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CANCER
55
CYTOPATHOLOGY
Cytologic Differential Diagnosis among Reactive
Mesothelial Cells, Malignant Mesothelioma, and
Adenocarcinoma
Utility of Combined E-Cadherin and Calretinin Immunostaining
Hiromi Kitazume, C.T.1
Kazuhisa Kitamura, C.T.1
Katsuhiko Mukai, M.D.1
Yoshiaki Inayama, M.D.1
Naomi Kawano, M.D.1
Nobuo Nakamura, M.D.2
Jinyu Sano, M.D.2
Kunihiro Mitsui, C.T.2
Sachiko Yoshida, M.D.3
Yukio Nakatani, M.D.1
1
Division of Anatomic and Surgical Pathology,
Hospital of Yokohama City University, Yokohama
City University School of Medicine, Yokohama, Japan.
2
Department of Pathology, Yokohama Citizens
Hospital, Yokohama, Japan.
3
Department of Pathology, Yokohama Kowan Hospital, Yokohama, Japan.
BACKGROUND. The differential diagnosis between reactive mesothelial cells (RMs),
malignant mesotheliomas (MMs), and adenocarcinomas (ACs) is often difficult in
cytologic specimens, and the utility of various immunohistochemical markers have
been explored. Because recent immunohistologic studies have suggested that
E-cadherin (E-cad) and calretinin (Cal) may be useful markers for epithelial and
mesothelial differentiations, respectively, the authors investigated their utility in
cytologic diagnosis.
METHODS. In this retrospective study, immunostaining was performed on smears
retrieved from Papanicolaou-stained slides of effusions using the labeled streptavidin-biotin method. Sixteen cases of RM, 9 cases of MM, and 52 cases of AC from
various sites, including 13 pulmonary primaries, were examined with primary
antibodies against E-cad and Cal.
RESULTS. The positive rates for E-cad and Cal, respectively, were as follows: RM,
0/16 (0%) and 16/16 (100%); MM, 9/9 (100%) and 8/8 (100%); and AC, 45/52
(86.5%) and 0/51 (0%). The E-cad expression by neoplastic cells was strongest in
the intercellular junctions, and poorly differentiated neoplastic cells in the single
cell form showed the weakest expression.
CONCLUSIONS. In contrast to the results of previous immunohistochemical studies,
the current study indicates that MMs constantly express E-cad, whereas RMs lack
its expression in cytologic specimens, which would be useful in the differential
diagnosis between the two. On the other hand, E-cad expression is not reliable for
distinguishing AC from MM. The Cal expression can be a very useful marker for the
distinction between AC and the mesothelial lineage. The combined immunostaining for E-cad and Cal has utility in differential diagnosis among RM, MM, and AC.
Cancer (Cancer Cytopathol) 2000;90:55– 60. © 2000 American Cancer Society.
KEYWORDS: immunostaining, mesothelial cell, mesothelioma, adenocarcinoma, Ecadherin, calretinin, cytologic diagnosis.
Presented in part at the 39th General Meeting of
the Japanese Society of Clinical Cytology, Sapporo,
Japan, June 19, 1998.
Address for reprints: Hiromi Kitazume, M.T., Division of Anatomic and Surgical Pathology, Hospital
of Yokohama City University, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-8566, Japan.
Received February 1, 1999; revision received June
11, 1999; accepted June 18, 1999.
