International Journal of Gynecological Pathology

32:3–14, Lippincott Williams & Wilkins, Baltimore

r 2012 International Society of Gynecological Pathologists

Original Article

Clear Cell Carcinomas of the Ovary: A Multi-Institutional

Study of 129 Cases in Korea With Prognostic Significance
of Emi1 and Galectin-3

Kyueng-Whan Min, M.D., Moon Hyang Park, M.D., Ph.D., Sung Ran Hong, M.D., Ph.D.,
Heejung Lee, M.D., Ph.D., Sun Young Kwon, M.D., Ph.D., Sook Hee Hong, M.D., Ph.D.,
Hee Jae Joo, M.D., Ph.D., In Ae Park, M.D., Ph.D., Hee Jung An, M.D., Ph.D., Kwang Sun Suh, M.D., Ph.D.,
Hoon Kyu Oh, M.D., Ph.D., Chong Woo Yoo, M.D., Ph.D., Mi Jin Kim, M.D., Ph.D.,
Hee Kyung Chang, M.D., Ph.D., Sun Young Jun, M.D., Ph.D., Hye Kyoung Yoon, M.D., Ph.D.,
Eun Deok Chang, M.D., Ph.D., Dong Won Kim, M.D., Ph.D., and Insun Kim, M.D., Ph.D.,
The Gynecologic Pathology Study Group of the Korean Society of Pathologists

Summary: Accurate diagnosis of ovarian clear cell carcinoma (CCC) is important because
of its poor prognosis with chemoresistance and a high recurrent rate. The clinicopathologic
characteristics and prognostic significance of the cell cycle regulator [early mitotic inhibitor-1
(Emi1)] and galactoside-binding protein (Galectin-3) were evaluated. Among 155 CCCs
from 18 hospitals in Korea between 1995 and 2006, 129 pure CCCs were selected with
consensus using immunohistochemical stains for hepatocyte nuclear factor-1b, Wilms’
tumor protein, and estrogen receptor. The expressions of Emi1, Galectin-3, p53, and Ki-67
labeling index were analyzed with clinicopathologic parameters and the patient’s survival.
The mean age of the patients was 49.6 yr; the tumors were bilateral in 10.9%, and the
average size was 12 cm. Adenofibromatous component was found in 7%, and endometriosis
in 48.1% of the cases. Psammoma body was seen in 16.3%. Disease-free survival and overall
survival rates were 78.3% and 79.1%, respectively. The International Federation of
Obstetrics and Gynecology (FIGO) stage was the most important prognostic indicator.
Emi1 expression (45%) was seen in 23.3% of CCCs, and associated with high FIGO grades
and poor overall survival (Po0.05). High Galectin-3 (Z80%) expression was seen in 59.7%
of CCCs, and associated with FIGO stages III and IV, and high Ki-67 labeling index. High
Ki-67 labeling index (Z50%) and p53 expression (Z50%) were seen in 27.1% and 18.6% of
CCCs, respectively, but there was no clinicopathologic and prognostic significance. On the

From the Department of Pathology (K-W.M., M.H.P.), Hanyang University, Seoul; Department of Pathology (S.R.H.), Kwandong
University, Seoul; Department of Pathology (H.L.), Bucheon St. Mary’s Hospital, Bucheon; Department of Pathology (S.Y.K.), Keimyung
University, Daegu; Department of Pathology (S.H.H.), Dong-A University, Busan; Department of Pathology (H.J.J.), Ajou University,
Suwon; Department of Pathology (I.A.P.), Seoul National University, Seoul; Department of Pathology (H.J.A.), CHA University, Bundang;
Department of Pathology (K.S.S.), Chungnam National University, Daejeon; Department of Pathology (H.K.O.), Catholic University of
Daegu, Daegu; Department of Pathology (C.W.Y.), National Cancer Center, Gyeonggi-do; Department of Pathology (M.J.K.), Yeungnam
University, Daegu; Department of Pathology (H.K.C.), Kosin University, Busan; Department of Pathology (S.Y.J.), Hallym University,
Gyeonggi-do; Department of Pathology (H.K.Y.), Inje University, Seoul; Department of Pathology (E.D.C.), Uijeongbu St. Mary’s Hospital,
Uijeongbu; Department of Pathology (D.W.K.), Soonchunhyang University, Seoul; and Department of Pathology (I.K.), Korea University,
Seoul, Korea.
K-W.M. and C.W.Y. have contributed equally.
The authors declare no conflict of interest.
Address correspondence and reprint requests to Insun Kim, MD, PhD, Department of Pathology, Korea University, Medical College,
126-1, 5Ka, Anam-Dong, Seongbuk-Gu, Seoul 136-705, Korea. E-mail: iskim@korea.ac.kr

