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Vol. 3, 1823-1829, October 1997 Clinical Cancer Research 1823

Abnormal bcl-2 and pRb Expression Are Independent Correlates of

Radiation Response in Muscle-invasive Bladder Cancer’

Alan Pollack,2 Catherine S. Wu,Bogdan Czerniak, Gunar K. Zagars,

William F. Benedict, and Timothy J. McDonnell

Departments of Radiation Oncology [A. P., C. S. W., G. K. Z.],

Pathology [B. C., T. J. M.], and Hematology [W. F. B.], TheUniversity of Texas M. D. Anderson Cancer Center, Houston, Texas

77030

ABSTRACT

The objective of this study was to determine whetherthe overexpression of bcl-2, a key protein governing theapoptotic response to radiation, adds to pRb status in esti-mating the propensity for radiation response in patientswith muscle-invasive bladder cancer.

Archival formalin-fixed, paraffin-embedded, pretreat-ment bladder tumor samples were available in 109 of 301patients treated preoperatively with 50 Gy in 25 fractionsfollowed by radical cystectomy 4-6 weeks later. Radiationresponse was assessed by clinical-to-pathological tumor

downstaging or upstaging. Altered expression of bcl-2 (47%of 107 patients), p53 (56% of 109 patients), and pRb (30% of98 patients ) was assessed by immunohistochemical staining.Morphological criteria were used to calculate the percentage

of apoptotic cells.bcl-2 staining correlated with tumor grade; all grade 2

tumors (n = 7) displayed normal bcl-2 expression (negativestaining). No correlations between bcl-2 staining and pre-

treatment apoptosis levels, p53 staining, and pRb stainingwere observed. In terms of the radiation response parame-ters, univariate analyses revealed that bcl-2 overexpression

was the only factor associated with upstaging. The main

predictor of downstaging was the loss of pRb expression(negative staining). Multivariate logistic regression con-firmed these findings and also showed that normal pRb

expression (positive staining) was significantly related to

upstaging. Patient outcome was adversely affected by bcl-2overexpression, because these patients experienced signifi-

cantly increased actuarial local failure rates. No difference

Received 3/27/97; revised 6/13/97; accepted 6/19/97.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 18 U.S.C. Section 1734 solely to

indicate this fact.I Supported in part by Grants CA 06294, CA 16672, and CA 54672from the National Cancer Institute, United States Department of Healthand Human Services, and by an American Cancer Society CareerDevelopment Award (to A. P.).

2 To whom requests for reprints should be addressed, at Department ofRadiotherapy (Box 97), The University of Texas M. D. Anderson

Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone:(713) 792-0781; Fax: (713) 792-3642.

in distant metastasis or survival rates by bcl-2 staining was

seen.

The strongest independent correlates of radiation re-sponse thus far identified in muscle-invasive bladder cancer

are from bcl-2 and pRb immunohistochemical staining. Theoverexpression of bcl-2 and the normal expression of pRb

seem to thwart the apoptotic response to radiation via inde-

pendent mechanisms. Abnormalities in the expression ofproteins that regulate apoptosis may prove to establish amolecular phenotype to characterize which patients shouldreceive radiotherapy.

INTRODUCTION

There is considerable heterogeneity in the response of

bladder cancers to external beam radiotherapy (I , 2). Unfortu-

nately, classical pretreatment clinical parameters, such as stage

and grade, do not provide any insight into whether a tumor will

respond to such treatment. Recent in vitro (3, 4) and in vivo (5)

data indicate that altered expression of key protein regulators of

apoptosis may affect radiation response. A number of studies

have documented that apoptosis is an important mechanism of

cell death from radiation (6, 7). Therefore, defects in the apop-

totic pathway should tip the balance between apoptosis and G1

arrest with repair of DNA damage. In the case of p53 mutations,

the potential for either of these mechanisms exists, and down-

stream factors such as p21 wafl/cipi and pRb may be more deter-

minant of cell fate. Indeed, the in vitro results concerning the

effects of altered p53 expression and radiation response have

been inconclusive (8-10). Only clinical studies will clarify the

true relationship between the altered expression of factors in the

apoptotic pathway and radiation response.

Three years ago, we began a systematic investigation of the

relationship between radiation response and pretreatment mo-

lecular and histological factors related to apoptosis in patients

with muscle-invasive transitional cell carcinomas of the bladder.