© 2000 American Cancer Society
I
t has long been a major diagnostic challenge to distinguish among
reactive mesothelial cells (RMs), malignant mesothelioma (MM),
and adenocarcinoma (AC) in both cytologic and surgical pathologic
specimens. For differential diagnosis between MM and AC, previous
immunohistochemical and immunocytochemical studies established
panels of useful antibodies against immunodeterminants, including
carcinoembryonic antigen (CEA), CD15 (recognized by the monoclonal antibody Leu M1), tumor-associated glycoprotein 72 (recognized
by the monoclonal antibody B72.3), BerEP4 glycoprotein, and
MOC-31 glycoprotein.1–5 Most of these are markers for AC and can be
56
CANCER (CANCER CYTOPATHOLOGY) February 25, 2000 / Volume 90 / Number 1
TABLE 1
Summary of Cases
Cytodiagnosis
Pleural fluids
Peritoneal
fluids
Total
Reactive mesothelial cells
Malignant mesothelioma
Adenocarcinoma
Lung
Breast
Stomach
Bile duct
Pancreas
Colon
Endometrium
Ovary
Total
16
9
31
13
4
4
0
3
1
2
4
56
0
0
21
0
0
4
2
2
3
4
6
21
16
9
52
13
4
8
2
5
4
6
10
77
useful in the setting of differential diagnosis between
RM and AC as well. For differentiation between RMs
and MMs, on the other hand, only a few markers have
been reported to be of utility. Among these are epithelial membrane antigen (EMA) and p53 protein,
which are often expressed in MMs but usually not in
RMs.6,7 Recently, two new immunohistochemical
markers have come into notice: E-cadherin for ACs8,9
and calretinin for the mesothelial lineage.10 Because
there are only a few reports of experience with the
immuonostaining of these new markers in cytologic
specimens,11,12 we investigated their utility in cytologic differential diagnosis.
MATERIALS AND METHODS
A total of 77 cytologic specimens of pleural or peritoneal fluids collected between 1991 to 1997 were retrieved from the cytology files of our laboratories (Table 1). They included 16 cases of RM, 9 cases of MM,
and 52 cases of AC. All cases of RM and MM were from
specimens of pleural fluids. Of 16 cases of RM, the
direct causes of the effusions were infectious in 9
cases, combined infectious and malignancy (lung involvement) in 3 cases, malignant (with lung involvement) in 1 case, traumatic in 1 case, and unknown in
2 cases. Of these, 12 cases had underlying malignant
disease at some site, but none of them showed malignant cells in the pleural effusions during their clinical
courses. The primary sites for the 52 cases of AC included 13 lungs, 4 breasts, 8 stomachs, 2 bile ducts, 5
pancreases, 4 colons, 6 endometria, and 10 ovaries.
The diagnoses of all neoplastic cases had been confirmed histopathologically. MMs were of either epithelial or biphasic type.
Immunocytochemical staining was performed using smears on the slides originally stained by Papani-
colaou dye and later decolorized with 0.5% hydrochloric acid in 95% ethanol for this study. The primary
antibody against E-cadherin (mouse monoclonal
HECD-1; Takara, Otsu, Japan) was diluted 1:400 in 20
mM Tris-buffered saline (TBS), pH 7.4, with 1.0% bovine albumin and 10mM CaCl2. The anticalretinin antibody (rabbit polyclonal; Swant, Bellinzona, Switzerland) was diluted 1:8000 in 10 mM phosphatebuffered saline (PBS) with 1.0% bovine albumin. The
decolorized slides were rinsed in tap water for 5
minutes. The slides were then incubated with 0.3%
hydrogen peroxide for 30 minutes to quench the endogenous peroxidase activity. Following overnight incubation with the primary antibodies at room temperature, the slides were rinsed twice in TBS for 5 minutes
each. The labeled streptavidin-biotin method (LSAB2
Kit; Dako, Carpinteria, CA) was used for the subsequent immunostaining procedure. Chromogen 3, 3⬘diaminobenzidine solution was used for detection of
the antigens. The smears were finally counterstained
with hematoxylin and coverslipped for examination.
Negative controls were run in parallel by replacing the
primary specific antibody with mouse immunoglobulin G1 (Dako, Glostrup, Denmark) or rabbit immunoglobulins.
The immunostaining was graded on a sliding scale
of ⫺ to 3⫹ according to the percentage of positive cells
(⫺, no positive cell; ⫹, 1–9%; ⫹⫹, 10 – 89%; ⫹⫹⫹,
90 –100%). In the final evaluation of the staining for
calretinin, there were not enough cells on the slides in
two cases (one mesothelioma and one ovarian adenocarcinoma), and these two cases were excluded from
the analysis of calretinin immunostaining.
