Carcinogenesis vol.19 no.10 pp.1755–1761, 1998
p21WAF1/CIP1 expression in primary lung adenocarcinomas:
heterogeneous expression in tumor tissues and correlation with
p53 expression and proliferative activities
Yukio Takeshima2, Masahiro Yamasaki,
Takashi Nishisaka1, Souichi Kitaguchi and Kouki Inai
Second Department of Pathology, Hiroshima University School of Medicine,
1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan
1Present
address: Department of Pathology and Clinical Laboratory,
Hiroshima Prefectural Hiroshima Hospital, 1-5-54 Ujina-Kanda, Minami-ku,
Hiroshima 734-8551, Japan
2To
whom correspondence should be addressed
Email: [email protected]
p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor,
discovered to be a downstream effector of p53-dependent
cell cycle regulation. In order to elucidate the significance
of p21 expression in lung adenocarcinomas, we performed
immunohistochemical analysis of p21, p53 and Ki-67
expression in surgically resected lung adenocarcinomas. In
non-neoplastic tissue, a few bronchial and bronchiolar
suprabasal and ciliated cells, and a few type II alveolar
cells and alveolar macrophages in the peripheral lung,
were p21 positive, but the positive rate in normal lung
tissue was very low (,1%). All 91 lung adenocarcinomas
examined showed p21 immunoreactivity: 39 cases (42.9%)
and 52 cases (57.1%) showed high and low p21 expression
levels, respectively. There was no significant correlation
between p21 expression and p53 expression, the loss of
heterozygosity status of the p53 gene, histological grade
determined by the predominant histology, lymph node
metastasis, pathological stage, tumor size, smoking history
or gender. A positive, not inverse, correlation between p21
and Ki-67 expression was observed. We also observed
heterogeneous expression of p21 in lung adenocarcinomas,
i.e. in about two-thirds of the tumors, the tumor cells in
the peripheral regions were p21 positive more frequently
than were those in the central regions. More intense p21
expression tended to occur in the more highly differentiated
areas. These results suggest that p21 is involved in tumor
cell differentiation and the physiological mechanism that
protects against tumor extension.
Introduction
Changes of many genes, including p53 (1), K-ras (2), RB (3),
p16 (4) and FHIT (5), in lung cancers have been reported.
Among these genes, p53 is well known to be a nuclear
phosphoprotein with tumor suppressive activity and to act as
a transcription factor. So far, p53 gene alteration is one of the
most frequent events in human cancers and p53 alterations in
lung cancers have been reported to be related to tumor
progression and the survival rate (6,7). The mutational spectrum
of p53 may be related to the carcinogen to which subjects
have been exposed. For example, the most common p53 gene
mutation in lung cancers in non-smokers is a G:C→A:T
Abbreviations: p21, p21WAF1/CIP1; LOH, loss of heterozygosity; PCR, polymerase chain reaction.
© Oxford University Press
transition (8), whereas in smokers G:C→T:A transversion
occurs more frequently (9).
Recently, p21WAF1/CIP1 (p21), an inhibitor of cyclin-dependent kinases, was isolated and found to be an effector of wildtype p53 (10). It regulates entry into DNA synthesis during
the cell cycle and is a potent inhibitor of the Cdk2–cyclin E
complex, which acts as an accelerator of cell cycle progression
from the G1 to S phase through the phosphorylation of
retinoblastoma protein, which in turn is a negative cell cycle
regulator (11). Wild-type p53 can activate p21, but recently, a
p53-independent p21 activation pathway was suggested to
operate in several human cancers (12–15).
Histological heterogeneity within one lung adenocarcinoma
is observed frequently, i.e. some areas have phenotypically
different components. Moreover, there are few reports on
correlations between p53, p21 and Ki-67 expression in relation
to the tumor heterogeneity. Also, there have been a few reports
available on p21 expression in primary lung adenocarcinoma
to date (16,17).
The aim of this study was to ascertain whether heterogeneous
expression of p21 in lung adenocarcinomas was associated or
correlated with p53 and Ki-67 expression and clinicopathological data.
Materials and methods
Tissue samples
Ninety-one primary lung adenocarcinomas were obtained from the surgical
file of the Second Department of Pathology, Hiroshima University School of
Medicine, during the period 1992–1997. The histological grade of each lung
adenocarcinoma was confirmed according to the ‘General Rules for Clinical
and Pathological Records of Lung Cancer of the Japan Lung Cancer Society’
(18). Histological grades were categorized as well, moderately and poorly
differentiated. Tumors showing atypical adenomatous hyperplasia in the
peripheral regions were excluded from this study.