The patient group studied was treated with preoperative radio-

therapy (50 Gy in 25 fractions), followed 4-6 weeks later with

cystectomy. None of the patients received chemotherapy. The

delay between the completion of radiotherapy and cystectomy

allowed time for the effects of treatment, such as clinical-to-

pathological downstaging, to be manifested. In many cases,

complete pathological tumor response (stage P0), another ob-

jective measure of radiation efficacy, was observed. The re-

moval of the bladder in these patients facilitated the evaluation

of radiation response using these truly objective parameters; this

type of an analysis is much less accurate in patients treated with

radiation alone, wherein clinical response must be used as an

end point.

Our initial evaluation of markers of radiation response

revolved around a determination of pretreatment apoptosis 1ev-

cbs using morphological criteria (1 1). Although a correlation

between radiation response and pretreatment apoptosis levels

Research. on June 28, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1824 bcl-2, pRb. and Bladder Cancer Radiation Response

was observed, the difference was negligible and was not clini-

cally relevant. An investigation of immunohistochemical p53

staining revealed that abnormal p53 expression also was not of

value in predicting radiation response ( 12). In contrast, altered

immunohistochemical pRb staining was strongly associated

with radiation response (5). The link between the absence (3) or

suppression (13, 14) of pRb function and radiation response is

fairly well supported and prompted us to further consider other

markers of apoptosis. The report described here evaluates the

relationship between bcl-2 expression and radiation response

and demonstrates that bcl-2 complements pRb in predicting

radiation response.

PATIENTS AND METHODS

Patient Characteristics. Between 1960 and 1983, a totalof 301 patients were treated with preoperative radiotherapy (50

Gy in 25 fractions), followed 4-6 weeks later by radical cys-

tectomy (2). Archival pretreatment tissue blocks were obtained

in 109 patients, and of these, the immunohistochemical staining

of bcl-2 was deemed satisfactory in 107 patients. These patients

represent the main study group. Patient age ranged from 35-80

years, with mean and median ages of 61 and 62 years, respec-

tively. There were 25 women and 82 men. The median fol-

low-up for those living (n = 33) was 91 months.

Staging. The workup has been detailed previously for the

parent cohort (2). All patients were staged clinically by biman-

ual exam at examination under anesthesia, which was done

before and after TURBT.3 Tumor characteristics including size,

location, number, and morphology were also recorded on a

cystoscopy record sheet. Because the 1988 and 1992 American

Joint Committee on Cancer bladder staging systems are largely

pathological, the 1983 classification system was used: stage T,,

tumor invades muscle, and bimanual examination after TURBT

does not reveal any residual induration or mass; stage T3, tumor

invades muscle, and bimanual examination after TURBT re-

veals residual induration (T3a) or a mobile mass (T3b); stage T4,

tumor invades muscle and adjacent structures, i.e. , prostate,

uterus, or vagina (T4a) or abdominal wall or pelvic sidewalls

(T4b). The tumors were graded on a 1-3 scale ( 15) by the study

pathologist (B. C.). No patient had evidence of lymph node

metastasis or distant metastasis at the initiation of treatment.

Treatment. Radiotherapy was delivered using a four-

field technique with 18-MV photons to a dose of 50 Gy in 25

fractions (2). The radiation was given over 5 weeks, and 4-6

weeks later, radical cystectomy was performed. Formal lymph

node dissection was not undertaken, although suspicious lymph

nodes were biopsied. Lymph node involvement was found in

four patients. Neoadjuvant or adjuvant chemotherapy was not

administered to any patient.

Definition of Disease End Points. Recurrence in the

pelvic surgical bed was classified as a local failure. Pelvic

failure was defined as any local failure and/or pelvic lymphad-

enopathy below the bifurcation of the common iliac vessels.

3 The abbreviations used are: TURBT, transurethral resection of thebladder tumor; APAAP, alkaline phosphatase antialkalinc phosphatase;

BUN. blood area urea nitrogen: IVP, iv. pycbogram.

Lymph node involvement above this level or in the inguinal

regions or hematogenous metastases were defined as distant

failures.