The relation between cytologic differentiation and
the degree of expression for E-cadherin in ACs was
analyzed by the Mann–Whitney U test. For this purpose, all ACs of the digestive organs were classified as
poorly, moderately, or well differentiated based on the
degree of cohesiveness of neoplastic cells in the cytologic specimens, and also were scored from 0 to 3,
with these scores corresponding to grades of immunoreactivity.
RESULTS
For the immunostaining of E-cadherin (Table 2), the
positivity rate in each category of the cases was as
follows: RM, 0 of 16 cases (0%); MM, 9 of 9 cases
(100%); and AC, 45 of 52 cases (87%). All 13 ACs of the
lung primary were positively stained. The positive
rates for ACs of the other sites varied from 63% (stomach primary) to 100% (bile duct and colon primaries).
Three negative cases of the gastric ACs were all poorly
differentiated adenocarcinoma or signet-ring cell carcinoma. When the cytologic differentiation was eval-
E-Cadherin and Calretinin Immunostaining/Kitazume et al.
57
TABLE 2
Immunoreactivity for E-Cadherin
E-cadherin immunoreactivity
Cytodiagnosis
No. of positive cases/
no. of cases (%)
Reactive mesothelial cells
Malignant mesothelioma
Adenocarcinoma
Lung
Breast
Stomach
Bile duct
Pancreas
Colon
Endometrium
Ovary
0/16 (0)
9/9 (100)
45/52 (87)
13/13 (100)
3/4 (75)
5/8 (63)
2/2 (100)
4/5 (80)
4/4 (100)
5/6 (83)
9/10 (90)
ⴙⴙⴙ
ⴙⴙ
7
20
11
2
9
ⴙ
ⴚ
16
1
3
1
4
3
1
2
2
1
16
2
3
2
7
1
2
2
4
1
1
3
1
1
⫺: no positive cell; ⫹: positive cell ⬍10%; ⫹⫹: 10% ⱕ positive cell ⬍ 90%; ⫹⫹⫹: positive cell ⱖ 90%.
TABLE 3
A Correlation Analysis between Cytologic Differentiation and Ecadherin Immunoreactivity in Adenocarcinomas of Digestive Organs
E-cadherin immunoreactivity
mesothelial cells in the background of adenocarcinomas were consistently positive for calretinin (Fig. 1I).
DISCUSSION
Cytologic differentiation
ⴙⴙⴙ
ⴙⴙ
ⴙ
ⴚ
Total
Poor
Moderate
Well
Total
0
3
16
19
1
5
1
7
3
6
3
12
3
1
0
4
7a,b
15a,c
20b,c
42
⫺: no positive cell; ⫹: positive cell ⬍10%; ⫹⫹: 10% ⱕ positive cell ⬍ 90%; ⫹⫹⫹: positive cell ⱖ 90%
Mann–Whitney U test: aP ⫽ 0.0376; bP ⫽ 0.0006; cP ⫽ 0.0035.
uated in relation to the expression of E-cadherin in
ACs of the digestive organs (stomach, bile duct, and
pancreas primaries combined) (Table 3), cytologically
well-differentiated ACs showed significantly higher expression of E-cadherin than moderately differentiated
ACs (P ⫽ 0.0035) or poorly differentiated ACs (P ⫽
0.0006). The localization of the E-cadherin staining
was generally membranous and was especially intense
along the intercellular junctions (Fig. 1E, H; Fig. 2A, B).
Poorly differentiated ACs in the single cell form, including signet-ring cell carcinoma, often showed dotlike cytoplasmic staining, which often appeared to be
perinuclear (Fig. 2C). Reactive mesothelial cells in the
backgrounds of adenocarcinomas were consistently
negative for E-cadherin.
