318
Association of Bcl-2 Protein Expression with
Gallbladder Carcinoma Differentiation and Progression
and Its Relation to Apoptosis
Tetuo Mikami, M.D.1
1
Nobuyuki Yanagisawa, M.D.
Hiroyuki Baba, M.D.2
Morio Koike, M.D.2
Isao Okayasu, M.D.1
1
Department of Pathology, School of Medicine,
Kitasato University, Sagamihara, Japan.
2
Department of Pathology, Tokyo Metropolitan
Komagome Hospital, Tokyo, Japan.
The authors thank Ms. K. Hana, Ms. Y. Numata,
and Ms. J. Hashimoto for their expert technical
assistance and Dr. T. Kayano for providing histo-
logic materials.
Address for reprints: Tetuo Mikami, Department of
Pathology, School of Medicine, Kitasato University,
1-15-1, Kitasato, Sagamihara-si, Kanagawa, 228-
8555, Japan.
Received March 24, 1998; revision received June
22, 1998; accepted June 22, 1998.
© 1999 American Cancer Society
BACKGROUND. Bcl-2 protein is believed to play a role in neoplasia by inhibiting
tumor cell apoptosis. To assess its contribution to gallbladder tumorigenesis and
cancer progression, an immunohistochemical study was performed.
METHODS. Fifteen adenomas and 68 adenocarcinomas were immunohistochemi-
cally and histopathologically investigated for the relation of Bcl-2 expression to p53
status, apoptosis (apoptotic index, AI), and proliferation activity (mitotic index, MI;
Ki-67 labeling index, Ki-67 LI).
RESULTS. The Bcl-2 score, based on intensity and extent, decreased in the order of
adenoma, well-differentiated, and moderately to poorly differentiated adenocar-
cinoma. Early stage carcinomas demonstrated significantly higher Bcl-2 scores
than their advanced counterparts (P ⬍ 0.05). On the other hand, p53 score, MI,
Ki-67 LI, and AI increased in the same order. The Bcl-2 negative adenocarcinomas
displayed higher AI and AI-to-MI ratios than the Bcl-2 positive group, especially in
the early stage, well-differentiated lesions. A significantly positive correlation be-
tween MI(r ⫽ 0.549) or Ki-67 LI(r ⫽ 0.446) and AI was observed. In early stage
carcinomas, adenomatous components in the lesions were found more frequently
in the polypoid lesions than in the nonpolypoid lesions (P ⬍ 0.05).
CONCLUSIONS. Expression of Bcl-2 protein in gallbladder tumors appears to be
positively associated with tumor cell differentiation and inversely with tumor
progression. It may thus play a role in regulating carcinoma growth, especially in
the early stage of tumorigenesis. It is believed that the polypoid carcinomas may
arise from preexisting adenomas but the nonpolypoid carcinomas may arise as de
novo carcinoma. Cancer 1999;85:318 –25. © 1999 American Cancer Society.
KEYWORDS: gallbladder neoplasms, Bcl-2, p53, immunohistochemistry, apoptosis.
A lteration of bcl-2, the B-cell leukemia and lymphoma 2 gene, was
first detected in B-cell lymphomas/leukemias accompanying the
t(14;18) chromosomal translocation. The bcl-2 mRNA encodes a 26
kilodalton (kD) protein that is known to inhibit apoptosis, thus pro-
longing the cell life span. Its aberrant expression can thereby modify
tumor progression or influence biologic behavior of cancers.1 Ele-
vated expression has been reported for a variety of human neo-
plasms.2–15
In addition to the effect of Bcl-2 protein on apoptosis, other roles
related to tumor cell differentiation have been proposed. For exam-
ple, Bcl-2 expression was found to be related to differentiation of
colorectal adenocarcinoma.5 It is expressed prominently in intestinal
type gastric adenocarcinomas but rarely in the diffuse type.6 To
investigate the greater contribution of Bcl-2 protein to gallbladder
tumorigenesis, we analyzed the expression in relation to histologic

type, histologic stage, proliferation activity (mitotic
index [MI] and Ki-67 labeling index [Ki-67 LI]), and
apoptosis (apoptotic index [AI]). Because it has been
reported that the bcl-2 gene transcription is down-
regulated by the p53 protein, a tumor suppressor gene
product,16,17 we also investigated p53 expression im-
munohistochemically for comparison.