Immunohistochemical Staining of bcl-2, pRb, and p53.The overexpression of bcl-2 was identified using immunohisto-

chemical staining. Archival formalin-fixed, paraffin-embedded

tissue in 3-5-jim sections on silane-coated slides was deparaf-

finized as described previously (5). The slides were overlain

with 10 mM citrate buffer (pH 6.0) and heated in a microwave at

high power for 3 mm and then at 70% for 3 mm, with a 5-mm

rest interval between boiling. When the slides and buffer were at

room temperature, the slides were rinsed in distilled water and

incubated for I h in anti-bcl-2 antibody (M0887; DAKO,

Carpinteria, CA; Ref. 16) diluted 1:80 in Tris-buffered saline

(pH 7.4-7.6). The slides were further incubated for 30 mm with

antimouse IgG (Z259; DAKO) and for 30 mm in APAAP

(D65l ; DAKO; Ref. 17), with rinsing between the incubations.

The second antibody and APAAP steps were repeated twice,

reducing the incubation times to 10 mm each. Thus, the second

antibody and APAAP steps were repeated a total of three times.

New fuchsin substrate (K698; DAKO) was applied for 20 mm in

the dark, followed by hematoxylin counterstaining and coverslip

placement with an aqueous-based mounting solution.

The staining intensity of bcl-2 was graded on a 0-4+

scale. Staining intensity was judged relative to infiltrating lym-

phocytes, which were used as internal controls and considered

4+ . Negative controls, consisting of normal skin tissue samples

kept in Tris-buffered saline during the first antibody incubation

period, were included with each batch of slides stained. Two

tumor samples were considered degraded because the infiltrat-

ing lymphocytes did not stain, leaving 107 samples for the

analysis.

Immunohistochemical staining of pRb was accomplished

using polyclonal antibody WL-l as described previously (5, 18).

Tissue sections on polylysine-coated slides were treated for 30

mm with 0.3% hydrogen peroxide in absolute methanol to block

endogenous peroxidase activity. After antigen retrieval, the sec-

tions were incubated with WL-l antibody (0.5 �i.g/ml), followed

by staining using the Vector ABC Elite Kit protocol. Tumor pRb

expression was classified as aberrant if no tumor cells showed

nuclear staining and if some adjacent nonneoplastic cells had

obvious nuclear staining. Cytoplasmic staining was disregarded.

Immunohistochemical staining of p53 using DO1 antibody

has been detailed previously ( I 2). The tumor was considered

positive for p53 when nuclear staining was seen in over 20% of

the tumor nuclei. The bcl-2, p53, and pRb stained tissue sections

were scored at blinded sessions with four of the coauthors

(B.C., A.P., C.S.W.,andW.F.B.)

Apoptotic Index. Apoptosis was identified using mor-

phological criteria such as nuclear condensation and fragmen-

tation (1 1). The apoptotic index was obtained by dividing the

number of apoptotic tumor cells by the total number of tumor

cells, multiplied by 100. A total of 500 tumor cells were counted

in five high-powered fields.

Statistical Analysis. The differences between propor-

tions were assessed using the x2 test (19). The independence of

correlates of radiation response (downstaging or upstaging) was

accomplished using multivariate logistic regression (20). Actu-

anal curves were calculated from the completion of radiother-



Lymph-vasc mv.”

Gender

Age (yrs)

HGB (gni/dlY

BUN (mg/dI)

Creatininc

(mg/dl)

IVP

49 (28)

28 (16)

23 (13)

12 (7)

88 (50)

26(10)

74(28)

85 (17)15 (3)

75 (43)

25 (14)

88 (50)

12 (7)

79(45)

21 (12)

75 (43)

25(14)

7 (4)

93 (52)

81 (46)

19(11)

94(47)

6(3)

74(34)

26(12)

20(10)

57 (29)

23 (12)

40(23)

60(34)

25 (13)

75 (40)

68 (39)

25 (14)

7 (4)

68 (39)

32 (18)

7 (4)

93 (53)

47 (27)

53 (30)

40 (20)

28 (14)

32(16) 0.52

0 (0)

100(50) 0.01

22(8)78 (28) 0.68

71 (15)

29 (6) 0.29

76(38)

24(12) 0.95

82 (40)

18(9) 0.38

74(37)

26(13) 0.55

60(30)

40(20) 0.09

10(5)

90(45) 0.60

84(42)

16(8) 0.66

85 (35)15(6) 0.17

58 (25)

