Bcl-2 proto-oncogene expression in low- and high-gradeprostatic intraepithelial neoplasiaS. BALTACI, D. ORHAN*, G. OÈ ZER, OÈ . TOLUNAY* and O. GOÈ GÏ UÈ S

Departments of Urology and *Pathology, University of Ankara School of Medicine, Ankara, Turkey

Objective To determine the incidence of bcl-2 protein

expression in low- and high-grade prostatic intra-

epithelial neoplasia (PIN) lesions, and to explore the

role of bcl-2 in prostatic tumorigenesis.

Materials and methods Immunoreactivity for bcl-2 was

examined in 10 samples of benign prostatic hyper-

plasia (BPH), 13 of primary prostatic adenocarci-

noma, 15 of high-grade PIN and 18 of low-grade PIN.

All immunostaining results were scored for the

approximate percentage of positive tumour cells and

relative immunostaining intensity (score range 0±12).

Results In all BPH samples, bcl-2 staining was detected

consistently in the basal cell layer of the ducts and

acini, but no staining was ever apparent in luminal

cells. The immunoreactivity for bcl-2 was hetero-

geneous in the prostatic carcinomas and bcl-2 protein

expression was present in six samples. In these six bcl-

2-positive tumours, the mean (range) staining score

was 1.15 (1±6). There was detectable expression of

bcl-2 in low- and high-grade PIN in all cell layers;

immunoreactivity was present in 10 of 15 high-grade

PIN lesions, with a mean (range) score of 1.14 (1±4),

and in 12 of 18 samples of low-grade PIN, with a

mean (range) score of 1.77 (1±6).

Conclusions The high incidence of bcl-2 protein expres-

sion in low- and high-grade PIN lesions suggests that

bcl-2 protein expression is associated with early

prostate tumorigenesis.

Keywords Bcl-2, prostatic intraepithelial neoplasia,

prostate cancer, immunohistochemistry

Introduction

The bcl-2 oncoprotein is known to inhibit programmed

cell death or apoptosis [1]. Within benign or normal

prostatic epithelium the proto-oncogene bcl-2 is normally

expressed in androgen-independent basal cells but not in

the differentiated luminal secretory cells that are andro-

gen-dependant [2,3]. In prostate cancer, it has been

suggested that bcl-2 plays a role in hormone resistance

and several studies have shown enhanced expression of

bcl-2 in hormone-refractory prostatic adenocarcinomas

[3±5]. Bcl-2 immunoreactivity has been reported in 25±

62% of untreated prostate cancers and staining in the

cancer areas is heterogeneous [3±6]. However, con¯icting

results have been reported for the incidence of bcl-2

immunoreactivity in high-grade prostatic intraepithelial

neoplasia (PIN), which has been shown by several

authors to be a precursor of prostatic adenocarcinoma

[3,6±9]. In two studies strong bcl-2 immunoreactivity

was observed in all foci of high-grade PIN [3,7] but in

three studies, only 17±35% of high-grade PIN lesions

were bcl-2 immunopositive [6,8,9]. However, there are

no reports of the expression of bcl-2 protein in low-grade

PIN. The purpose of the present study was to investigate

the incidence of bcl-2 staining in low- and high-grade PIN

lesions, as bcl-2 protein expression may be important in

prostate tumorigenesis.

Materials and methods

Formalin-®xed, paraf®n-embedded tissue samples from

10 cases of BPH, 18 low-grade PIN lesions, 15 high-grade

PIN lesions and 13 primary prostatic adenocarcinomas

were examined. The BPH samples were obtained from 10

suprapubic or retropubic prostatectomies performed for

BPH. The adenocarcinomas were removed by radical

prostatectomy from 13 patients who had received no

prior therapy. The 18 cases of low-grade and seven of

high-grade PIN were identi®ed in needle-biopsy speci-

mens. Because eight of the radical prostatectomy

specimens also contained high-grade PIN coexisting

with carcinoma, there were 15 high-grade PIN lesions

available. Tissue samples were re-examined by one

pathologist and the tumours graded according to the

Gleason system. PIN was classi®ed according to nomen-

clature described by an international consensus con-

ference held in 1989 [10].