3 DOI: 10.1097/PGP.0b013e31825554e9
4 K-W. MIN ET AL.
basis of the fact that the expression of Emi1 in CCC was correlated with a high histologic
grade and worse overall survival, target therapy using inhibitors of Emi1 may be tried in the
management of CCC patients with Emi1 expression. Key Words: Clear cell carcinoma—
Emi1 protein—Galectin-3—p53 antigen—Ki-67 antigen—Ovarian cancer.
Clear cell carcinoma (CCC) of the ovary comprises
about 4% to 12% of surface epithelial ovarian
cancers in western countries. CCC is known to be a
highly aggressive and chemotherapy-resistant neo-
plasm associated with poor prognoses compared with
the high-grade serous carcinoma (HGSCA) in the
high stages (III/IV), although the overall survival of
CCC patients in low stage (I/II) is higher than in
HGSCA in the same stage (1–6). A recent study
showed that the doubling time for CCC cells was
significantly longer than that for serous carcinoma
(SCA) cells and the expression of Galectin-3 might
contribute to lower cell proliferation and lead to
cisplatin chemoresistance (3,7).
Galectin-3 is a galactoside-binding protein that
shows widespread distribution in human tissues
including epithelial, stromal, and inflammatory cells.
It could potentially influence many areas of tumor
development and progression including cellular
proliferation, intercellular adhesion, cell-matrix in-
teractions, and angiogenesis (8).
Early mitotic inhibitor-1 (Emi1) is an anaphase-
promoting complex/cyclosome (APC/C) inhibitor,
which is induced by E2F transcription factors in late
G1 after activation of the growth factor-responsive
D-type cyclins (cyclin D)/retinoblastoma protein
(pRb) pathway. Emi1 overexpression induces genetic
instability and mitotic catastrophe through APC/C
misregulation, and thus contributes to carcinogene-
sis (9,10). Emi1 overexpression has been found in a
variety of malignant tumors, specifically implicated in
CCC (9,11)
In contrast to HGSCA, TP53 is typically wild type,
whereas loss-of-function mutation of ARID1A and
activating mutation of PIK3CA are known to be
genetically linked with CCC (12,13)
CCC is histologically characterized by tubulocys-
tic, papillary, and solid growth patterns of clear or
hobnail cells, and is commonly accompanied with
adenofibromatous (AF) component. However, be-
cause the histologic features of ovarian CCC are
diverse and sometimes mixed with SCA or endome-
trioid carcinoma (ECA), the collection of pure CCC
cases with confidence is difficult (14–16).
In this study, ovarian CCCs diagnosed in 18
hospitals in Korea between 1996 and 2006 were
Int J Gynecol Pathol Vol. 32, No. 1, January 2013
collected and the histologic diagnosis, grade, pattern,
and cell type were reevaluated by consensus among the
pathologist members of the Gynecological Pathology
Study Group of the Korean Society of Pathologists.
The diagnosis was enforced by immunohistochemical
(IHC) stains with WT-1, estrogen receptor (ER), and
hepatocyte nuclear factor-1b (HNF-1b) to exclude
other histologic types, especially HGSCA and ECA.
The information for clinical stage and follow-up was
obtained from the medical records of the patients. The
IHC expressions of Emi1, Galectin-3, p53, and Ki-67
were evaluated with various clinicopathologic para-
meters in pure CCC cases.
MATERIALS AND METHODS
Selection of the Cases
A total of 160 cases, which were diagnosed at 18
hospitals in Korea between 1995 and 2006, were
collected. For a central review, hematoxylin and
eosin-stained slides were sent to the Gynecological
Pathology Study Group of the Korean Society of
Pathologists, and reviewed by at least 6 pathologists.
The diagnosis, predominant histologic patterns and
cell types, histologic and nuclear grades, and the
presence of psammoma body and necrosis were
decided by consensus. The cell type was classified as
clear, hobnail, cuboidal, oxyphilic, or signet-ring cell
type, and the growth pattern was grouped into cystic,
solid, and mixed cystic and solid pattern (Figs. 1–3).
In addition, the association with AF component was
investigated. International Federation of Obstetrics
and Gynecology (FIGO) grading and the Silverberg
universal grading system were adopted for histologic
grading (17). Other clinicopathologic information
such as age, laterality, tumor size, the presence of
endometriosis, treatment modality, and follow-up
were obtained from the pathologic and hospital
records. Five cases were excluded because of no
available paraffin block and inadequate clinical
history.
Construction of Tissue Microarray (TMA)
The hematoxylin and eosin-stained slide was re-
viewed and the 2 appropriate areas were marked by 1
 EMI1 EXPRESSION IN OVARIAN CLEAR CELL CARCINOMA
FIG. 1. Representative histologic features of glandular (A), papillary (B), and solid (C) patterns of ovarian clear cell carcinoma. Psammoma
body is seen (D).
pathologist (K.I.). The corresponding formalin-fixed,
paraffin-embedded tissue block was retrieved, and the
selected areas were marked. The punched tissue core,
using a 3-mm punch instrument (UNITMA, Korea),
was inserted into the recipient block (UNITMA). For
each case, TMA included duplicate cores of tumor
tissue. The TMA block was left on a 601C oven for
30 min and embedded in paraffin block.
IHC Stain
Serial sections of 4 mm thickness were cut from the
array block and deparaffinized by the routine
technique. For IHC stain, the sections were deparaffi-
nized by heating at 551C for 30 min and by washing
with xylene 3 times for 5 min each. Sections were
rehydrated by a series of 5-min washes in 100%,
90%, and 70% ethanol. Antigen was retrieved by
microwaving the sections for 4 min 20 sec in 250 mL
of 10 mM sodium citrate (pH 6.0). Endogenous
5
peroxidase activity was blocked with 0.3% hydrogen
peroxidase for 20 min. The immunostain for WT-1,
ER, Galectin-3, p53, and Ki-67 was performed
with the Dako Autostainer plus Universal Staining
System (DakoCytomation, Carpinteria, CA) using
the ChemMate DAKO EnVision Detection Kit. The
primary antibodies for WT-1 (1:50), ER (1:100), Ki-
67 (1:400), and p53 (1:4000) were obtained from
Dako Corp. For Galectin-3, the affinity-purified
mouse anti-human Galectin-3 monoclonal antibody
(1:100, Clone 9C4, Novocastra, UK) was used. The
primary polyclonal antibody for HNF-1b was obtained
from ProteinTech Group Inc. (1:200, Chicago, IL), and
the affinity-purified rabbit anti-human Emi1 polyclonal
antibody (1:100, ab18341, Abcam, Cambridge, UK)
was used for Emi1. The detection for HNF-1b and
Emil was performed with the Ultra Tech HRP
Strepavidin-Biotin Detection System, using an auto-
matic stainer (Bond-Max, Vision Biosystem Leica
Biosystems, Melborne, Australia).
Int J Gynecol Pathol Vol. 32, No. 1, January 2013
6 K-W. MIN ET AL.