MATERIALS AND METHODS
Tissue samples from 68 patients with carcinoma and
15 patients with adenoma of the gallbladder were col-
lected from the files of surgery patients at Kitasato
University Hospital, Kitasato University East Hospital,
Tokyo Metropolitan Komagome Hospital, and Kudan-
zaka Hospital. The World Health Organization (WHO)
histologic typing was applied, and the American Joint
Committee on Cancer (AJCC) pT classification for in-
vasion was determined as follows: pTis, carcinoma in
situ; pT1, tumor invading the mucosa or muscular
layer; pT2, tumor invading the perimuscular connec-
tive tissue; pT3, tumor invading beyond the serosa
and/or into one adjacent organ; pT4, tumor extending
more than 2 cm into the liver and/or into two or more
adjacent organs.18 –20 The pTis and pT1 tumors were
grouped as early stage carcinomas, and the pT2, pT3,
and pT4 tumors were grouped as advanced stage car-
cinomas, after Mizumoto et al.21 When a tumor had
replaced the epithelium of the Rokitansky–Aschoff si-
nus but had not invaded the stroma, it was classified
as pTis. Cases of dysplasia and adenoacanthoma were
excluded.
Tissue slices were fixed routinely in 10% formalin
and embedded in paraffin. Four-micrometer-thick
sections were cut and used for hematoxylin and eosin
staining. For counting apoptotic figures and mitotic
figures, because of intratumoral heterogeneity, atten-
tion was concentrated on the intramucosal or luminal
surface areas. Areas of severe inflammatory cell infil-
tration and necrosis were excluded. The distinctive
morphologic features of apoptotic cells, marked con-
densation of chromatin and cytoplasm with or with-
out nuclear fragments, as described by Kerr et al.,22
were applied for their detection in hematoxylin and
eosin-stained sections under high power magnifica-
tion (Fig. 1). The percentage counts of mitotic figures
and apoptotic bodies were defined as the mitotic in-
dex (MI) and the apoptotic index (AI), respectively,
after examining at least 2000 nuclei for each case.
Immunohistochemistry
Immunohistochemical staining of 4-␮m-thick paraffin
sections was achieved by using a commercial kit
(DAKO, Glostrup, Denmark) for the labeled streptavi-
din-biotin-peroxidase complex method. The primary
Bcl-2 Expression in Gallbladder Tumor/Mikami et al. 319
FIGURE 1. Apoptotic cells (black arrow) identified in a well-differentiated
gallbladder adenocarcinoma. Note the condensation of chromatin separated
from the surrounding cells by a clear halo. The white arrow indicates a mitotic
figure (H&E; original magnification, ⫻400).
antibodies applied were anti-Bcl-2 (1/50 diluted,
monoclonal, clone 124; DAKO), anti-p53 (1/200 di-
luted, monoclonal, clone Do-7; Novocastra, Newcastle
upon Tyne, United Kingdom), and anti-Ki-67 (1/200
diluted, polyclonal; DAKO). After blocking endoge-
nous peroxidase with 0.3% hydrogen peroxide for 15
minutes, a 15-minute microwave pretreatment in ci-
trate buffer was performed for the retrieval of the three
antigens.23 Then, the sections were incubated with the
primary antibodies for overnight at 4°C. 3,3⬘-Diamino-
benzidine (DAB) was used as the final chromogen, and
nuclei were counterstained with hematoxylin or with
aqueous methyl green.
