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Cytogenetic analyses of hepatocellular carcinoma by in situ hybridization with a chromosome-specific DNA probe

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255
Apoptosis and lmmunohistochemical bcl-2 Expression
in Colorectal Adenomas and Carcinomas
Aspects of Carcinogenesis and Prognostic Significance
Gustavo Bruno Baretton, M.D.'
Joachim Diebold, M.D.'
Gregor Christoforis, M.D.'
Michael Vogt, M.o.'
Christian Miiller, M.D.'
Karin Dopfer, M.D.'
Karin Schneiderbanger, M.D.'
Michael Schmidt, M.O?
Udo Lohrs, M.D.'
' Institute of Pathology, University of Munich,
Munich, Germany
Department of Surgery, Klinikum Grosshadern University of Munich, Munich, Germany.
Tumor Register, Munich, Germany
BACKGROUND. The Ocl-2 oncoprotein confers a survival advantage to cells by inhibiting programmed cell death (PCD) or apoptosis. Overexpression of bcl-2 probably
plays a role in colorectal carcinogenesis. The aims of our study were to determine
bcl-2 expression and PCD index in colorectal adenomas and carcinonias in correlation with p53 expression, Ki-67 index, and histopathology, and to test their prognostic significance in patients with colorectal carcinomas,
METHODS. Immunohistologic staining for bcl-2 (MoAb clone 124), the proliferationassociated Ki-67 antigen (MoAb MIBl), and p53 (MoAb D01) was performed on
archival material from 44 colorectal adenomas and 95 adenocarcinomas (TNM
classifications pT2 and -3, pNO, and MO). The PCD was visualized by enzymatic
detection of DNA fragmentation.
RESULTS. bcl-2 was expressed in 86% of the adenomas and 67% of the carcinomas.
Mean PCD and Ki-67 rates were 1.7 ? 0.14% and 35 2 13% in adenomas and 1.9
t 0.15% and 28 2 14% in carcinomas, respectively. In carcinomas, Ocl-2 expression
was correlated with a low PCD index (< 1.5%; P = 0.005). Furthermore, a high Ki67 index (225%) was associated with a high PCD index (21.5%; P < 0.0001). p53
accumulation was seen in 16% of adenomas and in 42% of carcinomas, and did
not correlate with bcl-2 expression or PCD index. In the univariate analyses, significantly longer disease free survival intervals were observed in three groups: all
patients with bcl-2-positive carcinomas ( P < 0.05); the subgroup of carcinomas
with Ocl-2 expression and low PCD index ( P = 0.037); and the subgroup of OcI-2positive and p53-negative carcinomas ( P = 0.021). In the multivariate analysis,
however, only tumor stage and p53 expression were independent risk factors for
prognosis.
CONCLUSIONS. Our data indicate that Ocl-2 expression is characteristic of the early
phase of colorectal carcinogenesis. Its physiologic function as an inhibitor of PCD
is preserved in most colorectal carcinomas. whereas p53 is apparently not involved
in the regulation of PCD in colorectal neoplasias. bcl-2 expression in colorectal
carcinomas is associated with a better clinical course. This correlation became
even more evident in the subgroups of patients with carcinomas that also had low
PCD index or lacked p53 iminunoreactivity. Cancer 1996; 77955-64.
(0 1996 American Caricer Society.
This work contains parts of Gregor Christoforis'
doctoral thesis.
KEYWORDS colon, rectum, carcinoma, adenoma, bcl-2, protooncogene, apoptosis,
proliferation, Ki-67, p53, immunohistochemistry, prognosis.
Addrlsss for reprints: Dr Gustavo B. Baretton,
Pathologisches lnstitut der Universitat, Thalkirchnerstr. 36, [I-80337 Munchen, Germany.
T
Received June 6, 1995; ievision received September 1, 1995; accepted September 1, 1995.
