Background: Differentiating reactive mesothelial cells from metastatic carcinoma in effusion cytology is a challenging task. The application of at least 4 monoclonal antibodies including 2 epithelial markers (Ber-EP4, MOC-31, CEA, or B72.3) and 2 mesothelial markers (calretinin, WT-1, CK5/6, or HBME-1) are often useful in this distinction; however, it is not readily available in many resource-limited developing countries. Aberrant immunoexpression of enhancer of zeste homolog 2 (EZH2), a transcriptional repressor involved in cancer progression, is observed widely in various malignancy. In this study, we evaluate the diagnostic value of EZH2 as a single reliable immunomarker for malignancy in effusion samples. Methods: A total of 108 pleural, peritoneal, and pericardial effusions/washings diagnosed as unequivocally reactive (n = 41) and metastatic carcinoma (n = 67) by cytomorphology over 18 months were reviewed. Among the metastatic carcinoma cases, 54 were adenocarcinoma and others were squamous cell carcinoma (n = 1), carcinosarcoma (n = 1), and carcinoma of undefined histological subtypes (n = 11). Cell block sections were immunostained by EZH2 (Cell Marque, USA). The percentages of EZH2-immunolabeled cells over the total cells of interest were calculated. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off score to define EZH2 immunopositivity. Results: A threshold of 8% EZH2-immunolabeled cells allows distinction between malignant and reactive mesothelial cells, with 95.5% sensitivity, 100% specificity, 100% positive predictive value, and 93.2% negative predictive value (p < 0.0001). The area under the curve was 0.988. Conclusion: EZH2 is a promising diagnostic biomarker for malignancy in effusion cytology which is inexpensive yet trustworthy and could potentially be used routinely in countries under considerable economic constraints.

1.
Tabatabai
ZL
,
Nayar
R
,
Souers
RJ
,
Crothers
BA
,
Davey
DD
.
Performance characteristics of body fluid cytology analysis of 344 380 responses from the College of American Pathologists Interlaboratory Comparison Program in nongynecologic cytopathology
.
Arch Pathol Lab Med
.
2018
Jan
;
142
(
1
):
53
8
.
[PubMed]
0003-9985
2.
Cakir
E
,
Demirag
F
,
Aydin
M
,
Unsal
E
.
Cytopathologic differential diagnosis of malignant mesothelioma, adenocarcinoma and reactive mesothelial cells: A logistic regression analysis
.
Diagn Cytopathol
.
2009
Jan
;
37
(
1
):
4
10
.
[PubMed]
8755-1039
3.
Bedrossian
CW
.
Diagnostic problems in serous effusions
.
Diagn Cytopathol
.
1998
Aug
;
19
(
2
):
131
7
.
[PubMed]
8755-1039
4.
Lin
O
.
Challenges in the interpretation of peritoneal cytologic specimens
.
Arch Pathol Lab Med
.
2009
May
;
133
(
5
):
739
42
.
[PubMed]
1543-2165
5.
Motherby
H
,
Nadjari
B
,
Friegel
P
,
Kohaus
J
,
Ramp
U
,
Böcking
A
.
Diagnostic accuracy of effusion cytology
.
Diagn Cytopathol
.
1999
Jun
;
20
(
6
):
350
7
.
[PubMed]
8755-1039
6.
Loveland
P
,
Christie
M
,
Hammerschlag
G
,
Irving
L
,
Steinfort
D
.
Diagnostic yield of pleural fluid cytology in malignant effusions: an Australian tertiary centre experience
.
Intern Med J
.
2018
Nov
;
48
(
11
):
1318
24
.
[PubMed]
1444-0903
7.
Fetsch
PA
,
Abati
A
.
Immunocytochemistry in effusion cytology: a contemporary review
.
Cancer
.
2001
Oct
;
93
(
5
):
293
308
.
[PubMed]
0008-543X
8.
Bibbo
M
,
Draganova-Tacheva
R
,
Naylor
B
. Pleural, peritoneal and pericardial effusions. In:
Wilbur
D
,
Bibbo
M
, editors
.
Comprehensive Cytopathology
.
Elsevier
;
2015
. pp.
403
49
.
9.
Hjerpe
A
,
Ascoli
V
,
Bedrossian
CW
,
Boon
ME
,
Creaney
J
,
Davidson
B
, et al;
International Mesothelioma Interest Group
;
International Academy of Cytology
;
Papanicolaou Society of Cytopathology
.
Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology
.
Acta Cytol
.
2015
;
59
(
1
):
2
16
.
[PubMed]
0001-5547
10.
Simon
JA
,
Lange
CA
.
Roles of the EZH2 histone methyltransferase in cancer epigenetics
.
Mutat Res
.
2008
Dec
;
647
(
1-2
):
21
9
.
[PubMed]
0027-5107
11.
Chase
A
,
Cross
NC
.
Aberrations of EZH2 in cancer
.
Clin Cancer Res
.
2011
May
;
17
(
9
):
2613
8
.