Immunoreactivity for Bcl-2 and p53 was quantified
according to the classification of Sinicrope et al.2 to
evaluate both of the staining intensity and the amount of
stained cells. The staining intensity was scored as fol-
lows: 1, weak; 2, moderate; and 3, intense. In the Bcl-2
staining, the internal controls, infiltrating lymphocytes,
were set at a staining intensity of 3. In the p53 staining,
a p53 positive esophageal carcinoma sample was em-
ployed as a positive control. Positive tumor cells were
expressed as the percentage of the total number of tu-
mor cells and assigned to one of five categories: 0, ⬍5%;
1, 5–25%; 2, 25–50%; 3, 50 –75%; and 4, ⬎75%. Immuno-
reactive scores for each tumor were calculated by mul-
tiplication of the values for the two parameters (Figs. 2,
3). To generate the Ki-67 LI, more than 1000 tumor cells
were examined per case, and those that were unequiv-
320 CANCER January 15, 1999 / Volume 85 / Number 2

FIGURE 2. Serial sections through a well-differentiated gallbladder adenocarcinoma. (a) H&E-stained section (original magnification, ⫻400). (b) Bcl-2 is expressed
in the cytoplasm (scored at an intensity of 2). Lymphocytes showed stronger expression than carcinoma cells. (c) Positivity for p53 is only scattered and faint in
carcinoma cell nuclei (scored at an intensity of 0).

FIGURE 3. Serial sections through a moderately differentiated gallbladder adenocarcinoma. (a) H&E-stained section (original magnification, ⫻300). (b) Bcl-2 is
negative for carcinoma cells, whereas lymphocytes were stained positively. (c) p53 is diffusely positive in the carcinoma cell nuclei (scored at an intensity of 3).

ocally positive for nuclei staining were counted and cal-
culated as percentage values.
Statistical Analysis
Data are given as the mean standard ⫾ deviation.
Statistical analyses were performed by using the Fish-
er’s PLSD test as a post-hoc test for comparison of
three or more groups, Student’s t test for comparison
of two groups, chi-square test, and Pearson’s correla-
tion coefficient test with the computer software Stat-
view (Abacus Concepts, Berkeley, CA). P ⬍ 0.05 was
regarded as statistically significant.

TABLE 1
Comparison between Polypoid and Nonpolypoid, Well-Differentiated Early Adenocarcinoma of the Gallbladder
Carcinoma
Type No. only
Polypoid 5 2*
Nonpolypoid 16 14*
a
Values are expressed as mean ⫾ standard deviation.
b
Not significant.
* P ⬍ 0.05.
RESULTS
The 15 collected adenomas were all tubular adeno-
mas. The collected carcinoma lesions comprised 54
well-differentiated, 9 moderately differentiated, and 5
poorly differentiated adenocarcinomas. The 54 well-
differentiated lesions consisted of 21 early stage cases
(18 pTis and 3 pT1) and 33 advanced stage cases (28
pT2 and 5 pT3). Of the 21 early stage adenocarcino-
mas, 5 contained adenomatous components in the
carcinoma lesions, which consisted of 3 polypoid le-
sions and 2 nonpolypoid lesions. On the other hand,
16 early carcinomas without an adenomatous compo-
nent consisted of 2 polypoid lesions and 14 nonpol-
ypoid lesions (Table 1). Statistically, the adenomatous
components were found more frequently in polypoid
early stage adenocarcinomas than in their nonpol-
ypoid counterparts (P ⬍ 0.05). The moderately and
poorly differentiated adenocarcinomas, 14 in total,
were all advanced stage lesions (4 pT2, 6 pT3, and 4
pT4).
The Bcl-2 scores, p53 scores, Ki-67 LI, MI, and AI
were compared with the histologic type and stage (Fig.
4). The Bcl-2 scores decreased in the order of adenoma
(3.8 ⫾ 3.8 [mean ⫾ standard deviation]), well-differenti-
ated adenocarcinoma (1.7 ⫾ 2.3), and moderately to
poorly differentiated adenocarcinoma (0.1 ⫾ 0.3), the
difference among the three being significant (P ⬍ 0.05).