8 1996 American Cancer Society
issue growth depends on both cell proliferation and the rate of cell
death.',2Thus it is conceivable that neoplastic growth may be caused
or promoted by factors inhibiting cell death. Apoptosis [programmed cell
death (PCD)]is defined as a morphologic change culminating in cell death
by a process clearly distinct from necrosis.3 The regulation of apoptosis
256
CANCER January 15,1996 I Volume 77 I Number 2
is central to morphogenesis during fetal development and
to maintenance of tissue homeostasis during adulthood
and seems to be of importance for neoplastic transformation in some
bcl-2 is a protooncogene that is
involved in the regulation of cell death by inhibiting
apoptosis in many cell systems in physiologic and neoplastic condition^.'^^^' Its role as a new kind of oncogene
that provides a growth advantage to a tumor cell by
blocking PCD has been seen primarily in follicular B-cell
lymphomas. In these lymphomas the bcl-2 gene usually
located at 18q21 is moved into juxtaposition with powerful enhancer elements in the immunoglobulin heavy
chain (IgH) locus at 14q32; the result is overproduction
of bcl-2 mRNAs and their encoded proteins.*-“’ Increased
bcl-2 expression has recently been reported also in epithelial malignancies, e.g., carcinomas of the lung,””2thyroid,I3 breast,I4 stomach,I5and ovaries.16
Colorectal cancer is one of the most common malignancies in western industrialized countries including the
United States” and Germany.’* In these countries colorectal carcinoma is the second leading cause of cancer
mortality. Pathogenetically, an adenoma-carcinoma sequence can be postulated for most colorectal carcinomas.’q-2’This concept has been supported by the discovery of molecular genetic changes that were correlated
with certain histologic stages of this sequence.”.’3 As one
important consequence of these genetic aberrations, cell
proliferation is abnormally increased in these premalignant and malignant lesions of the colorectal epithe1ium,24-27
By contrast, only scarce information is available
concerning the importance of perturbations in the regulation of cellular loss in colorectal carcinogenesis. In the
normal colonic mucosa, cellular shedding is the principal
physiologic mechanism of epithelial cell
Before
mechanical cell elimination takes place, however,
apoptosis seems to be triggered in the uppermost nuclei
at the edge of the crypts facing the lumen.2HA putative
link between the current concept of molecular colonic
carcinogenesis and the regulation of apoptosis might be
found in the physiologic function of the TP53 tumor suppressor gene. The most important function of wild-type
p53 protein is the maintenance of genome integrity by
preventing a cell with DNA damage from entering the cell
cycle.’”Subsequently, DNA repair can take place, or PCD
will be induced if the DNA damage is irreversible. This
concept was confirmed by the results of a recent study
from our own group showing statistically significant correlations between p53 accumulation and DNA nondiploidy in adenomas and carcinomas, as well as between
p53 immunoreactivity and an unfavorable prognosis in
nonmetastatic colorectal carcinomas.”
Recently, Sinicrope et al.32reported the first data concerning bcZ-2-expression and apoptosis in colorectal tumorigenesis: in 17 of 24 (71%) colonic adenomas and
14 of 21 (67%) adenocarcinomas bcl-2 immunoreactivity
could be detected. However, no study has investigated
the prognostic importance of bcl-2-expression, PCD index, and proliferation in colorectal carcinomas. By
searching for correlations between these parameters and
p53 accumulation, a better understanding of growth regulation including apoptosis in colorectal neoplasias
might be achieved. We also looked for the prognostic
significance of these parameters in nonmetastatic adenocarcinomas of tumor stages pT2 and pT3 according to
the Union International Centre le Cancer (UICC) classification.33
MATERIALS AND METHODS
Materials
Formalin fixed and paraffin embedded tumor tissue from
44 colorectal adenomas (polypectomies) and 95 primary
colorectal carcinomas was studied. Histologic classifications were carried out on hematoxylin and eosin (H & Elstained slides. The adenomas were randomly chosen
from the files of the Institute of Pathology, University of
Munich, and classified according to the following criteria:
(1) size [<1 cm, n = 26 (59%),or 2 1 cm, n = 18 (41%)];
(2) histologic growth type according to the World Health
Organization (WHO) classification34 [tubular, n = 19
(43%); tubulo-villous, n = 18 (41%); or villous, n = 7
(16%)];and (3) grade of atypia [low, n = 6 (14%);moderate, n = 23 (52%); or severe, n = 15 (34%)],or dysplasia
[low, n = 29 (66%), comprising the cases with low and
moderate atypia, or high, n = 15 (34%) according to the
WHO clas~ification”~1.
By applying these criteria, villous growth patterns (in
tubulo-villous and villous adenomas) correlated in a statistically significant manner with tumor diameter 2 1 cm
( P < 0.01) and increasing grade of atypia ( P < 0.001) or
dysplasia ( P < 0.001). The carcinomas comprised only
cases with tumor stages pT2 (n = 49) and pT3 (n = 46)
according to UICC with no evidence of lymphogenous or
hematogenous metastases (pNO, MO).33All patients had
undergone tumor resection at the Department of Surgery,
Klinikum Grosshadern, University of Munich, between
1986 and 1988. From all cases clinical follow-up data were
available up to 8 years after operation. The mean age of
the patients at time of operation was 63.8 2 12.5 years,
and the male/female ratio was 1.2:l. Thirty-one tumors
were localized in the right colon, and 64 carcinomas in
the left colon and rectum.