Sections of 5 mm were mounted on poly-L-lysine-

coated slides and deparaf®nized. Antigens were retrievedAccepted for publication 31 August 1999

BJU International (2000), 85, 155±159

# 2000 BJU International 155

by subjecting the slides to microwaves (700 W) for

4r5 min in 1 L of citrate solution. Sections were then

exposed to anti-bcl-2-124 monoclonal primary antibody

(Biogenex, Santa Barbara, CA, USA; dilution 1 : 20) for

1 h. After washing with PBS, the sections were incubated

with biotinylated secondary antibody (Dako, Carpenteria,

USA) for 15 min. After incubation for 15 min with

streptavidin-peroxidase complex (Dako), sections were

exposed to AEC chromogen (Dako) for 5 min; they were

counterstained with Mayer's haematoxylin. Sections of

human tonsil tissue were concurrently immunostained

with the anti-bcl-2 antibody to provide a positive control

and substitution of the anti-bcl-2 antibody with normal

mouse serum was used as a negative control.

The immunostaining results were scored as described

by Krajewska et al. [6]; the percentage of positive tumour

cells was graded as: 0, none; 1, 25%; 2, 26±50%; 3, 51±

75%; and 4, 76±100%. The immunostaining intensity

was rated as: 0, none; 1, weak; 2, moderate; and 3,

intense. Specimens were considered immunopositive

when o1% of the tumour cells had clear evidence of

immunostaining. As the tumours were sometimes

heterogeneous, for some analyses a score was calculated

in which the percentage positive rating was multiplied by

the intensity rating (score range 0±12). The Kruskal±

Wallis ANOVA was used to compare the groups, with

P<0.05 considered to indicate signi®cant differences in

all tests applied.

Results

In the 10 samples of BPH, positive staining of bcl-2 was

always present in the basal cells of the prostatic ducts and

acini; in contrast, there was no staining in luminal cells

in any of the tissues examined (Fig. 1). Of the 13 samples

of prostatic adenocarcinoma, six showed at o1% bcl-2-

positive tumour cells. Staining for bcl-2 was hetero-

geneous in these tumours, and intensely stained and

entirely negative areas were recorded in each tumour

(Fig. 2). The immunostaining intensity and percentage of

immunopositive cells are shown in Table 1. There was no

correlation between the Gleason score and bcl-2 score, as

the Gleason scores of all tumours were 6±8.

Of the 18 samples of low-grade PIN, 12 were bcl-2

immunopositive; the expression of bcl-2 in these

samples was apparent in all cell layers (Fig. 3). The

immunostaining variables are shown in Table 1. Bcl-2

reactivity was detected in 10 of the 15 high-grade PIN

lesions; like the low-grade PIN lesions, bcl-2 was

expressed in all cell layers (Fig. 4). The immunostaining

variables are shown in Table 1; there were no

statistically signi®cant differences in the staining

scores among the three groups.

Discussion

The present study, like others, showed that bcl-2 is

normally expressed in the basal cell layer of hyperplastic

glands [3,7]. Considering previous reports that the basal

epithelial cells, in contrast to the secretory cells, are

resistant to the programmed cell death induced by

androgen withdrawal [11], the present results are

consistent with the interpretation that the basal cells

represent the stem cell layer in the adult prostate gland.

The present study, using an immunohistochemical

approach, is the ®rst to characterize the expression of bcl-

2 protein in low-grade PIN. Although ®ve studies have

reported the incidence of bcl-2 immunoreactivity in high-

grade PIN [3,6±9], there have been no reports to our

knowledge of bcl-2 expression in low-grade PIN.

In the present study, bcl-2 immunoreactivity was

detected in 12 of 15 high-grade PIN samples; in two other

studies strong bcl-2 immunoreactivity in all high-grade

PIN samples assessed [3,7] was reported, whereas in

another three studies, only 17±35% of high-grade PIN

Fig. 1. Immunostaining of bcl-21-24 in the basal cells of prostatic

acini in BPH.r100.

Fig. 2. Bcl-21-24 positive prostatic carcinoma cells.r100.

156 S. BALTACI et al.

# 2000 BJU International 85, 155±159

lesions were bcl-2 positive [6,8,9]. This variation might

be attributable to differences in the antibodies used and

the relative preservation of antigen epitopes. The

microwave treatment method for antigen unmasking

used in here may increase antigenic preservation,

resulting in higher bcl-2 positivity.

Of the 13 untreated primary prostatic adenocarci-

nomas, there was bcl-2 immunoreactivity in six; the

previously published incidence varies widely, at 25±62%

[3±6]. Some of this variation might be attributable to

the threshold level for positivity or to differences in the

patient populations presenting to different institutions.

Alternatively, it might also re¯ect differences in the

antibodies used and the relative preservation of antigen

epitopes. However, in all studies, including this, bcl-2

staining was always heterogeneous in the cancer areas.

There are con¯icting results about the relationship

between bcl-2 immunoreactivity and tumour grade. In

two studies the expression of bcl-2 tended to be more

frequent in high-grade tumours [6,7], but in two other

studies [3,12], there was no correlation between these

variables. In the present study it was not possible to

compare the Gleason score and bcl-2 immunoreactivity,

as the range of Gleason scores was too narrow.