Early carcinomas (2.5 ⫾ 2.8) showed significantly higher
Bcl-2 scores than their advanced counterparts (0.9 ⫾ 1.7;
P ⬍ 0.01). There was no significant difference between
polypoid and nonpolypoid early stage carcinomas in
Bcl-2 score; however, three of the five lesions of early
stage, well-differentiated adenocarcinoma with adeno-
matous component were stained positively stained at
the adenomatous area.
The scores of the background gallbladder muco-
sae could be investigated in 50 cases, the scores of
which (0.62 ⫾ 0.90) were significantly lower than those
of adenoma, well-differentiated adenocarcinoma, and
early stage adenocarcinoma (P ⬍ 0.01). Focal Bcl-2
positivity was observed, but a specific link with histo-
Bcl-2 Expression in Gallbladder Tumor/Mikami et al. 321
Carcinoma with
adenomatous component Bcl-2 Scorea p53 Scorea
3* 2.60 ⫾ 3.29b 1.80 ⫾ 2.68b
2* 2.44 ⫾ 2.68b 3.94 ⫾ 4.73b
logical findings was not found except for intestinal
metaplasia.
Inversely, p53 scores increased in the order of
adenoma (0.6 ⫾ 1.6), well-differentiated adenocarci-
noma (4.3 ⫾ 4.6), and moderately to poorly differen-
tiated adenocarcinoma (6.4 ⫾ 5.5) and were higher in
advanced stage (5.3 ⫾ 5.0) than in early stage (3.4 ⫾
4.4) carcinomas, although the difference was not sig-
nificant.
Regarding proliferation activity, Ki-67 LI and MI
similarly increased in the order of adenoma (1.7 ⫾ 3.0
and 0.034 ⫾ 0.032, respectively), well-differentiated
adenocarcinoma (13.3 ⫾ 6.9 and 0.286 ⫾ 0.215, re-
spectively), and moderately to poorly differentiated
adenocarcinoma (19.7 ⫾ 12.6 and 0.425 ⫾ 0.248, re-
spectively). The MI was also significantly higher for
advanced stage (0.364 ⫾ 0.234) than for early stage
(0.202 ⫾ 0.168) carcinomas (P ⬍ 0.01).
Regarding apoptosis, AIs of well-differentiated tu-
mors (0.212 ⫾ 0.208) and moderately to poorly differ-
entiated tumors (0.350 ⫾ 0.233) were significantly
higher than AIs of adenomas (0.022 ⫾ 0.025; P ⬍ 0.05).
The difference between moderately to poorly differen-
tiated and well-differentiated tumors also was signifi-
cant (P ⬍ 0.05).
The carcinoma cases then were divided into two
groups, Bcl-2 positive and negative. A Bcl-2 score less
than 1 was judged as Bcl-2 negative, and a Bcl-2 score
more than 2 was judged as Bcl-2 positive. The Bcl-2
positive group consisted of 11 early stage and 10 ad-
vanced stage well-differentiated adenocarcinomas.
The Bcl-2 negative group consisted of 10 early stage
and 23 advanced stage well-differentiated adenocarci-
nomas and all 14 moderately to poorly differentiated
adenocarcinomas. The p53 score and the proliferation
activity assessed by Ki-67 LI showed no significant
difference between the two groups. However, the
Bcl-2 negative group demonstrated significantly a
higher AI, AI-to-MI ratio, and MI (Table 2). In early
stage, well-differentiated adenocarcinomas, both the
AI and the AI-to-MI ratio were higher in the Bcl-2
322 CANCER January 15, 1999 / Volume 85 / Number 2
FIGURE 4. Data for Bcl-2 scores, p53 scores, Ki-67 labeling indices
(Ki-67 LI), mitotic indices (MI), and apoptotic indices (AI) of gallbladder
tumors. n, Number of cases; AC, adenocarcinoma. (a) P ⬍ 0.05. (b) P ⬍
0.01.
negative group (0.317 ⫾ 0.282 and 1.503 ⫾ 1.100, re-
spectively) than in the Bcl-2 positive group (0.074 ⫾
0.117 and 0.443 ⫾ 0.579, respectively; P ⬍ 0.05). The AI
in the Bcl-2 negative (0.272 ⫾ 0.193), advanced stage,
well-differentiated adenocarcinomas was significantly
higher than that in the Bcl-2 positive group (0.132 ⫾
0.137), whereas the AI-to-MI ratio was not signifi-
cantly different (Fig. 5).