[PubMed]
1078-0432
12.
Rao
ZY
,
Cai
MY
,
Yang
GF
,
He
LR
,
Mai
SJ
,
Hua
WF
, et al
EZH2 supports ovarian carcinoma cell invasion and/or metastasis via regulation of TGF-β1 and is a predictor of outcome in ovarian carcinoma patients
.
Carcinogenesis
.
2010
Sep
;
31
(
9
):
1576
83
.
[PubMed]
0143-3334
13.
Fluge
Ø
,
Gravdal
K
,
Carlsen
E
,
Vonen
B
,
Kjellevold
K
,
Refsum
S
, et al;
Norwegian Gastrointestinal Cancer Group
.
Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis
.
Br J Cancer
.
2009
Oct
;
101
(
8
):
1282
9
.
[PubMed]
0007-0920
14.
Choi
JH
,
Song
YS
,
Yoon
JS
,
Song
KW
,
Lee
YY
.
Enhancer of zeste homolog 2 expression is associated with tumor cell proliferation and metastasis in gastric cancer
.
APMIS
.
2010
Mar
;
118
(
3
):
196
202
.
[PubMed]
1600-0463
15.
Jiang
H
,
Gupta
R
,
Somma
J
.
EZH2, a unique marker of malignancy in effusion cytology
.
Diagn Cytopathol
.
2014
Feb
;
42
(
2
):
111
6
.
[PubMed]
8755-1039
16.
Sadullahoglu
C
,
Nart
D
,
Veral
A
.
The importance of EZH2 and MOC-31 expression in the differential diagnosis of benign and malignant effusions
.
Diagn Cytopathol
.
2017
Feb
;
45
(
2
):
118
24
.
[PubMed]
8755-1039
17.
Shinozaki-Ushiku
A
,
Ushiku
T
,
Morita
S
,
Anraku
M
,
Nakajima
J
,
Fukayama
M
.
Diagnostic utility of BAP1 and EZH2 expression in malignant mesothelioma
.
Histopathology
.
2017
Apr
;
70
(
5
):
722
33
.
[PubMed]
0309-0167
18.
Jiang
T
,
Wang
Y
,
Zhou
F
,
Gao
G
,
Ren
S
,
Zhou
C
.
Prognostic value of high EZH2 expression in patients with different types of cancer: a systematic review with meta-analysis
.
Oncotarget
.
2016
Jan
;
7
(
4
):
4584
97
.
[PubMed]
1949-2553
19.
Park
CK
,
Malinowski
DP
,
Cho
NH
.
Diagnostic algorithm for determining primary tumor sites using peritoneal fluid
.
PLoS One
.
2018
Jul
;
13
(
7
):
e0199715
.
[PubMed]
1932-6203
20.
Selves
J
,
Long-Mira
E
,
Mathieu
MC
,
Rochaix
P
,
Ilié
M
.
Immunohistochemistry for diagnosis of metastatic carcinomas of unknown primary site
.
Cancers (Basel)
.
2018
Apr
;
10
(
4
):
108
.
[PubMed]
2072-6694
21.
Raaphorst
FM
,
Otte
AP
,
van Kemenade
FJ
,
Blokzijl
T
,
Fieret
E
,
Hamer
KM
, et al
Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation
.
J Immunol
.
2001
May
;
166
(
10
):
5925
34
.
[PubMed]
0022-1767
22.
Nautiyal
N
,
Bhardwaj
A
,
Acharya
S
,
Kishore
S
,
Kudesia
S
.
Diagnostic utility of epithelial membrane antigen (EMA) and calretinin (CAL) in effusion cytology
.
J Clin Diagn Res
.
2017
May
;
11
(
5
):
EC36
9
.
[PubMed]
2249-782X
23.
Woo
CG
,
Son
SM
,
Han
HS
,
Lee
KH
,
Choe
KH
,
An
JY
, et al
Diagnostic benefits of the combined use of liquid-based cytology, cell block, and carcinoembryonic antigen immunocytochemistry in malignant pleural effusion
.
J Thorac Dis
.
2018
Aug
;
10
(
8
):
4931
9
.
[PubMed]
2072-1439
24.
Arora
R
,
Agarwal
S
,
Mathur
SR
,
Verma
K
,
Iyer
VK
,
Aron
M
.
Utility of a limited panel of calretinin and Ber-EP4 immunocytochemistry on cytospin preparation of serous effusions: A cost-effective measure in resource-limited settings
.
Cytojournal
.
2011
;
8
(
1
):
14
.
[PubMed]
1742-6413
25.
Kim
JH
,
Kim
GE
,
Choi
YD
,
Lee
JS
,
Lee
JH
,
Nam
JH
, et al
Immunocytochemical panel for distinguishing between adenocarcinomas and reactive mesothelial cells in effusion cell blocks
.
Diagn Cytopathol
.
2009
Apr
;
37
(
4
):
258
61
.
[PubMed]
8755-1039
26.
