexes; thesecomplexes are inhibited by several cyclin-dependent kinase inhib

itors, including p21. p27, and pl6 (18, 19). It has been shown thatloss of expression of these inhibitors may be associated with tumorprogression (7). It is therefore tempting to speculate that abnormalities of one or more upstream regulators of pRb phosphoryla-tion might result in constitutive hyperphosphorylation (and inac-

tivation) of pRb. Alternatively, it is possible that some mutationsof the Rb gene might lead to expression of a functionally inactiveaberrantly expressed protein, although this is not consistent withthe present data, because mutations in the Rb gene in the presenceof expressed protein would be expected to be rare (6). Finally,binding of pRb to other proteins such as MDM2 or certain DNAviral oncoproteins may also abrogate the function of pRb whilepreserving its expression. Whatever its basis, our results indicatethat the pattern of pRb reactivity must be taken into account whenevaluating the Rb status in bladder tumors. It is likely that this willbe the case for other tumor types as well.

Whereas alterations in p53 and pRb have been shown to beindependent predictors of bladder cancer progression, our resultsdemonstrate that concomitant alterations in both p53 and pRb havefurther negative effects on recurrence and survival compared toalterations in either one alone. Furthermore, when neither p53 norpRb is altered, patients demonstrate very low rates of recurrenceand increased survival, no matter what the stage of disease. Cor-don-Cardo et al. (20) have recently reported this for patients with

superficial bladder tumors. Of note is the extremely low rate ofrecurrence in patients with organ-confined tumors that show nei

ther p53 nor pRb alterations. It is of interest that patients withorgan-confined tumors showing alterations in either p53 or pRb

(but not both) also have low rates of recurrence and higher rates ofsurvival; however, those with tumors showing alterations in bothp53 and pRb have significantly increased recurrence and decreasedsurvival rates. These results indicate that alterations in both p53and Rb are important in the progression of early stage bladder

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Rb AND p53 ALTERATIONS IN BLADDER CANCER

tumors. Thus, knowledge of both the p53 and pRb status providesmore specific information about the most likely outcome for thepatient.

That alterations in p53 and pRb act in cooperative or synergisticways to promote bladder cancer progression is not entirely unexpected. Cancer is a multistep genetic process: the accumulation ofmultiple alterations produces the malignant phenotype ( 1). Furthermore, p53 and pRb have effects in the same phase of the cell cycle andact in separate but interconnected pathways. Finally, studies in micehave demonstrated that germ-line mutations in both p53 and Kbproduce tumors in mice at higher rates than it the mice had germ-line

mutations in either />53 or Rb, but not both (7). Thus alterations inboth p53 and Rb might be expected to be more deleterious thanalterations in one system alone.

In summary, our results indicate that the level of pRb expression,and not simply its presence or absence, should be determined whenassessing the impact of Rb on bladder cancer progression. The basisfor this is an important issue and is the topic of ongoing research.Furthermore, determination of alterations in more than one tumorsuppressor pathway or within the same pathway can provide increased

pRb[1+] (n=83)

pRb[0] (n=54)

pRb[2 +] (n 48)

6 8 10 12

Years Since Surgerx

B

> 0.8

0.6

o 0.4

pRb[1+] (n=83)

I

aLU

0.2

6 8

Years Since Surgery

12

Fig. 2. Probability of (Ai remaining relapse-free and (fl) survival in 185 patients withinvasive transitional cell carcinoma of the bladder according to pRb status. pRb 0. nodetectable nuclear reactivity; pRb I +, 1-50% nuclear reactivity; pRb 2+, >50% nuclear

reactivity.

CC

0

-Q

S

0.8

0.6

0.4

pRbwt p53wt (n=60)

pRbwt/p53altpRbalt/p53wt

and(n = 72)

.2Log-rank

P- 000010

2pRbalt

p53;ilt(n =53)4

6 810Years

Since Surgery12

B

> 0.8

M'S

>•0.6

0.4

0.2

Log-rankP<0.0001

pRbwt,p53wt (n-60)

pRbwt/p53alt and pRbalt/p53wt<n = 72)

pRbalt/p53alt(n = 53)

10 12

Years Since Surgery

Fig. .1.Probability of (A) remaining relapse-tree and (Hi survival in 1X5patients with

invasive transitional cell carcinoma of ine bladder according lo combined pRh/p.M status.Altered pRb (jiKhtiln tumors include those with pKh 0 and 2+ nuclear reactivity, and wlpRb (¡>Kh\\'titumors were those with pRb 1+ nuclear reactivity. Altered p53 (jrfjulti

tumors were those with ^:\iY7c nuclear reactivity, and wl p53 (/tf.fir/l tumors were thosewith <IO% nuclear reactivity.

information regarding prognosis. These findings may be relevant inother tumors as well. Finally, determining the status of a tumorsuppressor pathway may be important in making specific treatmentdecisions for patients with bladder cancer, a subject that can be furtheraddressed by large prospective clinical (rials (hat incorporate p53 andpRb status into the treatment protocol.

Acknowledgments

We (hank Dr. David Esrig for input into the study. Christina Yang for

technical assistance, and Ming Dickinson for assistance with statistical anal

ysis.

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1998;58:1090-1094. Cancer Res   Richard J. Cote, Matthew D. Dunn, Sunanda J. Chatterjee, et al.   p53Bladder Cancer Progression and Has Cooperative Effects with Elevated and Absent pRb Expression Is Associated with

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