The AI and MI of adenomas and adenocarcino-
mas showed a positive correlation, which is demon-
strated in Figure 6 (r ⫽ 0.549, P ⬍ 0.0001). The AI and
Ki-67 LI also showed a positive correlation (r ⫽ 0.446,
P ⬍ 0.0001).
DISCUSSION
The present study of different histologic types of gall-
bladder tumors demonstrated an inverse relation be-
tween tumor progression and Bcl-2 expression and a
positive link with p53, apoptosis, and proliferation
activity. To date, there has been one report on Bcl-2
expression in gallbladder cancer11 in which mainly
advanced stage cancer was analyzed. Among the ade-
nocarcinomas, sufficient early stage lesions were
available to allow demonstration of a significant de-
crease in Bcl-2 and corresponding increase in other
parameters with advance and deeper invasion.
A positive relation between Bcl-2 expression and
tumor cell differentiation has been reported for sev-
eral carcinomas, including colorectal5 and gastric car-
cinomas.6 Thus, our finding of a positive link with the
differentiation state is in line with the literature. Fur-
thermore, Bcl-2 was shown to be correlated with pro-
gression of gallbladder carcinomas, as shown previ-
ously for esophageal15 and gastric cancer.12 It is
interesting to note that gallbladder adenoma showed a
raised Bcl-2 score. It is believed that high expression in
adenomas also implies tumor cell differentiation. Al-
though the Bcl-2 score was very low in the background
mucosa, several positive foci were found, including
intestinal metaplasia. In the stomach, both intestinal
metaplasia and adenoma were Bcl-2 positive, and the
origin of the gastric adenoma is suggested to be intes-
tinal metaplastic epithelium.4,10 However, in the gall-
bladder, because the Bcl-2 positive adenoma in this
study did not show the morphological phenotype of
intestinal metaplasia (goblet cells or epithelial cells
with brush border), it is difficult to interpret the Bcl-2
positive findings in adenoma as a differentiation to-
ward intestinal metaplasia. It is believed that the Bcl-2
positive findings may suggest some unknown differ-
entiation of the tumor cells.
On the histogenesis of gallbladder carcinoma,
there have been two theories: adenoma-carcinoma
sequence and de novo carcinogenesis.24 –26 Because an
 Bcl-2 Expression in Gallbladder Tumor/Mikami et al. 323

TABLE 2
p53 Score, Apoptosis, and Proliferation Activity of Bcl-2 Positive and Negative Gallbladder Adenocarcinomas According to Bcl-2 Expression

Bcl-2a No. p53 Score AI AI/MI Ki-67 LI MI

Positive 22 4.1 ⫾ 4.9 0.103 ⫾ 0.128** 0.478 ⫾ 0.525** 13.1 ⫾ 5.6 0.235 ⫾ 0.133*
Negative 46 5.0 ⫾ 4.8 0.305 ⫾ 0.224** 1.058 ⫾ 0.791** 15.4 ⫾ 9.8 0.352 ⫾ 0.253*

Values are expressed as mean ⫾ standard deviation. AI: apoptotic index; MI: mitotic index; LI: labeling index.
a
Bcl-2 negative, score 1 or less; Bcl-2 positive, score 2 or more.
* P ⬍ 0.05.
** P ⬍ 0.01.

FIGURE 5. Apoptotic indices (AI) and ratios of apoptotic and mitotic indices
(AI/MI) of well-differentiated adenocarcinomas classified by Bcl-2 positivity. n,
Number of cases; asterisk, P ⬍ 0.05.