Immunohistochemistry and immunohistochemical scoring
Monoclonal anti-p21 (Ab-1; Oncogene Science, Cambridge, MA), anti-p53
(DO-7; Novocastra, Newcastle, UK) and anti-Ki-67 (MIB-1; Immunotech,
Marseilles, France) antibodies were used for immunohistochemistry. Sections
(5 µm) were cut from formalin-fixed and paraffin-embedded tissues and
immunostained using the avidin–biotin–peroxidase complex method with an
antigen retrieval procedure, after which all slides were counterstained weakly
with hematoxylin. Cells showing nuclear staining were considered to be
positively stained, and positive immunostaining by the three antibodies of the
peripheral and central regions of each tumor were scored. Representative
areas in the peripheral and central regions of each tumor were chosen and the
proportion, as percentages of positive cells were calculated. The half area
from the center of the tumor was designated as ‘central region’, and the rest
of the tumor was designated the ‘peripheral region’. One thousand tumor cells
per section were counted under a microscope and scored as follows: 0, no
immunopositive cells were present; 1, ,1% of the cells were immunopositive;
2, 1 to ,10% of the cells were immunopositive; 3, 10 to ,50% of the cells
were immunopositive; 4, 50 to ,90% of the cells were immunopositive; and
5, .90% of the cells were immunopositive. The immunohistochemical scores
0, 1 and 2 were considered to reflect low-level or no expression and 3, 4 and
5 were considered to reflect high expression levels. The representative score
for each tumor was determined by averaging the scores for its peripheral and
central regions. When the staining score for the peripheral region was higher
than that for the central region, we called the expression pattern ‘peripheral
dominant’, we called the converse a ‘central dominant pattern’ and when the
staining scores for the two regions were the same, we called it an ‘equal pattern’.
1755
Y.Takeshima et al.
Table I. Correlations between p21 expression and clinicopathological
factors in lung adenocarcinomas
p21 expression
Low
High
P-value
Tumor differentiation
Well
Moderate
Poor
16
22
11
7
20
12
0.28
Age
,59
.60
21
31
7
32
0.02*
Gender
Male
Female
26
26
13
26
0.10
Smoking history (pack-years of cigarette smoking)
0
25
27
,20
13
14
ù20
4
8
Fig. 1. p21 expression in non-neoplastic lung tissue. (A) Occasional
suprabasal or ciliated bronchial epithelium showed positivity for p21, but
basal cells were negative. (B) Occasional alveolar epithelial cells showed
positivity for p21. [(A) p21 expression, 3400; (B) p21 expression, 3400.]
Fig. 2. p21 expression in organizing pneumonia. Hyperplastic epithelial
cells and fibroblastic cells in organizing pneumonia showed increased
positivity for p21, compared with normal alveolar cells (p21 expression,
3200).
Analysis of loss of heterozygosity (LOH) analysis of the p53 gene
The TP53 locus was selected for the detection of allelic loss on 17p13. LOH
analysis of TP53 was carried out essentially as described by Takeshima et al.
(19). For informative cases, allelic loss was considered to have occurred if
the intensity of the autoradiographic signal of a given allele in the tumor
DNA was at least 50% lower than that of the corresponding normal allele.
Statistical analysis
The data were analyzed using chi-squared or Fisher’s exact test. P-values of
,0.05 were considered significant.
Results
Expression of p21, p53 and Ki-67 in non-neoplastic lung tissues
A few bronchial and bronchiolar suprabasal and ciliated cells
(Figure 1A) showed p21 immunopositivity, as did a few type
II alveolar cells (Figure 1B) and alveolar macrophages in the
peripheral lung tissue, but the positive rate in normal lung
tissue was very low (,1%). The presence of p21 expression
in non-neoplastic cells could be considered as an internal
positive control for the immunohistochemical procedure. When
background inflammatory changes were present, p21 expression levels appeared to be higher (Figure 2). Some of the basal
bronchial and bronchiolar cells were p53- and Ki-67-positive,
but their positive rates were also very low (,1%).