Histologically all tumors were adenocarcinomas. According to the WHO classification the grade of differentiation was high in four cases (Gl), moderate in 75 cases
(G2), and low in 16 cases (G3).R4
Methods
Detection of apoptotic cells in paraffin sections
DNA fragments of apoptotic cells were visualized by an
enzymatic reaction using modified protocols of Gavrieli
PCD and bcl-2 in Colorectal Neoplasias/Baretton et al.
et al.'" and Ansari et al.35Paraffin sections were cut at 34 pm, mounted on silane-coated slides, and dried overnight at 56 "C. After deparaffinization in xylene and ethanols in descending concentrations, the sections were
washed in double-distilled water (DDW) two times for
5 minutes. Subsequently, incubation with proteinase K
solution containing 400 pI proteinase stock solution 125
mg proteinase K (Sigma, Deisenhofen, Germany) in 2.5
ml DDW] in 200 ml Tris-HC1 buffer (pH 7.0) was performed for 15 minutes at room temperature.
After tour washes in DDW for 2 minutes the sections
were incubated with terminal transferase mixture under
a coverslip for 1 hour at 37 "C. The terminal transferase
reaction mixture for 15 slides consisted of 15 pl digoxigenein-dUTP, 2.7 p l dATP, 90 pl terminal transferase reaction
buffer containing cacodylate, 30 pl CoC12, 3 pl terminal
transferase (all from Boehringer, Mannheim, Germany),
and 306.3 p1 DPW. Approximately 25 pI/slide were applied.
After lhree washes in TB buffer (300 mM sodium
chloride and 30 mM sodium citrate in 1000 ml DDW, pH
7.5) for 5 minutes and one wash in buffer 1 (100 mM TrisHCI and 150 mM NaCl in 2000 ml DDW, pH 7.2) for 5
minutes, the sections were immersed in buffer 2 for 30
minutes. For preparation of this buffer 0.1 g of blocking
reagent 0.5% (Boehringer) was mixed for 1 hour at 60 "C
with 200 rril of buffer 1.
Subsequently, two washes in buffer 1 for 5 minutes
were followed by incubation with alkaline phosphataseconjugated Fab fragments of polyclonal sheep antidigoxigenin antibody (Boehringer) at a dilution of 1:ZOOO.
Three washes in buffer 1 for 5 minutes and one 5minute wash in a buffer containing 100 mM Tris-HC1, 100
mM NaCI, (and 50 mM MgCI2 in 1000 ml DDW, pH 9.5 (=
buffer 3 ) were then followed by incubation with nitroblue
tetrazolium (NBT) solution, first for 30 minutes at room
temperature in the dark and then overnight at 4 "C in the
dark. For preparation of NBT solution 22.5 pl NBT stock
solution containing 1 g NBT (Boehringer) in 9.33 ml 70%
dimethylformamide solution (Sigma, St. Louis, MO) was
mixed with 17.5 pl bromochloroindolylphosphate (Boehringer), 5 m M levamisole (Sigma), and 5000 pl buffer 3.
On the next day after a wash in running tap water for 10
minutes the slides were counterstained in nuclear fast
red for 10 minutts After two additional washes in DDW
the sections were mounted with Aquatex (Merck, Darmstadt, Germany).
As a positive control, sections of normal colonic mucosa or lymph nodes were used. Since the enzymatic reaction described labels both apoptotic cells and areas of
necrosis, only those labeled cells were regarded as positive that showed additional characteristics of apoptosis,
e.g., isolated localization within an intact cell complex
without an inflammatory reaction. At least 3000 tumor
257
cells/case were evaluated. The results were expressed as
positive cells/1000 tumor cells. As 1.5% represented the
median value of all tumorous samples under study, a PCD
index < 1.5% was considered low, and a PCD index >
1.5% high.
bcl -2 and Ki-67 immunohistochemistty
Sections (2-3 pm thick) were cut from the paraffin blocks
and mounted on Super-Frost/Plus microscope slides
(Menzel, Germany). After deparaffinization and rehydration the slides were placed in a Coplin jar of microwavecompatible plastic (TPX Brand, Germany) that was filled
with 10 mM citrate buffer (pH 6.0). The jar was heated
in a Panasonic microwave oven ("-4241)
at 750 W for
5 minutes. Then, the fluid level was checked and evaporated buffer was supplemented. Two additional heating
cycles of 5 minutes followed in the same way. The jar was
then allowed to cool for about 1 hour at room temperature.