Although interobserver variability with low-grade PIN

limits its use clinically utility, and many pathologists do

not report this ®nding [13], low-grade PIN corresponds to

`very mild' to `mild' dysplasia. The present results show

that two-thirds of low-grade PIN samples were bcl-2

immunoreactive and like high-grade PIN lesions, bcl-2

was expressed in all cell layers. However, the evidence

supporting a relationship between high-grade PIN and

invasive carcinoma of the prostate is strong, and is

supported by histological, immunohistochemical and

genetic studies. Several authors suggested high-grade

PIN to be a precursor of prostate cancer [14,15].

Although Myers and Grizzle [16] reported that strong

expression of bcl-2 typically occurs in advanced-stage

prostatic adenocarcinomas, and therefore probably

represents late events in the development of prostatic

adenocarcinoma, we suggest (as did Stattin et al. [7] and

Colombel et al. [3]) that bcl-2 expression, probably by

prolonging cell survival, renders the PIN cells more

vulnerable to other oncogenes, which can induce further

steps in tumorigenesis. Thus bcl-2 expression may be

involved in early prostate tumorigenesis.

Androgen deprivation has been shown to induce bcl-2

expression in the rat ventral prostate in the short-term

[4] and several studies have shown enhanced expression

of bcl-2 protein in hormone-refractory prostatic adeno-

carcinomas, suggesting a role in the development of

androgen insensitivity [3,4]. In contrast, HaÈggman et al.

Table 1 The immunostaining results for bcl-2 expression in primary prostate adenocarcinomas, and low- and high-grade PIN

Mean (SD)

Tissue Immunostaining intensity % of immunopositive cells Bcl-2 score

Primary prostatic carcinoma 0.92 (1.18) 7.69 (12.68) 1.15 (1.77)

Low-grade PIN 1.16 (0.98) 12.94 (18.21) 1.77 (1.98)

High-grade PIN 0.85 (0.89) 7.86 (10.75) 1.14 (1.46)

P 0.698 0.594 0.564

Fig. 3. Immunostaining for bcl-21-24 in epithelial cells of prostatic

acini showing low-grade PIN.r100.Fig. 4. Epithelial cells of high-grade PIN staining positively withanti-bcl-2124 monoclonal antibody.r100.

BCL-2 EXPRESSION IN PIN LESIONS 157

# 2000 BJU International 85, 155±159

[17] and Ferguson et al. [18] reported a reduced

frequency and extent of PIN after maximal androgen

blockade, which suggests that PIN is androgen-

dependent. However, we and others have shown that

17±100% of high-grade PIN lesions express bcl-2

immuno-reactivity [3,6±9]. Additionally, van der

Kwast [19] reported that PIN was still present in a few

prostatectomy specimens of patients treated for 3 months

with androgen-ablation therapy. Thus, as suggested by

Stattin et al. [7], the question of whether bcl-2-expressing

prostatic cancer cells are hormonally dependent seems to

be complex. These apparently contradictory results

suggest that the interaction between bcl-2 and other

anti-apoptotic genes, e.g. bcl-x and mcl-1, and the pro-

apoptotic gene bax, determines the difference in androgen

dependence in PIN and tumour cells [6].

In the present study, the expression of bcl-2 was not

evaluated by in situ hybridization. A recent study

described discrepancies between the detection of bcl-2

by in situ hybridization and immunocytochemistry in

human prostate cancer tissues [20], and this point should

be considered in future studies.

In conclusion, although the present study included

only relatively few specimens of low- and high-grade

PIN, the results suggested that bcl-2 is expressed in both

grades of PIN. The high incidence of bcl-2 immuno-

reactivity in PIN lesions suggests a role for bcl-2 in early

prostate tumorigenesis. Further studies with this proto-

oncogene and other bcl-2 family genes are needed to

understand the role of these genes in prostate

tumorigenesis. Such studies with a large series of

patients may also address the value of bcl-2 as a

prognostic marker.

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AuthorsS. Baltaci, MD, Associate Professor.

D. Orhan, MD, Assistant Professor.

G. OÈ zer, MD, Urologist.

OÈ . Tolunay, MD, Professor.

O. GoÈgÏuÈ s, MD, Professor.

Correspondence. Dr SuÈ mer Baltaci, Fahrettin Altay Cad.,

GuÈ zin Sok. No:50/6, 06130 Aydinlikevler, Ankara, Turkey.

e-mail: sbaltaci@hotmail.com

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