42(18) 0.12

21 (10)

60 (29)19(9) 0.8446(23)

54 (27) 0.56

36(15)

64 (27) 0.23

70(35)

6(3)

24(12) 0.004

70(35)

30(15) 0.86

24(12)

76(38) 0.01

46(23)

54(27) 0.89

Radiation response

Overall response

Clinical Cancer Research 1825

apy using the Berkson-Gage method, and statistical significance

was determined using log-rank analyses (20). Actuarial multi-

variate analyses were performed using the Cox proportional

hazards models (20). The cause of death was not known in one

patient, and this patient was excluded from all of the actuarial

calculations except for overall survival.

RESULTS

The expression of proteins that regulate apoptosis was

investigated by determining the immunohistochemical staining

of p53, pRb, and bcl-2. Positive nuclear staining for p53 in

bladder cancer usually indicates that the mutant form is being

expressed (21), whereas it is the loss of pRb nuclear staining that

is indicative of altered expression (22). In contrast, positive

staining for bcl-2 usually represents overexpression of the func-

tional protein (23). In the study cohort, altered p53 and pRb

expression was seen in 56 and 30% of the tumors, respectively.

Prior investigations have centered on the relationship of p53

(12) and pRb (5) expression to radiation response and patient

outcome, and in this report, the results with bcl-2 are integrated

with these other findings.

The overexpression of bcl-2 was observed in 47% of the

tumors (50 of 107 tumors). Table 1 displays the distribution of

patients by bcl-2 staining, other potential prognostic factors, and

radiation response. The only potential prognostic factor that

correlated with bcl-2 expression was tumor grade, although a

weak relationship was also seen with patient age. bcl-2 staining

was not associated with p53 or pRb staining status. The most

significant finding was that bcl-2 correlated with radiation

response.

Radiation response was evaluated using three variables.

Overall response was assessed by dividing patients into those

that exhibited clinical-to-pathological downstaging, no differ-

ence in the clinical and pathological stages, and clinical-to-

pathological upstaging. bcl-2 was a strong correlate of overall

radiation response. The main difference between those that were

bcl-2 negative and those that were bcl-2 positive was in the

proportion that were upstaged. Further evaluation was facilitated

by grouping the patients into dichotomous variables that meas-

ured downstaging (downstaged patients versus all others) and

upstaging (upstaged patients versus all others) separately. The

results in Table 1 demonstrate that the positive staining of bcl-2

correlated only with upstaging and not with downstaging. Thus,

the overexpression of bcl-2 seemed to promote radiation

resistance.

Other prospective factors that could affect radiation re-

sponse were also investigated. Table 2 shows that the only

factors identified in the univariate analyses were pretreatment

creatinine level, pRb expression, and bcl-2 expression. Whereas

bcl-2 staining status was the most significant correlate of up-

staging, pRb staining status and creatinine level were more

predictive of downstaging.

Multivariate logistic regression confirmed that bcl-2 cx-

pression correlated independently with upstaging (Table 3). The

only other factor predictive of upstaging was positive pRb

staining, and the relationship was considerably weaker. The

findings indicate that the combination of bcl-2 overexpression

and normal pRb expression (bcl-2-positive and pRb-positive

Table I Distribution of patients by bcl-2, other potential prog

factors, and radiation response

nostic

Percentage (n)

bcl-2 bcl-2

negative positive P’

Potential prognostic factors

Clinical stage T2

T3a

T3b

Grade 2

3

Morphology Papillary

Sessile/mixed

Tumor size (cm) <5

No. of tumors 1

>1

No

Yes

Male

Female

�65

>65

<12�l2

�20

>20

�; 1.5

>1.5NormalHydronephrosis

Apoptosis (%) �l

>l�3

>3

p53 staining Negative

Positive

pRb staining NegativePositive

parameters

Downstaged

Unchanged

Upstaged

Downstaging Yes

No

Upstaging Yes

No

Path-CR” Path P0Path >P0

(1 �2 analysis.I, Lymph-vasc mv.. lymphatic vascular invasion.

C HGB, hemoglobin.�1 Path-CR, pathological complete response.

staining) connotes a very poor response to irradiation. Table 4

shows the relationship of combined staining results to radiation

response and demonstrates this to be the case. For those with

bcl-2-positive and pRb-positive tumors, the rate of upstaging

was 33%, despite the fact that high-dose preoperative radiation

was administered. Multivariate logistic regression analysis also

showed that altered pRb expression (pRb-negative staining) was

the principal correlate of downstaging (Table 3).