FIGURE 6. Correlation between mitotic (MI) and apoptotic indices (AI) in
gallbladder tumors. The regression line is also shown (AI ⫽ 1.023 ⫻ MI ⫹
adenomatous component was found more frequently
0.075; r ⫽ 0.549; P ⬍ 0.0001). Solid diamonds, adenomas; open circles,
in the polypoid early stage carcinoma than in the
well-differentiated adenocarcinomas; crosses, moderately to poorly differenti-
nonpolypoid carcinoma in the present study, the his-
ated adenocarcinomas.
togenesis of the polypoid carcinoma can be explained
at least in part by the adenoma-carcinoma sequence
theory. The expression of Bcl-2 found at the adeno- significant relation between the levels of the two pro-
matous component is consistent with this theory. On teins was not found. The positive correlation between
the other hand, the nonpolypoid type adenocarci- Bcl-2 and p53 overexpression reported by Sasatomi et
noma may not arise from adenoma, because an ad- al.11 was not apparent, but this discrepancy could
enomatous component was found rarely in the non- have arisen because most of their cases were ad-
polypoid type carcinoma. Therefore, it can be vanced carcinoma. Reviewing their data, if the analy-
considered that the two pathways, the adenoma-car- sis was limited to early carcinomas, then no positive
cinoma sequence and de novo carcinogenesis, may relation was observed. In agreement with previous
exist in gallbladder carcinogenesis. Furthermore, the reports of p53 overexpression in 35–92%11,27–29 of gall-
majority of the nonpolypoid carcinomas are suggested bladder carcinomas, 42 of the 68 (61.8%) malignant
to arise by the latter theory. lesions investigated here showed p53 scores greater
It has been reported that bcl-2 gene transcription than 2.
is down-regulated by p53 protein.16,17 Our study dem- Although diffuse p53 overexpression detected in
onstrated that p53 scores increased, whereas Bcl-2 tumor by immunohistochemistry is interpreted as
scores decreased, in gallbladder tumors according to mutant p53,30 whether the mutant p53 can down-
histological type and tumor progression, although a regulate Bcl-2 expression is controversial. Haldar et
324 CANCER January 15, 1999 / Volume 85 / Number 2
al.31 reported that overexpression of a mutant p53
induced down-regulation of Bcl-2 both at protein
levels and at mRNA levels in a breast carcinoma cell
line. On the other hand, Delia et al.32 found that
mutant p53 expression induced by radiation was not
related to Bcl-2 protein expression in Li-Fraumeni
cells. Because Bcl-2 expression is affected by many
factors,1 not all tumors may show Bcl-2 down-reg-
ulation by p53 (wild or mutant type). For example,
lung small cell carcinomas showed high expression
of both p53 and Bcl-2.33 Although, at least immuno-
histochemically, gallbladder carcinoma showed a
regulation pattern similar to that of colorectal car-
cinoma,2 further molecular investigations are
needed to clarify the relation between the two pro-
teins in gallbladder carcinomas.
An inverse relation between Bcl-2 expression
and apoptosis was demonstrated clearly in the cur-
rent study, especially for early stage carcinomas.
Whereas Sasatomi et al. found no such link between
Bcl-2 and apoptosis in 49 gallbladder carcinomas,11
their analysis did not take into account histologic
typing. Generally, Bcl-2 protein is known to inhibit
apoptosis,1 and a number of authors have demon-
strated that this is the case in various human can-
cers.2,9,13 Tumors with a high proliferative activity
also appear likely to include large numbers of cells
undergoing apoptosis. This has been reported for
gastric13 and endometrial carcinomas,9 and our data
demonstrate that gallbladder tumors show a similar
positive correlation between proliferation activity
(MI and Ki-67 LI) and apoptosis (AI).
In conclusion, Bcl-2 expression in gallbladder tu-
mors is associated positively with tumor cell differen-
tiation and negatively with tumor progression. It may
contribute to carcinoma growth by inhibiting apopto-
sis, especially in the early stage of tumorigenesis. To
clarify the relation between p53 and Bcl-2 expression,
further investigations are needed.