As shown in Table 4, the positivity rate of calretinin expression was 16 of 16 cases (100%) for RM, 8 of
8 cases (100%) for MM, and 0 of 51 cases (0%) for AC.
All cases of RM and MM showed many positive cells
with strong staining intensity in the cytoplasm and
often in the nucleus as well (Fig. 1C, F). Reactive
E-cadherin is a member of the Ca2⫹-dependent cell
adhesion molecules that is specifically expressed on
all epithelial cells.13 Soler et al. studied the expression
of E-cadherin (and also N-cadherin and catenins) in
MMs and pulmonary ACs using immunohistochemical methods on frozen tissue sections.8 They found
that all 19 ACs expressed E-cadherin, whereas a minority of the cells in some MMs expressed weak and
mostly cytoplasmic staining for E-cadherin, leading
them to advocate the utility of E-cadherin in differentiating between pulmonary ACs and MMs. A few years
later, the same group reported similar results using
formalin fixed, paraffin embedded tissues.9 In an immunohistochemical study reported by Leers et al.,14 20
of 21 metastatic ACs of the pleura were positive for
E-cadherin, whereas only 3 of 20 MMs were immunoreactive for E-cadherin; and most were focal, with less
than 5% of tumor cells positive. In contrast to these
previous studies using tissue sections, the current
study indicates that E-cadherin is constantly and
clearly expressed in MMs and cannnot serve as a reliable marker for distinguishing ACs from MMs in
cytologic specimens. The discrepancy between the results of our study and the previous report by Leers et
al.14 may be due partly to the difference in the state of
the antigen preservation, because both studies used
the same monoclonal antibody (HECD-1) and immunostaining method (labeled streptavidin-biotin method). This view is supported by our recent observation
that most MMs expressed E-cadherin in formalin
fixed, paraffin embedded tissues when a highly sensi-
58
CANCER (CANCER CYTOPATHOLOGY) February 25, 2000 / Volume 90 / Number 1
FIGURE 1. (A) Papanicolaou staining of reactive mesothelial cells (RMs). (B) Immunostaining of RMs for E-cadherin. Note complete absence of staining. (C)
Immunostaining of RMs for calretinin. The cells show intense cytoplasmic staining. (D) Papanicolaou staining of malignant mesothelioma (MM) cells. (E)
Immunostaining of MM cells for E-cadherin. Intercellular junctions of a cell ball are strongly positive. (F) Immunostaining of MM cells for calretinin. Note diffuse
cytoplasmic staining. (G) Papanicolaou staining of adenocarcinoma (AC) cells. (H) Immunostaining of AC cells for E-cadherin. Note strong positivity localized in the
intercellular junctions. (I) Immunostaining of AC cells for calretinin. Note the sharp contrast between the negatively stained cluster of AC cells and the strongly positive
cluster of RMs.
tive detection method, the Catalyzed Signal Amplification (CSA) System (Dako, Carpinteria, CA), was used
in the immunohistochemical staining (Mukai et al.,
unpublished data). Since the original study by Solar et
al.8 used frozen tissues, it is hard to attribute the
difference in the results to the state of the antigen
preservation in this case. It may be of note that these
authors do admit that MMs sometimes express E-
cadherin and that its staining property alone should
not be relied upon in the differentiation between ACs
and MMs.8
Our study indicates that the expression of E-cadherin is a very useful marker for differentiating ACs or
MMs from RMs in cytologic specimens. Schofield et al.
demonstrated that E-cadherin immunostaining is useful in the former setting, ACs versus RMs.12 Our study
E-Cadherin and Calretinin Immunostaining/Kitazume et al.
59
FIGURE 2. Immunostaining for E-cadherin in various adenocarcinomas (ACs). (A) Well-differentiated pancreatic AC cells. (B) Ovarian clear cell AC cells. (C) Poorly
differentiated gastric AC cells. Note the dotlike cytoplasmic staining.