Fig. 1 illustrates the relationship of bcl-2 staining to actu-

anal patient outcome in terms of local control, distant metastasis

freedom, and overall survival. The overexpression of bcl-2 was



1826 bcl-2, pRb, and Bladder Cancer Radiation Response

Table 2 Factors predictive of radiation response: univariate analyses

A. Correlates of tumor response”

Percentage (n)

Downstaged Unchanged Upstaged P”

pRb staining status

bcl-2 staining status

NegativePositive

Negative

Positive

86 (24)61 (41)

68 (39)

70(35)

7 (2) 7 (2)19(13) 19(13)

25 (14) 7 (4)

6(3) 24(12)

0.OY

0.004

B. Correlates of downstaging”

Percentage (n)

P”Downstagcd Not downstagcd

Creatinine

pRb staining status

�1.5

> 1 .5

NegativePositive

73 (60)

44 (4)86 (24)61 (41)

27 (22)

56 (5)

14 (4)39 (26)

0.07

0.02

C. Correlates of upstaging”

Percentage (n)

P”Upstaged Not upstaged

pRb staining status


NegativePositive

Negative

Positive

7 (2)19(13)

7 (4)

24 (12)

93 (26)81 (54)

93 (53)

76 (38)

0.14

0.014

C’ Only factors with P < 0.15 are shown.

b �2 analysis.

C. �2 for linear association.

Table 3 Factors p

1

redictive of radiation response:

ogistic regression analyses

multivariate

End points Prognostic factors P

Downstaging Normal creatininc

Negative Rb staining

0.14

0.02

Upstaging Positive bcl-2 staining

Positive Rb staining

0.006

0.05

associated with significantly worse local control. Although

trends were seen for lower distant metastasis freedom and

overall survival rates when bcl-2 was overexpressed, the differ-

ences were not significant. An examination of other factors that

correlated with local control is shown in Table 5. In addition to

bcl-2 staining, serum creatinine level, IVP result, and the radi-

ation response parameters were significantly associated with

local control; the BUN level was of borderline significance.

Dividing bcl-2 staining into three groups (0, 1 + and 2+, and

3+ and 4+ staining) revealed that all five patients that had

tumors with the highest bcl-2 staining intensity failed locally.

This grouping of bcl-2 also correlated significantly with radia-

tion response.

Cox proportional hazards models were used to determine

the independence of bcl-2 as a predictor of actuarial local

control (Table 6). Inclusion of all of the significant factors in

Table 5, including bcl-2 as a dichotomous covariate (no staining

versus any staining), revealed that bcl-2, downstaging, and BUN

levels were independently associated with local control. In fact,

when only pretreatment factors were considered, bcl-2 was the

dominant correlate of local control. These data suggest that

bcl-2 overexpression led to reduced radiation response and,

consequently, to lower local control rate.

Table 4 Impact of combined bcl-2 and pRb staining on radiation

response

Percentage (n)

Down-bcl-2/pRb staining staged Unchanged

Up-staged Response”

bcl-2 neg”/pRb neg 85 (1 1) 15 (2) 0 (0) Favorable

bcl-2 ncg/pRb posc or 69 (38) 20 ( I 1) 1 1 (6) Intermediate

bcl-2 pos/pRb neg

bcl-2pos/pRbpos 59(16) 7(2) 33(9) Poor

C, p < 0.015 trended x2.1’ Neg, negative.C Pos, positive.

DISCUSSION

bcl-2 is instrumental in governing the response of cells to

irradiation. bcl-2 expression is elevated after irradiation (24),

and the apoptotic response to irradiation is ablated by bcl-2 (4,

25, 26). The action of bcl-2 in the latter is multifaceted and

seems to involve p53-dependent and -independent mechanisms.

When bcl-2 is overexpressed, p53-dependent apoptosis is re-

duced (25, 27), and nuclear accumulation of p53 may be

blocked (28). bcl-2 also affects cell death through interference

with p53-induced cell cycle arrest (28), the reduction of gener-

ated reactive oxygen species (29), and the blocking of the

protease cell death pathway (30).