REFERENCES
1. Lu QL, Abel P, Foster CS, Lalani EN. bcl-2: Role in epithelial
differentiation and oncogenesis. Hum Pathol 1996;27:102–
10.
2. Sinicrope FA, Ruan SB, Cleary KR, Stephens LC, Lee JJ, Levin
B. bcl-2 and p53 oncoprotein expression during colorectal
tumorigenesis. Cancer Res 1995;55:237– 41.
3. Bronner MP, Culin C, Reed JC, Furth EE. The bcl-2 proto-
oncogene and the gastrointestinal epithelial tumor progres-
sion model. Am J Pathol 1995;146:20 – 6.
4. Saegusa M, Takano Y, Okayasu I. Bcl-2 expression and its
association with cell kinetics in human gastric carcinomas
and intestinal metaplasia. J Cancer Res Clin Oncol 1995;121:
357– 63.
5. Bosari S, Moneghini L, Graziani D, Lee AKC, Murray JJ,
Coggi G, et al. bcl-2 oncoprotein in colorectal hyperplastic
polyps, adenomas, and adenocarcinomas. Hum Pathol
1995;26:534 – 40.
6. Lauwers GY, Scott GV, Karpeh MS. Immunohistochemical
evaluation of bcl-2 protein expression in gastric adenocar-
cinomas. Cancer 1995;75:2209 –13.
7. Jiang SX, Kameya T, Sato Y, Yanase N, Yoshimura H, Ko-
dama T. bcl-2 protein expression in lung cancer and close
correlation with neuroendocrine differentiation. Am J
Pathol 1996;148:837– 46.
8. Diebold J, Raretton G, Felchner M, Meier W, Dopfer K,
Schmidt M, et al. bcl-2 expression, p53 accumulation, and
apoptosis in ovarian carcinomas. Am J Clin Pathol 1996;105:
341–9.
9. Saegusa M, Kamata Y, Isono M, Okayasu I. bcl-2 expression
is correlated with a low apoptotic index and associated with
progesterone receptor immunoreactivity in endometrial
carcinomas. J Pathol 1996;180:275– 82.
10. Uesugi H, Saegusa M, Takano Y, Okayasu I. Different ex-
pression of bcl-2 protein in gastric adenomas and carcino-
mas. Pathol Int 1996;46:274 – 80.
11. Sasatomi E, Tokunaga O, Miyazaki K. Spontaneous apopto-
sis in gallbladder carcinoma: relationships with clinicopath-
ologic factors, expression of E-cadherin, bcl-2 protoonco-
gene, and p53 oncosuppressor gene. Cancer 1996;78:2101–
10.
12. Saegusa M, Takano Y, Kamata Y, Okayasu I. bcl-2 expression
and allelic loss of the p53 gene in gastric carcinomas. J
Cancer Res Clin Oncol 1996;122:427–32.
13. Koshida Y, Saegusa M, Okayasu I. Apoptosis, cell prolifera-
tion and expression of bcl-2 and Bax in gastric carcinomas:
immunohistochemical and clinicopathological study. Br J
Cancer 1997;75:367–73.
14. Seagusa M, Okayasu I. Down-regulation of bcl-2 expression
is closely related to squamous differentiation and proges-
terone therapy in endometrial carcinomas. J Pathol 1997;
182:429 –36.
15. Ohbu M, Saegusa M, Kobayashi N, Tsukamoto H, Mieno H,
Kakita A, et al. Expression of bcl-2 protein in esophageal
squamous cell carcinomas and its association with lymph
node metastasis. Cancer 1997;79:1287–93.
16. Miyashita T, Hagigai M, Hanada M, Reed JC. Identification
of a p53-dependent negative response element in the bcl-2
gene. Cancer Res 1994;54:3131–5.
17. Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK,
Liebermann DA, et al. Tumor suppressor p53 is a regulator
of bcl-2 and bax gene expression in vitro and in vivo. On-
cogene 1994;9:1799 – 805.