FIG. 2. Representative cell types of hobnail (A), cuboidal (B), oxyphilic (C), and signet-ring cell types (D) of ovarian clear cell carcinoma.

Interpretation of IHC Stain previous study, a tumor that showed no immuno-

The stain for Emi1 showed immunoreactivity in the reactivity or focal stain in <5% of the entire tumor
cytoplasm and/or nucleus of the neoplastic cells, but area in the TMA section of each case was regarded as
only cytoplasmic expression was evaluated. As in the negative for Emi1 (Fig. 4). Two pathologists (M.K.H.

FIG. 3. The tumor associated with endometriosis (A) and the tumor with adenofibroma component (B).

Int J Gynecol Pathol Vol. 32, No. 1, January 2013

 EMI1 EXPRESSION IN OVARIAN CLEAR CELL CARCINOMA 7

types were analyzed by the w2 test. For the univariate

analysis, categorical variables (clinical parameters
such as age, FIGO stage, bilaterality, tumor size,
growth pattern, necrosis, lymph node metastasis, and
endometriosis, and pathologic parameters such as cell
type, FIGO grade, universal grade, and the presence
of psammoma body) were compared using the w2 test,
and continuous variables such as Ki-67 labeling index
(LI) were analyzed using Student t test.
Disease-free survival and overall survival curves
were generated using the Kaplan-Meier method and
compared by the Breslow test. Multivariate analysis
was performed to identify independent prognostic
markers for overall survival using a Cox multistep
regression model. A 2-tailed P value of <0.05 was
FIG. 4. The expression of Emi1 in ovarian clear cell carcinoma.
considered statistically significant. All data were
and P.M.H.) evaluated the stain separately. In the
cases with a discrepancy, a decision was made by TABLE 1. Clinicopathologic characteristics of 129 patients
with clear cell carcinoma of the ovary
consensus (11). For determination of the optimal cut-
off values of Galectin-3, receiver-operating character- Clinicopathologic parameters Case no. Percentage
istic curves, plotting sensitivity versus 1 – specificity, Age (range: 28–77 yr)
45 and younger 48 37
were used. In reference with the receiver-operating Older than 45 81 63
characteristic curve, the cytoplasmic expression of Size (range: 3–30 cm)
Galectin-3 in neoplastic cells was divided into high- r12 80 63
412 49 38
expression (Z80%) and low-expression (o80%) Laterality
groups (Fig. 5). For p53 and Ki-67, the nuclear Unilateral 115 89
staining in tumor cells was scored from 0 to 10, and Bilateral 14 11
Growth patterns
classified into high-expression (Z50%) and low- Cystic 66 51.2
expression (o50%) groups (12). Solid 20 15.5
Mixed (cystic and solid) 43 33.3
Cell types
Clear 70 54.3
Statistical Analysis Hobnail 39 30.2
Cuboidal 7 5.4
The distribution of patients in each tumor stage Oxyphilic 8 6.2
and histologic findings of growth pattern and cell Signet-ring 5 3.9
FIGO grade
1 32 24.8
2 48 37.2
3 49 38
Universal grade
1 61 47.3
2 59 45.7
3 9 7
FIGO stage
I 82 63.6
II 14 10.9
III 31 24
IV 2 1.6
Therapy
Only radical surgery 10 7.8
Adjuvant chemotherapy 119 92.2
Lymph node metastasis 9 7
Adenofibroma 9 7
Endometriosis 62 48.1
Psammoma body 21 16.3
FIG. 5. The expression of Galectin-3 in ovarian clear cell FIGO indicates International Federation of Obstetrics and
carcinoma. Gynecology.