The monoclonal bcl-2-antibody (clone 124) was purchased from Dianova (Hamburg, Germany). The lyophilisate was dissolved in Tris buffer (pH 7.5), and the antibody was used at a dilution of 1:60. The tissue sections
were incubated with the primary antibody overnight at
4 "C.
For immunohistochemistry of Ki-67, a proliferationassociated antigen, the slides were incubated with the
monoclonal antibody MIB 1 (Dianova; dilution 1:15 for
30 minutes at room temperature) after 2x 10-minute microwave pretreatment."," The reactions were visualized
by the APAAP-method, i.e., the employment of immune
complexes of alkaline phosphatase and monoclonal antialkaline phosphatase.3' For this purpose the sections were
incubated with the second rabbit/anti-mouse antibody
(Dakopatts, Glostrup, Denmark) at a dilution of 1:25 for
30 minutes at room temperature and subsequently with
the APAAP complex (Dakopatts) at a 1:50 dilution for 30
minutes at room temperature. To enhance the intensity
of the final staining, these two steps were repeated twice.
Fast blue-naphthole AS-MX phosphate (Sigma, Deisenhofen, Germany) served as chromogene. Finally, the
slides were counterstained with nuclear fast red and
mounted in Aquatex (Merck). Controls were performed
by replacing the primary monoclonal antibodies with
RPMI.
Immunohistologic detection of p53 protein and interpretation of the results were performed as described
previously using the MoAb DO 1 (Dianova).3'
Analysis of the immunohistochemical reactions
bcl-2. The immunohistochemical results for bcl-2 were
recorded as positive if the tumor cells showed an unequivocally strong cytoplasmic immunoreaction. Cases
258
CANCER January 15,1996 I Volume 77 I Number 2
1.38%. The PCD index was graded as low in 22 cases (51%)
and as high in 21 adenomas (49%). In nearly all cases
single apoptotic cells were seen in the surface epithelium
of nonneoplastic colonic mucosa. Moreover, scarce apoptotic cells were also detected by terminal transferase reaction in the germinal centers of intramucosal lymphoid
follicles.
bcl-2 immunohistochemistry
FIGURE 1. Tubular colorectal adenoma showing single apoptotic nuclei
marked enzymatically by labeling of DNA fragmentation using terminal
transferase reaction (arrows). The labeled cells exhibit typical morphologic
signs of apoptosis with condensation and margination of nuclear chromatin without associated inflammatory reaction (PCD index 6%; original magnification x400).
with only equivocally faint staining were regarded as negative.
Ki-67. According to the immunoreaction in at least 300
tumor cells/tumor the percentage of nuclei with a positive immunoreaction was determined (= Ki-67 index). A
Ki-67 index was classified as high if it was >25%, which
represented the median value in the carcinoma group.
1753. The results of the p53 imniunoreaction were recorded as positive it' a distinct nuclear staining was seen
in at least 20% of the tumor cells. Cases with no or only
single positive cells were regarded as negative. This classification was used because there is increasing evidence
that p53 immunoreactivity in only a few tumor cells or
even normal cells does not correlate with a TP53 mutation, but represents enhanced p53 activation due to physiologic DNA repair.
Statistical analyses
Mean parameter values ? standard error of the mean
(SEM)were statistically compared by chi-square analysis.
Survival analyses were performed by Kaplan-Meier
curves and the Log rank test using the SAS/STAT statistical software (SAS Institute, Cary, NC). Multivariate analysis was carried out by the Cox model. P < 0.05 was regarded as statistically significant.
RESULTS
Colorectal Adenomas and Adjacent Nonneoplastic
Tissues
Apoptosis
In 43 of 44 adenomas apoptotic cells could be visualized
enzymatically (Fig. 1). The mean PCD index was 1.69 &
Thirty-eight of 44 adenomas (86%) exhibited a positive
cytoplasmatic immunohistochemical reaction for bcl-2
(Fig. 2A). The remaining six cases (14%) were bd-2 negative. In all positive cases more than 30% of the cells were
immunolabeled. The pattern of immunostaining was not
uniform within the adenomas; clusters of cells that reacted positively alternated with only faint staining areas
within an individual case without correlation to tubular or
villous architecture or histologic grade of the adenomas.
Moreover, variations in staining intensity were also noted
from case to case, although the reactivity of lymphocytes
within the lamina propria or in the mantle zones of intraniucosal lymphoid follicles was similarly strong in all
specimens. A positive cytoplasmatic bcl-2 reaction was
also present in nonneoplastic epithelia at the bases of
preserved crypts in the adjacent mucosa corresponding
with the physiologic proliferative zone and in ganglion
cells of the intramural neural plexus of the intestine.