Ko
EC
,
Jhala
NC
,
Shultz
JJ
,
Chhieng
DC
.
Use of a panel of markers in the differential diagnosis of adenocarcinoma and reactive mesothelial cells in fluid cytology
.
Am J Clin Pathol
.
2001
Nov
;
116
(
5
):
709
15
.
[PubMed]
0002-9173
27.
Wang
B
,
Li
D
,
Ou
X
,
Yi
Q
,
Feng
Y
.
Diagnostic accuracy of Ber-EP4 for metastatic adenocarcinoma in serous effusions: a meta-analysis
.
PLoS One
.
2014
Sep
;
9
(
9
):
e107741
.
[PubMed]
1932-6203
28.
Ordóñez
NG
.
Value of claudin-4 immunostaining in the diagnosis of mesothelioma
.
Am J Clin Pathol
.
2013
May
;
139
(
5
):
611
9
.
[PubMed]
0002-9173
29.
Saleh
HA
,
El-Fakharany
M
,
Makki
H
,
Kadhim
A
,
Masood
S
.
Differentiating reactive mesothelial cells from metastatic adenocarcinoma in serous effusions: the utility of immunocytochemical panel in the differential diagnosis
.
Diagn Cytopathol
.
2009
May
;
37
(
5
):
324
32
.
[PubMed]
8755-1039
30.
Lauritzen
AF
.
Diagnostic value of monoclonal antibody B72.3 in detecting adenocarcinoma cells in serous effusions
.
APMIS
.
1989
Aug
;
97
(
8
):
761
6
.
[PubMed]
1600-0463
31.
González-Lois
C
,
Ballestín
C
,
Sotelo
MT
,
López-Ríos
F
,
García-Prats
MD
,
Villena
V
.
Combined use of novel epithelial (MOC-31) and mesothelial (HBME-1) immunohistochemical markers for optimal first line diagnostic distinction between mesothelioma and metastatic carcinoma in pleura
.
Histopathology
.
2001
Jun
;
38
(
6
):
528
34
.
[PubMed]
0309-0167
32.
Hall
PA
,
D’Ardenne
AJ
.
Value of CD15 immunostaining in diagnosing Hodgkin’s disease: a review of published literature
.
J Clin Pathol
.
1987
Nov
;
40
(
11
):
1298
304
.
[PubMed]
0021-9746
33.
Jo
VY
,
Cibas
ES
,
Pinkus
GS
.
Claudin-4 immunohistochemistry is highly effective in distinguishing adenocarcinoma from malignant mesothelioma in effusion cytology
.
Cancer Cytopathol
.
2014
Apr
;
122
(
4
):
299
306
.
[PubMed]
1934-662X
34.
Facchetti
F
,
Lonardi
S
,
Gentili
F
,
Bercich
L
,
Falchetti
M
,
Tardanico
R
, et al
Claudin 4 identifies a wide spectrum of epithelial neoplasms and represents a very useful marker for carcinoma versus mesothelioma diagnosis in pleural and peritoneal biopsies and effusions
.
Virchows Arch
.
2007
Sep
;
451
(
3
):
669
80
.
[PubMed]
0945-6317
35.
Oda
T
,
Ogata
S
,
Kawaguchi
S
,
Minabe
S
,
Dokyu
M
,
Takahashi
H
, et al
Immunocytochemical utility of claudin-4 versus those of Ber-EP4 and MOC-31 in effusion cytology
.
Diagn Cytopathol
.
2016
Jun
;
44
(
6
):
499
504
.
[PubMed]
8755-1039
36.
Dinu
M
,
Ciurea
RN
,
Stefan
M
,
Georgescu
AC
.
The role of immunohistochemistry in the diagnosis of neoplastic pleural effusions
.
Rom J Morphol Embryol
.
2012
;
53
(
3
Suppl
):
817
20
.
[PubMed]
1220-0522
37.
Rahmani
A
,
Dehghani
MZ
,
Afshar
NM
,
Heidarian
H
,
Tahririan
R
.
HBME-1 immunostaining in reactive mesothelial versus metastatic adenocarcinoma cells in serous fluid
.
Indian J Pathol Microbiol
.
2011
Jul-Sep
;
54
(
3
):
460
3
.
[PubMed]
0377-4929
38.
Lozano
MD
,
Panizo
A
,
Toledo
GR
,
Sola
JJ
,
Pardo-Mindán
J
.
Immunocytochemistry in the differential diagnosis of serous effusions: a comparative evaluation of eight monoclonal antibodies in Papanicolaou stained smears
.
Cancer
.
2001
Feb
;
93
(
1
):
68
72
.
[PubMed]
0008-543X
39.
Zhao
L
,
Guo
M
,
Sneige
N
,
Gong
Y
.
Value of PAX8 and WT1 immunostaining in confirming the ovarian origin of metastatic carcinoma in serous effusion specimens
.
Am J Clin Pathol
.
2012
Feb
;
137
(
2
):
304
9
.
[PubMed]
0002-9173
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