1756
0.91
Tumor size
ø3cm
.3cm
38
14
31
8
0.48
Lymph node metastasis
(–)
(1)
32
20
22
17
0.77
Stage
I, II
III, IV
32
20
23
16
0.80
p53 expression
Low
High
25
27
24
15
0.80
LOH of TP53
(–)
(1)
20
15
7
3
0.84
Ki-67 expression
Low
High
27
25
12
27
0.04*
*Significantly different.
Table II. Heterogeneous expression of p21, p53 and Ki-67 in lung
adenocarcinomas
p21 expression
p53 expression
Ki-67 expression
P . Ca
P 5 Cb
P , Cc
59 (65)d
37 (41)
54 (59)
22 (24)
46 (51)
26 (29)
10 (11)
8 (8)
11 (12)
. C, peripheral dominant pattern; bP 5 C, equal pattern; cP , C, central
dominant pattern.
dCases (cases with P . C of p21/total cases3100%).
aP
Expression of p21 in lung adenocarcinomas
All 91 lung adenocarcinomas examined showed some degree
of p21 immunoreactivity. The representative expression score
for each tumor was determined by averaging the scores for its
peripheral and central regions. Thirty-nine (43%) and 52 (57%)
tumors showed strong and weak expression, respectively. There
was no significant correlation between the p21 expression and
the histological grade (determined according to the predominant histology), lymph node metastasis, smoking history, pathological stage, tumor size, gender, p53 expression or the presence
of LOH at the TP53 locus (Table I). However, there was a
significant correlation between the p21 expression level and
p21WAF1/CIP1 expression in primary lung adenocarcinomas
Fig. 3. Heterogeneous expression of p21 in a lung adenocarcinoma (case F9). The p21 and Ki-67 expression levels in the peripheral area were higher than
those in the central area. Most of the Ki-67-positive cells were positive for p21. A small number of tumor cells in the peripheral area were p53-positive, but
no p53 expression was observed in the central area. No LOH of TP53 was detected in this tumor (not shown). Left, the peripheral area of the tumor:
(A) histological appearance (hematoxylin and eosin); (B) p21 expression; (C) Ki-67 expression; (D) p53 expression. Right, the central area of the tumor:
(E) histological appearance (hematoxylin and eosin); (F) p21 expression; (G) Ki-67 expression; (H) p53 expression (3200).
patient age. A positive, not inverse, correlation (P , 0.05)
between p21 and Ki-67 expression was observed. Elevated
expression levels of both p21 and Ki-67 occurred in the same
areas, and most of the Ki-67 positive cells were positive for
p21 (Figure 3).
Heterogeneous expression of p21, p53 and Ki-67 in lung
adenocarcinomas
We observed peripheral dominant localization of p21-positive
cells in 65% (59/91) of the tumors: 24% (22/91) showed
equal expression pattern and 11% (10/91) showed the central
dominant pattern (Table II). Expression of p21 tended to be
more intense in the highly differentiated areas of the same
tumors (Figures 3–5), in particular in non-sclerosing or sclerosing bronchiolo-alveolar carcinomas, although poorly differentiated adenocarcinomas also showed the same tendency.
Thirty-seven (41%), 46 (51%) and eight (9%) of the 91
tumors showed the peripheral dominant, equal and central
dominant patterns of p53 expression, respectively (Table II).
Tumors showing strong p53 immunoreactivity frequently
showed a diffuse p53 expression pattern (Figure 4), whereas
weak p53 expression, was frequently associated with the
peripheral dominant p53 expression pattern (Figure 3). Overall,
tumors showing the peripheral dominant pattern of p21 expression associated with the peripheral dominant (27/91; 30%) or
equal (27/91; 30%) pattern of p53 expression were predominant
(Table III).
Fifty-four (59%), 26 (29%) and 11 (12%) of the 91 tumors
showed the peripheral dominant, equal and central dominant
Ki-67 expression pattern, respectively (Table II). Overall,
tumors showing the peripheral dominant pattern of both
1757
Y.Takeshima et al.
Fig. 4. Heterogeneous expression of p21 in a lung adenocarcinoma (case M15). The p21 expression level in the peripheral, well-differentiated area was much
higher than that in the central, more atypical area. The same diffuse p53 expression pattern was observed in both central and peripheral regions. This tumor
showed LOH of TP53 (not shown). Left, the peripheral region of the tumor: (A) histological appearance (hematoxylin and eosin); (B) p21 expression;
(C) p53 expression. Right, the central region of the tumor: (D) histological appearance (hematoxylin and eosin); (E) p21 expression; (F) p53 expression
(3200).