Ki-67
The mean Ki-67 index in adenomas was 34.9 2 13%. According to the defined classes of proliferation (see Methods section), in 9 of 44 cases (20%) the Ki-67 index was
graded as low and as high in 37 of 44 cases (80%; Fig.
2B). N o statistically significant correlations could be established between Ki-67 index and size or histomorphologic features of the adenomas.
In adjacent nonneoplastic intestinal mucosa a positive Ki-67 immunoreaction was seen in the proliferative
zone of the crypts (mean Ki-67 index, 16.4 ? 6.7%). The
differentiated surface epithelium never showed nuclear
staining with the MIBl antibody. Correspondingly, the Ki67 index was graded as low in 90% of the norineoplastic
colonic inucosa adjacent to the adenomas or carcinomas.
In 10% of the cases, however, a high Ki-67 index with
values up to 35% MIBl reactivity was observed in the
nonneoplastic mucosa.
P53
Thirty-seven of 44 adenomas (84%) were immunohistochemically p53 negative, whereas in the remaining 7
cases (16%) a nuclear p53 accumulation could be observed. In all positive adenomas, however, parts of the
neoplasms remained negative. Nonetheless, in the immunoreactive areas staining was quite homogeneous and
PCD and bcl-2 in Colorectal Neoplasias/Baretton et al.
259
FIGURE 2. (A) Tubulo-villous colorectal adenoma showing a positive cytoplasrnatic bcl-2 reactivity of different intensity in
more than 30% of tumor cells (MoAb clone 124; APAAP, original magnification x100). (B) High Ki-67 index (42%) in the
:same case, with nuclear staining for MoAb MI61 (APAAP, original magnification x200).
reaction in a colonic adenocarcinorna with high PCD index (8.2%) (terminal
traiisferase reai:tion, original magnification x400).
FIGURE 4. Strong positive cytoplasmatic staining for bcl-2 in a moderately differentiated colonic adenocarcinorna with interspersed lymphocytes
showing also a positive reaction (MoAb clone 124; APAAP. original magnification x400).
diffuse. Statistically significant correlations could be observed between p53 expression and occurrence of villous
growth pattern in the adenomas ( P <: 0.05), and histologic
grade of atypia ( P < 0.01) or dysplasia (P < 0.001). The
preserved nonneoplastic intestinal tissues showed no p53
immunoreactivity.
tioned above, the PCD index was classified as low in 44
cases (47%) and high in 49 cases (53%; Fig. 3).
FIGURE 3. Apoptotic nuclei labeled enzymatically by terminal transferase
Colorectal Carcinomas
Apoptosis
The mean PCD index in the 93 carcinomas suitable for
evaluation was 1.9 ? 1.45%. According to the limits men-
bcl-2 immunohistochemistry
Sixty-four of the 95 colorectal carcinomas analyzed (67%)
showed a positive cytoplasmatic immunoreactivity for
bcl-2 (Fig. 4). The rate of bcl-2-expressing cases was significantly lower in carcinomas than in adenomas (86%;
P < 0.04). As in adenomas, the immunohistological bcl-
260
CANCER January 15,1996 / Volume 77 / Number 2
TABLE 1
bcl-2-ExpressionlPCDRate and Tumor Progression in Colorectal
Carcinomas"
TABLE 2
bcl-2 Expression and Apoptosis (PCD Rate) in Colorectal Carcinomas"
PCD rate
No
progression
Progression
Total
bcl-2 negiPCD rate 1.5%
bcl-2 pos./PCD rate < 1.5%
9
26
13
10
22 (3R)
36 (62)
Total
35 (60)
23 (40)
58 (100)
Colorectal carcinomas (tumor stages pT2 and pT3, pN0, MO): Statistically significantl) l o w rate of
tumor progression in the subgroup of patients harboring tumors with bci-2-expression (MoAb clime
124) and low rate of apoptosis (PCD < 1.5%) compared with patients with carcinomas lacking bcl-2
and hiEh rate of apoptosis IP = 0.0181. Data are numbers or numbers with perceni in parrntheses
'I
TABLE 3
Ki-67 Index and PCD Rate in Colorectal Carcinomas
PCD rate
Total
<1.5%
>1.5%
Ki-67 index < 25%
Ki-67 index > 25%
27
16
13
36
40 143)
52 (57)
Total
43 (47)
49 (53)
92 (1001
Colorectal carcinonias Itunior stages pT2 and pT3 pNO, MOI. Statistically significanr correlation lietween inimunohistochemical brl-2 expression IMoAb clone 1211 and Ki-6; index IMoAb MlBll IP <
0.0001 I.
2-staining features in carcinomas were heterogeneous.