Int J Gynecol Pathol Vol. 32, No. 1, January 2013

8 K-W. MIN ET AL.

analyzed using SPSS software for Windows 13.0
(SPSS Inc, Chicago, IL).
RESULTS
Clinicopathologic Characteristics of CCCs
Among 155 cases, 144 cases were classified as pure
CCC and 14 cases were mixed CCC or other
histologic types, on the basis of the histologic and
IHC findings of WT-1, ER, and HNF-1b. After the
exclusion of 14 mixed tumors or other histologic
types, and 12 pure CCCs, of which IHC results were
missing, 129 pure CCCs were analyzed in this study.
The clinicopathologic findings for 129 patients with
ovarian CCC are summarized in Table 1.
The mean age of the patients was 49.6 yr (range;
28–77 yr), and the tumor was found in 81 patients
older than 45 yr of age (62.8%). The tumors were
bilateral in 14 (10.6%) patients. The average size was
12 cm (range; 3–30 cm), and it was >12 cm in
diameter in 49 (38%) patients. The tumor was grossly
cystic in 66 (51.2%), solid in 20 (15.5%), and solid
and cystic in 43 (33.3%) patients. Histologically, AF
component was found in 9 (7%), and endometriosis
in 62 (48.1%) patients. Two cases had both AF
component and endometriosis. Psammoma body was
observed in 10 of 49 glandular patterns, 5 of 48
papillary patterns, and 6 of 32 solid patterns.
The clear, hobnail, cuboidal, oxyphilic, and signet-
ring cell types were found in all tumors in various
proportions. However, the predominant tumor cell
types were clear cell (54.3%) and hobnail cell
(30.2%). Oxyphilic, signet-ring, and cuboidal cells
were minor cell types. The tumors were FIGO grades
2 and 3 in 75.2%, whereas 52.7% of the tumors were
classified as grades 2 and 3 in the universal grading
system.
The distribution of the FIGO stage was as follows:
82 patients at stage I, 14 at stage II, 31 at stage III,
and 2 at stage IV. In addition, the lymph node
metastasis revealed 9 of 23 patients with FIGO IIIc.
Except 9 of 82 patients in stage I, and one of 2
patients in stage IV, 119 patients were treated with
surgery and adjuvant chemotherapy. The chemo-
therapeutic regimen consisted of paclitaxel and
platinum in 80 patients, platinum-based regimens in
23, a combination of topoisomerase I inhibitor and
platinum in 8, paclitaxel only in 3, platinum only in 3,
a combination of paclitaxel and cyclophosphamide in
1, and melphalan only in 1 patient.
The clear cell type was more frequently observed in
FIGO grades 2 and 3 compared with other cell types
(Po0.001). Although the solid gross pattern seemed
to be associated with a high universal grade, there
was no statistical significance (Table 2). The histo-
logic pattern was glandular in 49 (38%), papillary in
48 (37.2%), and solid in 32 (24.8%) patients. The
solid histologic pattern and the high nuclear grade
were significantly associated with FIGO grades 2 and
3 (Po0.001 and 0.001, respectively), but not with the
mitotic count (Table 3).
Except 3 patients who were lost during follow-up
(mean; 69.9 mo, range; 3–196 mo), 25 patients had
recurrence (disease-free survival rate 78.3%) and 23
patients died (overall survival rate 79.8%). The
overall survival rates of the patients in FIGO stages
I (91.5%) and II (92.9%) were better than those of
the patients in stages III (45.2%) and IV (0%)
(Po0.001). CCC patients with endometriosis were
more commonly younger than 45 yr of age