Our data corroborate the premise that bcl-2 is central in the

response of muscle-invasive bladder cancer to irradiation. Con-

sistent with reports that bcl-2 negates the apoptotic response to

irradiation, the tumors that overexpressed bcl-2 were found to

have a substantially increased rate of clinical-to-pathological

upstaging. Indeed, bcl-2 was the most significant independent



InLocal Control

. p0.03-.- bcl-2 Negative-B-. bcl-2 Positive

0.8

0.6

0.4

0.2

An

,l Percentage P”

Potential prognostic factors

BUN

Creatinine

IVP



Radiation response parameters

Overall response

MONTH:

bcl-2 Neg:

bcl-2 Pos:

0 60 96

56 30 14

50 19 8

0 � 1’O � 20 � 30 40 50 60 70 80 90 I#{212}O

Freedom From Distant Metastasis

Downstaging

Upstaging

p0.l9

MONTH:

bcl-2 Neg:

bcl-2 Pos:

0 60 96

56 30 14

50 17 8

PC

0.03

0.0060.(X)5

0.07

0.02

I � 10 � 20 � 30 40 50 60 70 80 90 i#{244}o

Overall Survival

0 60 96

57 30 14

50 19 81’I’I’I’I,I’

40 50 60 70 80 90 100

MONTH:

bcl-2 Neg:

bcl-2 Pos:

predictor of upstaging, which is a relatively infrequent event in

the setting of 50-Gy preoperative radiotherapy administered

4-6 weeks before cystectomy. Of the 107 patients analyzed,

only 16 were upstaged. and 75% ofthese were bcl-2 positive by

immunohistochemical analysis. Yet only 24% of those that

exhibited bcl-2 overexpression were upstaged. suggesting that

although bcl-2 is important, it is not the sole determinant of

radiation response. The results with pRb establish that abnor-

malities in other proteins that regulate apoptosis may comple-

ment hcb-2 in identifying patients with a high likelihood of

responding or not responding to irradiation.

The data presented verify our prior conclusions that pRb is

a valuable marker of radiation response (5). Whereas the ab-

sence of detectable pRb expression by immunohistochemistry is

a strong correlate of clinical-to-pathological downstaging, bcl-2

overexpression was a strong correlate of clinical-to-pathological

upstaging. Some overlap was evident, however, because func-

tional pRb expression was also significantly aligned with up-

staging. This apparent incongruity is clarified by examining the

characteristics of the associations of pRb to the radiation re-

sponse parameters. The loss of pRb immunohistochemical stain-

ing was significantly related to downstaging. These results are


Table 5 Actuarial 5-year correlates of local control

:�� 0.8.

� 0.6

{_ 0.4

S

,- 0.2.

� ISA

Months after cystectomy

Fig. I Actuarial local control. freedom from distant metastasis. and

overall survival for the entire study cohort. The numbers of patients

available for the analyses at 60 and 96 months are shown in the table

inserts. #{149},bcl-2-negative staining; EL bcl-2-positive staining.

�20 87 95

>20 19 81 0.06�l.5 81 93>1.5 9 77 0.05Normal 58 96

Hydrone- 30 85 0.05

phrosis

No 57 98Yes 50 86 0.03

None 56 98

Intermediate 45 90

Strong 5 0 <0.0001

Downstaged

Unchanged

Upstaged

Yes

No

No

Yes

74 97

16 88

16 78 0.02

74 97

32 83 0.008

90 95

16 78 0.01

C, Log-rank test: only correlations with P < 0.15 are shown.

Table 6 Cox proportional hazards multivariate analysis of factors

predictive of local control

x-All significant prognostic factors

BUN 4.7

Downstaging 7.7

bcl-2 staining status (2 groups) 7.8

Significant pretreatment prognostic factors

BUN 3.3

bcl-2 staining status (2 groups) 5.7

(C Cox regression: degrees of freedom = I for all variables.



1828 bcl-2, pRb, and Bladder Cancer Radiation Response

analogous to several in vitro studies that indicate that when pRb

function is absent (3) or suppressed (13, 14), apoptosis is en-

hanced in response to irradiation.