Specific topographic staining patterns, however, were
missing. Moreover, there were no correlations between
the results of bcl-2 immunostaining and patient age or
sex, carcinoma site, tumor stage pT2 or pT3, or grading.
Ki-67
The mean value of the Ki-67 index was 28 ? 14% in colorectal carcinomas. In 41 cases (44%) the Ki-67 index was
classified as low and as high in 53 cases (56%).One case
could not be evaluated. Thus, the rate of carcinomas with
a high Ki-67 index (56%)was significantly lower than the
rate of adenomas with a high Ki-67 index (80%;P < 0.01).
P53
In 40 carcinomas (42%) a nuclear p53 accumulation could
be observed. The remaining 55 cases (58%) showed no
imniunohistochemical staining for p53. In comparison
with adenomas (16%) the rate of aberrant immunohistochemical p53 expression in carcinomas was statistically
significantly higher ( P < 0.005).
Prognostic significance
I n the univariate analysis patients with bcl-2-positive carcinomas showed a statistically significant longer disease
Total
4.5%
>1.5%
bcl-2 negative
bcl-2 positive
8
36
22
26
62 (67)
Total
44 (48)
48 (52)
92 (100)
30 (33)
' Colorectal carcinomas (tumor stagespT2 and pT3, pNO, MOI. Correlation between immunohistothemical Drl-2 expression (MoAb clone 1211 and rate at apoptotic cells (PCDI determined by enzymatic
detection of DNA fragmentation using terminal transferase (P= 0.005). Data are numbers or numbers
with percent in parentheses.
free time without evidence of tumor progression (no recurrence, no metastasis, no death of disease) than those
with bcl-2-negative tumors ( P = 0.047; Fig. 1). In patients
with bcl-2-expression and a low PCD index (n = 36), the
tumors progressed less frequently (Table 1) compared
with patients who had bcl-2-negative tumors and a high
PCD index (n = 22). Moreover, these cases and patients
harboring bcl-2-positivelp53-negative carcinomas exhibited significantly longer disease free intervals ( P = 0.037
and P = 0.0205; Fig. 5B,C)than the rest of the study group.
In the stepwise multivariate analysis using the Cox
model and including the parameters age, pT stage, and
bcl-2 and p53 expression, however, only tumor stage ( P
= 0.0011; risk ratio, 3.1) and p53 expression ( P = 0.0051;
risk ratio, 2.6) proved to be independent prognostic risk
factors.
Correlations Between Results From the Different Methods
in Adenomas and Carcinomas
Neither in adenomas nor in carcinomas were statistically
significant correlations observed between bcl-2 expression, Ki-67 index, or PCD index and morphologic features
of the tumors or p53 expression. Whereas in adenomas
only a trend toward a negative correlation between bcl2-expression and PCD index could be noted ( P = 0.068),
bcl-2-expression in carcinomas was negatively correlated
with PCD index in a statistically significant manner (n =
86; P = 0.005; Table 2). Furthermore, in carcinomas the
grade of Ki-67 index correlated highly significantly with
the grade of PCD ( P < 0.0001; Table 3).
DISCUSSION
The purpose of our investigation was to look for a possible
relation between programmed (PCD) and immunohistochemical bcl-2 expression in colorectal adenomas and
carcinomas. Moreover, correlations of these parameters
with p53 accumulation and proliferative activity (Ki-67
PCD and bcl-2 in Colorectal Neoplasias/Baretton et al.
100% '1
90%
80%
70%
261
contrast, only scarce data concerning the importance of
bcl-2 and apoptosis for gastrointestinal carcinogenesis
are available: Lauwers et
were able to show t7cl-2
overexpression in 13 of 16 cases (81%)with gastric epithelial dysplasia and 46 of 74 (72%) gastric adenocarcinomas." In gastric cancer bcl-2 expression was highly significantly associated with intestinal p h e n ~ t y p e . ' Sini~
crope et a1" recently described increased bcl-2 expression
normal
adenoma
carcinoma
in 17 of 24 (71%) colonic adenomas and 14 of 21 (67%)
mucosa
adenocarcinomas. To the best of our knowledge, howFIGURE 5. Percent frequencies of immunohistochemical bcl-2-expresever, ours is the first study to investigate systematically
sion (>3O%), aberrant p53 imrnunoreactivity (>20%), high Ki-67 index
the role of bcl-2 expression and enzymatically determined
(>25%), and high rate of apoptosis (PCD index > 1.5%) in nonneoplastic
PCD index in colorectal adenomas and carcinomas in
colorectal rnucosa, colorectal adenomas, and nonmetastatic colorectal adcorrelation with proliferation (Ki-67 index), p53 accumuenocarcinomas (tumor stages pT2 and pT3).