Fig. 5. Heterogeneous expression of p21 according to the tumor differentiation status in a lung adenocarcinoma (case F4). p21 expression level in the
peripheral, well-differentiated area was higher than that in the central, poorly differentiated, area. Left, the peripheral region of the tumor: (A) histological
appearance (hematoxylin and eosin); (B) p21 expression. Right, the central region of the tumor: (C) histological appearance (hematoxylin and eosin); (D) p21
expression (3200).
1758
p21WAF1/CIP1 expression in primary lung adenocarcinomas
Table III. Correlations between p21 expression pattern and those of p53
and Ki-67 in lung adenocarcinomas
p21 expression (%)
P . Ca
p53 expression
P
P
P
Ki-67 expression P
P
P
.
5
,
.
5
,
C
C
C
C
C
C
27
27
5
38
13
8
(30)d
(30)
(5)
(42)
(15)
(9)
P 5 Cb
P , Cc
7 (8)
15 (17)
0 (0)
10 (11)
11 (12)
1 (1)
3
4
3
6
2
2
(3)
(4)
(3)
(7)
(2)
(2)
. C, peripheral dominant pattern; bP 5 C, equal pattern; cP , C, central
dominant pattern.
dCases (cases with P . C of p21/total cases3100%).
aP
Table IV. Correlations between p21/p53 expression and pathological stage
in lung adenocarcinomas
Pathological stage
I, II
III, IV
aCase
p21 low
p21 high
p53 low
p53 ligh
p53 low
p53 high
16 (64)a
9 (36)
16 (59)
11 (41)
16 (67)
8 (33)
7 (46)
8 (54)
(%).
p21 and Ki-67 expression were predominant (38/91; 42%)
(Table III).
Correlations between pathological stage and p21/p53
expression
Table IV shows the correlations between pathological stage
and p21/p53 expression: the p531/p211 group had a higher
percentage of high-stage tumors (54%) than the other groups,
but there were no significant differences between any of
the groups.
Discussion
In this study, we demonstrated that p21 expression in nonneoplastic lung tissues and adenocarcinomas correlated with
the differentiation status of non-neoplastic and neoplastic cells.
Expression of p21 was observed in the nuclei of non-neoplastic
cells, as described in the Results. Expression in the normal
bronchial mucosa clearly showed topographical distributions,
i.e., p21 was expressed only in suprabasal cells and ciliated
cells, whereas Ki-67 expression was observed only in basal
cells. Therefore, p21 expression may be involved in the
differentiation of bronchial epithelium. When background
inflammation was present, the p21 and Ki-67 expression levels
increased, which suggests that their expression may be induced
by genotoxic agents, such as free radicals, induced by inflammation. Guinee et al. demonstrated that enhanced p53 and
p21 expression in diffuse alveolar damage reflected normal
physiological up-regulation in response to cellular and DNA
damage, and was associated with apoptosis of type II pneumocytes (20), and immunohistochemical and genetic analyses
revealed that p21 expression in reactive lung lesions depended
on wild-type p53 expression (17).
Lung adenocarcinomas usually show histological heterogeneity, and consequently it is not always easy to evaluate
immunohistochemical staining patterns, especially those of the
cell cycle related proteins. In this study, we divided the
immunostained sections into peripheral and central regions,
and counted the immunohistochemically stained cells in each.
About two-thirds of the tumors showed the peripheral dominant
pattern of p21 expression. Usually, the adenocarcinoma cells
in the peripheral area were more highly differentiated (i.e.
they showed less cellular and/or structural atypia) than those
in the central region, especially in non-sclerosing and sclerosing
bronchiolo-alveolar carcinomas. These findings indicate that
p21 may be involved in the differentiation of tumor cells, even
though no significant correlation between p21 expression and
tumor differentiation, according to the predominant histology,
was demonstrated. Marchetti et al. reported that p21 immunoreactive cells were usually seen in foci that were more highly
differentiated than the rest of the tumor and p21 expression
tumors occurred more frequently in well than poorly differentiated non-small lung cancers (16), and speculated that p21 may
be related to inhibition of DNA synthesis in relation to
differentiation. This phenomenon was also observed in terminally differentiated cells of embryonic and adult mouse tissue
(21). Differentiation and permanent cell cycle arrest of skeletal
murine myocytes caused by MyoD expression was reported to
be associated with induction of p21 RNA and protein expression (22), whereas the association between p21 expression and
differentiation of breast carcinomas was less strong (23,24).