TABLE 4
Immunoreactivity for Calretinin
Calretinin
immunoreactivity
Cytodiagnosis
No. of positive cases/
no. of cases (%)
Reactive mesothelial cells
Malignant mesothelioma
Adenocarcinoma
Lung
Breast
Stomach
Bile duct
Pancreas
Colon
Endometrium
Ovary
16/16 (100)
8/8 (100)
0/51 (0)
0/13 (0)
0/4 (0)
0/8 (0)
0/2 (0)
0/5 (0)
0/4 (0)
0/6 (0)
0/9 (0)
ⴙⴙⴙ
ⴙⴙ
ⴙ
ⴚ
16
8
51
13
4
8
2
5
4
6
9
⫺: no positive cell; ⫹: positive cell ⬍10%; ⫹⫹: 10% ⱕ positive cell ⬍ 90%; ⫹⫹⫹: positive cell ⱖ 90%.
further suggests that it would be especially useful in
the latter setting as well, as there have been only a few
markers available, such as EMA and p53, to distingusish MMs from RMs.6,7,15 It was reported by van der
Kwast et al. that of 25 serous fluid smears from patients with MM, 23 cases showed varying numbers of
EMA-stained cells, whereas reactive mesothelial cells
in 56 smears were virtually all negative.6 The finding
that EMA and E-cadherin, both normally expressed in
epithelial cells, are detected in MMs suggests that
mesothelial cells, which are of mesodermal origin,
may acquire epithelial characters when they are transformed. Our study also demonstrates that poorly differentiated ACs, in the single cell form or in a few cell
clusters, show less expression of E-cadherin than welldifferentiated ACs in tight, large clusters. Because Ecadherin plays a central role in epithelial cell– cell
adhesions, this observation is not unexpected and actually is in accordance with the results of immunohis-
tochemical examination.16 E-cadherin immunostaining thus may be more informative and of practical
diagnostic value in a situation where it is necessary to
distingush well-differentiated ACs, rather than poorly
differentiated ACs, from RMs. It is noteworthy that
some poorly differentiated gastric AC cells showed
dotlike cytoplasmic immunostaining for E-cadherin.
Some previous immunohistochemical studies17,18
have described the “disorganized” pattern of cytoplasmic staining for E-cadherin in carcinomas of advanced stage or of invasive nature. Our finding also
suggests that the disorganized cytoplasmic staining
pattern may be related to poor cohesiveness of the
neoplastic cells. In this regard, it is interesting that
E-cadherin gene mutations have been detected in gastric signet-ring cell carcinomas.19
Calretinin is a 29 kD calcium-binding protein that
normally is expressed in neuronal cells. Recent immunohistochemical studies10,20 have suggested that it is a
very useful marker for cells of mesothelial lineage,
both reactive and neoplastic. Barberis et al. have reported recently that calretinin is just as useful in evaluating cytologic specimens as it is in the histologic
differential diagnosis between cells of the mesothelial
lineage and ACs.9 Observations in our study support
the results of these studies, indicating the utility of
calretinin as a good mesothelial marker. Although the
current series of ACs in cytologic specimens included
no positive cases, a small number of calretinin positive
ACs have been noted in some previous studies.10,11,21
Our own experience also suggests the presence of a
few calretinin positive ACs in immunohistochemical
staining (Mukai et al., unpublished data). Thus, just as
is the case with other immunocytolochemical markers, the results of immunostaining for calretinin
should be evaluated in the whole context of examination with a panel of antibodies.
60
CANCER (CANCER CYTOPATHOLOGY) February 25, 2000 / Volume 90 / Number 1
TABLE 5
Typical Immunoreactivity for E-cadherin and Calretinin
9.
Cytodiagnosis
E-cadherin
Calretinin
Reactive mesothelial cell
Malignant mesothelioma
Adenocarcinoma
⫺
⫹
⫹
⫹
⫹
⫺
10.
11.
In summary, the current study indicates that the
combined immunostaining of E-cadherin and calretinin is a useful adjunct to the panels of antibodies for
immunocytochemical differentiation among RMs,
MMs, and ACs (Table 5).
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