lation, and prognosis. For this purpose, adenomas with
different histopathologic features (size, growth pattern,
and grade of atypia/dysplasia) and nonmetastatic adenoindex) were to be tested as well as their prognostic sigcarcinomas of tumor stages pT2 and pT3 according to
UICC" were investigated. Locally confined tumors were
nificance.
selected to exclude cases with probably more secondary
bcl-2 is a recently recognized protooncogene that
bcl-2 expression is a common
genetic alterations. In good accordance with the observaregulates cell
finding in low grade follicular B-cell lymph om as."^'" In
tions published recently by Bosari et al.,4bour results
these tumors a 14:18 translocation leads to bcl-2 overexshowed bcl-2 expression in 38 of the 44 (86%) adenomas
pression. bcl-2 overexpression has also been found in
and 64 of the 95 (67%) adenocarcinomas under study.
and epithelial malignancies''-I6 lacking the
This may indicate that bcl-2 activation is an early event
14: 18 translocation. The molecular mechanism underlyin colorectal carcinogenesis followed by other genetic
ing bcl-2 overexpression in cells without t(14:18), howchanges, e g , p53 accumulation. I n carcinomas a highly
ever, is still unknown. bcl-2 must form heterodimers with
significant correlation between bcl-2 expression and a low
PCD index was found ( P = 0.005; Table 2), whereas in
other proteins, e.g., Bax, to carry out its physiologic functliom4"Thus, a disturbance of the posttranslational regulaadenomas only a trend ( P = 0.065) was noted. Thus, our
tion of the bcl-2 gene might be present in carcinomas.
data assessed by enzymatic labeling of apoptotic cells
This theory is further supported by the finding of a dissoconfirm the results published by Sinicrope et al.,j2who
ciation of Ocl-2 mRNA level and bd-2 protein expression,
determined apoptosis by microscopic examination of
which is present in reactive lymphocytes but not in neoconventionally H & E-stained slides.
plastic conditions.' 1,39,41
These observations suggest that the physiologic funcIn the gastrointestinal tract the physiologic exprestion of bcl-2 for the regulation of apoptosis is preserved
sion of bcl-2 protein is confined to the stem cells and the
in most of the colorectal carcinomas under study. Howproliferative zone, i.e., the lower crypt of the intestinal or
ever, in 36 of our cases (38%) such a close correlation
colonic miicosa."~"' Obviously, the function of bcl-2 here
was not recorded, supporting the notion that regulation
is to protect the regenerative epithelial compartment
of apoptosis is complex and bcl-2 is not the only factor
from cell death. Although shedding of differentiated and
involved in this process. For example, under certain cirmature cells to i.he luminal surface is the major mechacumstances the proliferation-stimulating oncogen c- myc
nism of cellular loss in the gastrointestinal t r a ~ t , ~ " ~ ' is also known to stimulate apoptosis."' Furthermore, actiapoptosis seems to be triggered before mechanical cell
vation of a cyclin-dependent kinase at an inappropriate
elimination takes place. Thus, apoptosis also contributes
time during the cell cycle can have the same
physiologically to the normal intestinal cell turnThese observations support the notion that apoptosis
over ,2H.:F.-I-I
may also be initiated by incompatible or conflicting
In the gastrointestinal tract carcinomatous growth
growth signal^.^ This is in keeping with the finding of a
is frequently preceded by preinvasive or preneoplastic
highly significant correlation between a high proliferation
epithelial lesions characterized by autonomic hyperprolirate (Ki-67index) and a high PCD index ( P < 0.0001, Table
feration.'4-2' This observation led to the concept of the
3) in the carcinomas analyzed. Conversely, although bcladenoma-carcinoma sequence that can be postulated for
2 expression paralleled Ki-67 index, no significant correlamost colorectal
Thus, most investigation between these parameters could be established
tions have focused on this aspect of tuniorigenesis. By
(Fig. 5).
.
-
-
CANCER January 15,1996 / Volume 77 / Number 2
262
'"1
btl-
L
4c-
50 40
30
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n = 11
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2
4
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5
4
<
8
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Months
A
9op-I,
B
0
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12
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24
30
36
42
40
54
60
Months
%
1W
.......... . . . . . . . . . . . . . . . . . . . . . . . . . . .
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70
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The surprising result of a significantly lower proliferative activity in carcinomas than in adenomas is in contrast to our observations reported previously but may be
explained by the selection of only nonmetastatic carcinomas of tumor stages pT2 and pT3 in the present study.'"'