18. Albores-Saavedra J, Henson DE, Sobin LH. WHO histologi-
cal typing of tumours of the gallbladder and extrahepatic
bile ducts. In: World Health Organization International his-
tological classification of tumours. 2nd Ed. Berlin: Springer-
Verlag, 1991.
19. Albores-Saavedra J, Henson DE, Sobin LH. The WHO histo-
logical classification of tumors of the gallbladder and extra-
hepatic bile ducts: a commentary on the second edition.
Cancer 1992;70:410 –14.
20. Beahrs OH, Henson DE, Jutter RVP, Myers M, editors (Amer-
ican Joint Committee on Cancer). Manual for staging of
cancer. 3rd edition. Philadelphia: J.B. Lipincott, 1988.
21. Mizumoto R, Ogura Y, Kusuda T. Definition and diagnosis of
early cancer of the biliary tract. Hepatogastroentelology
1993;40:69 –77.

22. Kerr JFR, Winterford CM, Harmon BV. Apoptosis: its signif-
icance in cancer and cancer therapy. Cancer 1994;73:2013–
26.
23. Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA, Cote RJ.
Strategies for improving the immunohistochemical staining
of various intranuclear prognostic markers in formalin-par-
affin sections: androgen receptor, estrogen receptor proges-
terone receptor, p53 protein, proliferating cell nuclear anti-
gen, and ki-67 revealed by antigen retrieval techniques.
Hum Pathol 1994;25:263–70.
24. Kozuka S, Tsubone M, Yasui A, Hachisuka K. Relation of
adenoma to carcinoma in the gallbladder. Cancer 1982;50:
222634.
25. Albores-Saavedra J, Angeles-Angeles A, de Jesus Manrique J,
Henson DE. Carcinoma in situ of the gallbladder: a clinico-
pathologic study of 18 cases. Am J Surg Pathol 1984;8:323–
33.
26. Takei K, Watanabe H, Itoi T, Saito T. p53 and Ki-67 immu-
noreactivity and nuclear morphometry of ’carcinoma-in-
adenoma’ and adenoma of the gall-bladder. Pathol Int 1996;
46:908 –17.
27. Teh M, Wee A, Raju GC. An immunohistochemical study of
p53 protein in gallbladder and extrahepatic bile duct/amp-
ullary carcinomas. Cancer 1994;74:1542–5.
Bcl-2 Expression in Gallbladder Tumor/Mikami et al. 325
28. Wistuba II, Sugio K, Hung J, Kishimoto Y, Virmani AK, Roa I,
et al. Allele-specific mutations involved in the pathogenesis
of endemic gallbladder carcinoma in Chile. Cancer Res 1995;
55:2511–5.
29. Wistuba II, Gasdar AF, Roa I, Albores-Saavedra J. p53 pro-
tein overexpression in gallbladder carcinoma and its pre-
cursor lesions: an immunohistochemical study. Hum Pathol
1996;27:360 –5.
30. Baas IO, Mulder J-WR, Offerhaus GJA, Vogelstein B, Hamil-
ton SR. An evaluation of six antibodies for immunohisto-
chemistry of mutant p53 gene product in archival colorectal
neoplasms. J Pathol 1994;172:5–12.
31. Haldar S, Negrini M, Monne M, Sabbioni S, Croce CM.
Down-regulation of bcl-2 by p53 in breast cancer cells. Can-
cer Res 1994;54:2095–7.
32. Delia D, Goi K, Mizutani S, Yamada T, Aiello A, Fontanella E,
et al. Dissociation between cell cycle arrest and apoptosis
can occur in Li-Fraumeni cells heterozygous for p53 gene
mutations. Oncogene 1997;14:2137– 47.
33. Brambilla E, Negoescu A, Gazzeri S, Lantuejoul S, Moro D,
Brambilla C, et al. Apoptosis-related factors p53, bcl2, and
Bax in neuroendocrine lung tumors. Am J Pathol 1996;149:
19 –1952.