In our study, the p21 and Ki-67 expression levels in both
well and poorly differentiated adenocarcinomas correlated
significantly. The functions of p21 demonstrated previously
(11) suggest that cells that express p21 have low proliferative
activities, i.e. low Ki-67-labeling indices. However, our results
discussed above conflict with these and indicate that p21
expression is also induced by the physiological protection
mechanism that protects against the actions of some proliferation factors and/or by some inhaled cytotoxic and/or genotoxic
agents that reach the most proliferative areas of the alveoli, and
that p21 is not the only factor that controls lung adenocarcinoma
proliferation. Recently, we detected an inverse correlation
between p21 and Ki-67 expression levels in lung tissue
showing atypical adenomatous hyperplasia (unpublished data).
Therefore, escape from p21-dependent growth inhibition may
be related to the proliferation status of adenocarcinoma cells.
We found that it is not always easy to visualize co-expression
of p21 and Ki-67 antigens in the same tumor cells, but serial
sections showed that most of the Ki-67 positive cells were
positive for p21 as presented in Figure 3. However, further
study including double immunohisto-staining is necessary.
Expression of p21 and Ki-67 in colonic adenomas is reported
to be reciprocal (25); p21-positive cells were frequently clustered in the superficial areas and p21 expression in adenomas
was tentatively related to differentiation in the more superficial
areas of the tumors. However, p21 expression in colonic
adenocarcinomas was found to be heterogeneous and occasionally related to topographical or morphological features of
differentiation (25). In endometrial hyperplasias and adenocarcinomas, an inverse relationship between p21 and Ki-67
expression has been reported (26), and p21 expression levels
in brain tumors were found to be higher than those in nontumorous brain tissue (27). These different p21 expression
patterns in various human cancers may result from organspecific regulation of p21 expression.
Our results show that p21 expression is not always related
to p53 expression or LOH of the p53 gene. It has been
suggested that there is a close correlation between the presence
of missense mutation of p53 gene and p53 immunopositivity
1759
Y.Takeshima et al.
(28). Comparison of the LOH and p53 immunohistochemical
results showed a close relationship with a few exceptions in
our study (data not shown). Absence or a low frequency of
somatic p21 gene mutation in most human cancers, including
lung cancers, has been reported (29). Even though sequence
analysis of the p53 gene is necessary, these findings suggest
that p21 can be expressed by p53-dependent and -independent
pathways. Expression of p21 in ovarian, colonic and pancreatic
cancers did not necessarily correlate with p53 gene mutation
(13,30). Michieli et al. observed that growth factors, such as
platelet-derived growth factor, fibroblast growth factor and
epidermal growth factor, triggered the induction of p21 expression in p53-deficient cells (12) and transforming growth factorβ is known to be associated with p53-independent induction
of p21 expression (31). In view of these findings, further
examination of the correlations between expression of p21 and
these growth factors is needed.
Recently, many data about the significance of p21 expression
as a prognostic indicator have been reported. Expression of
p21 protein may be a better prognostic factor rather than that
of p53 protein in patients with gastric carcinomas (32), whereas
other investigators showed that high p21 expression levels
correlated with deep invasion and lymph node metastasis (15).
High p21 expression levels were reported to be associated
with short relapse-free survival of patients with breast cancer
(23), but the converse has also been reported (33). High p21
expression levels in colonic adenocarcinomas were associated
with lower tumor stages (25,34) and p21 expression has been
reported to be associated significantly with the early clinical
stages of pancreatic adenocarcinomas (35). The carcinomas
with the p53 high/p21 low phenotype may show the highest
stage and, therefore, were associated with a poor prognosis
(24), but we found there was no significant correlation between
p53/p21 expression and the pathological stage. Therefore, p21/
p53 expression alone may not be a prognostic indicator in
patients with lung adenocarcinomas. Further studies, including
long-term follow-up, is necessary to enable definite conclusions
concerning the prognostic significance of p21 expression in
lung adenocarcinomas to be drawn.
Acknowledgements
We thank Mr Fumio Nakayori and Mr Tomohiro Nakano for technical
assistance.
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Received on March 18, 1998; revised on June 26, 1998; accepted on July
2, 1998
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