Prognostically, patients with bcl-2-positive nonmetastatic
colorectal carcinomas showed a better clinical course in
the univariate analysis than those with bcl-2-negative tumors ( P < 0.05; Fig. 6A). The difference in disease free
interval was even greater between the subgroups bcl-2positivellow PCD index and bcl-2-negativelhigh PCD index ( P = 0.037; Fig. 6B) where the physiologic situation
is obviously preserved. These data agree with observations in other malignancies showing that bcl-2 expression
is associated with a less malignant behavior. Also, among
nonsmall cell lung cancer patients those with tumors expressing bcl-2 had a better prognosis than patients with
bcl-2-negative carcinomas,'' and in B-cell lymphomas
bcl-2-expression is frequently lost during transformation
from low grade into high grade lymphomas.4' Almost all
thyroid cancers showing at least poor differentiation are
Ocl-2 positive, whereas undifferentiated thyroid carcinomas, which have the worst prognosis, are mostly bcl-2
negative.13 In ovarian carcinomas bcl-2 immunohistochemistry identified a subgroup of patients with p53- and
FIGURE 6. Colorectal carcinomas (pT2 and pT3, pNO, MO): Kaplan-Meier
curves showing significantly longer disease free survival rates for patients
with bcl-2-positive carcinomas versus patients with bcl-2-negative tumors
( P < 0.05) (A); bcl-2-positive tumors with low PCD index versus patients
with bcl-2-negative tumors with high PCD index ( P = 0.037) (B); and bcl2-positive and p53-negativetumors versus the rest of the group ( P = 0.021);
1 , bcl-2 positive and p53 positive (n = 28); 2, bcl-2 negative and p53
negative (n = 19); 3, bcl-2 negative and p53 positive ( n = 12).
bcl-2-positive tumors who had a significantly better outcome than patients with p53-positive and bcl-2-negative
carcinomas." I n the present study, patients with colorectal carcinomas showing bcl-2 expression but lacking
p53 immunoreactivity exhibited the best clinical course
( P = 0.0205; Fig. 6C). In the multivariate analysis, however, only tumor stage and p53 expression were independent prognostic risk factors. In contrast to bcl-2 overexpression, which was particularly frequent in adenomas
and decreased in carcinomas, p53 immunoreactivity
showed an inverse behavior, indicating that loss of TP53
is obviously a later event in the adenoma-carcinoma sequence.
In accordance with results from ovarian carcinomas,
we found no correlation between bcl-2 and the PCD index
on the one hand and p53 accumulation on the other hand
in colorectal adenomas and carcinomas." Thus, according to our data there is no evidence for the hypothesis
that functional p53 loss is linked to perturbations in the
regulation of apoptosis in colorectal neoplasias."' This
agrees with the findings of Bosari et aL4' but is contradictory to the data from Sinicrope et al.,3' who report a significantly inverse correlation between bcl-2 and p53 in
immunostaining in adenomas.
All these findings indicate that apoptosis plays a role
PCD and b d 2 in Colorectal NeoplasiadBaretton et al.
in tumor progression but that the regulation of apoptosis
is complex and cannot simply be reduced to assessment
of bcl-2 grne expression or p53 a c c ~ m u l a t i o nThe
. ~ coniplete elucidation of these mechanisms remains an issue
for further investigation. Furthermore, future research
will have to show whether the pathways regulating
apoptosis can be used as a target for novel approaches
to anticancer therapy.
In conclusion, our results clearly show that bcl-2 is
strongly expressed in most colorectal adenomas and carcinomas, with the highest incidence in adenomas. bcl-2
expression is evidently characteristic of early stages in
colorectal carcinogenesis followed by other genetic
(changes like TP53 loss. Thus, apart from genetic changes
leading to deregulation of proliferation, the inhibition of
;apoptosis by bcl-2 expression may also promote tumor
:growth at this stage of carcinogenesis. Since bcl-2 expres'sion was correlated significantly with a low rate of
,apoptosis in the carcinomas investigated, it can be conNcluded that in these tumors apoptosis is regulated in a
way similar to that in physiologic conditions. However,
this pathway seemingly was not preserved in all tumors
analyzed. Furthermore, the correlation between high proliferation rate and a high rate of apoptosis in carcinomas
suggests that an excess of putatively inappropriate or conflicting growth signals may also trigger apoptosis. N o correlations between p53 accumulation and hcl-2 or
apoptosis could be found in our cases. I n accordance
with results reported from other malignancies, Dcl-2-expression was associated prognostically with a less malignant behavior of the colorectal carcinomas in our study.
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