The contrary scenario we describe is the association of

wild-type pRb expression with clinical-to-pathological upstag-

ing after preoperative radiotherapy. The expression of pRb

mediates p53 effects downstream through p21waf�� and

the cyclin-dependent kinases (3 1). Recent evidence from

McConkey et a!. (32) points to pRb as a critical constituent in

the regulation of apoptosis in human bladder tumor cells via the

ceramide pathway. They observed that wild-type pRb expres-

sion reduced the induction of apoptosis from ceramide. An

ostensible downstream target for ceramide is the ceramide-

activated protein phosphatase that dephosphorylates pRb, block-

ing cells in G0-G1 (33). Under these circumstances, it seems that

pRb functions to limit apoptosis by promoting cell cycle arrest

and DNA damage repair, although additional studies are needed

to confirm this mechanism. Because ceramide production is

enhanced by ionizing radiation and seems to be a consequential

contributor to the apoptotic response to ionizing radiation (34),

the clinical-to-pathological upstaging we describe when wild-

type pRb is expressed could be accounted for in part by effects

on the ceramide pathway. Whether the ceramide pathway is

independent of p53 remains to be determined.

The multivariate logistic regression analyses presented

(Table 3) demonstrate that the relationships of bcl-2 and pRb to

radiation response are independent. The composite bcl-2/pRb

staining profiles (Table 4) affirmed that patients with the worst

radiation response were those having bcl-2-positive/pRb-posi-

tive tumors, in which only 59% were downstaged and 33% were

upstaged. Those with bcl-2-negative/pRb-ncgative tumors had

the best radiation response, with downstaging in 85% and up-

staging in none.

The impact of bcl-2 on radiation response was mirrored to

a degree in patient outcome. Local control was significantly

inferior when bcl-2 was overexpressed. No significant differ-

ences in distant metastasis freedom or overall survival based on

bcl-2 staining status were seen, although trends were evident.

The influence of radiation therapy on the end points of distant

metastasis freedom and overall survival resides with the effect

on local control. Whereas we have established that local failure

in bladder cancer is significantly aligned with the development

of distant metastasis (35) and that preoperative radiation has a

significant effect on this process (36), many patients will have

micrometastasis at presentation and will not benefit from im-

proved tumor control locally. The propensity for muscle-inva-

sive bladder cancer to metastasize distantly early dilutes the

contribution of local control to freedom from distant metastasis,

and as such, larger patient numbers would be needed to dem-

onstrate a significant correlation of bcl-2 staining with this end

point. As has been described previously, pRb was a correlate of

distant metastasis freedom and overall survival only for patients

in this cohort with stage T3b disease (5).

The findings of Glick et al. (37) in invasive transitional cell

carcinomas of the bladder are in line with ours in that bcl-2

expression did not correlate with survival. King et a!. (38) did

not observe any relationship between the degree of bcl-2 cx-

pression and tumor progression, yet they found a relationship

with tumor grade; higher tumor grade was significantly associ-

ated with a higher degree of bcl-2 expression and increased

proliferative indices. We also found that high-grade tumors

more frequently overexpressed bcl-2 (Table 1 ). These data,

taken together with the radiation response results presented,

indicate that bcl-2 overexpression in transitional cell carcinomas

of the bladder is a predictor of more aggressive tumor behavior.

However, bcl-2 overexpression has also been found to be pre-

dictive of improved freedom from progression and/or survival in

carcinomas originating at some sites other than the bladder

(39-42). In these studies, radiotherapy was not a major com-

ponent of treatment. Because our findings show that bcl-2

overexpression dampened radiation response and consequently

lessened local control, it is probable that under similar treatment

conditions, bcl-2 expression may be an equally adverse prog-

nostic factor for tumors of these other sites as well.

In conclusion, bcl-2 expression as measured by immuno-

histochemical staining proved to be a significant marker of

impaired radiation response that complemented the loss of pRb

staining, a significant marker of improved radiation response.

These two proteins contribute to the regulation of the apoptotic

response to ionizing radiation, and it is hypothesized that char-

acterization of additional factors (e.g., bax, cyclin Dl) in the

apoptotic pathway will lead to defining a molecular phenotype

that predicts for radiation response much more accurately than

classical clinical parameters.

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1997;3:1823-1829. Clin Cancer Res A Pollack, C S Wu, B Czerniak, et al. of radiation response in muscle-invasive bladder cancer.Abnormal bcl-2 and pRb expression are independent correlates

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