TABLE 2. Cell type and gross pattern of ovarian clear cell carcinoma according to the FIGO grade and the universal grade
Cell type Gross pattern
Clear (%) Hobnail (%) Cuboid (%) Oxyphilic (%) Signet (%) Cystic (%) Solid (%) Mixed (%)
FIGO grade*
Grade 1 10 (31.3 14 (43.8 6 (18.8) 2 (6.3) 0 (0) 19 (59.4) 3 (9.4) 10 (31.3)
Grade 2 22 (45.8) 20 (41.7) 1 (2.1) 3 (6.3) 2 (4.2) 21 (43.8) 9 (18.8) 18 (37.5)
Grade 3 38 (77.6) 5 (10.2) 0 (0) 3 (6.1) 3 (6.1) 26 (53.1) 8 (16.3) 15 (30.6)
Universal gradew
Grade 1 28 (45.9) 21 (34.4) 7 (11.5) 3 (4.9) 2 (3.3) 33 (54.1) 8 (13.1) 20 (32.8)
Grade 2 35 (59.3) 17 (28.8) 0 (0) 4 (6.8) 3 (5.1) 28 (47.5) 10 (16.9) 21 (35.6)
Grade 3 7 (77.8) 1 (11.1) 0 (0) 1 (11.1) 0 (0) 5 (55.6) 2 (22.2) 2 (22.2)
Total no. 70 (54.3) 39 (30.2) 7 (5.4) 8 (6.2) 5 (3.9) 66 (51.2) 20 (15.5) 43 (33.3)
Po0.05 are in bold.
*Clear type versus other types (hobnail, cuboid, oxyphilic, and signet), Po0.001 (w2 test); Solid pattern versus other patterns (cyst and
mixed), P = 0.515 (w2 test).
wClear type versus other types (hobnail, cuboid, oxyphilic, and signet), P = 0.115 (w2 test); Solid pattern versus other pattern (cyst and
mixed), P = 0.715 (w2 test).
FIGO indicates International Federation of Obstetrics and Gynecology.

Int J Gynecol Pathol Vol. 32, No. 1, January 2013

 EMI1 EXPRESSION IN OVARIAN CLEAR CELL CARCINOMA 9

TABLE 3. Correlation between the FIGO grade and the universal grading system in terms of histologic pattern, nuclear grade,
and mitotic count
Histologic pattern* Nuclear gradew Mitotic count (/10 HPF)z
Glandular (%) Papillary (%) Solid (%) Mild (%) Moderate (%) Marked (%) r9 (%) 10–24 (%) Z25 (%)
FIGO grade
Grade 1 11 (34.4) 21 (65.6) 0 (0) 0 (0) 32 (100) 0 (0) 24 (75) 6 (18.8) 2 (6.3)
Grade 2 28 (58.3) 19 (39.6) 1 (2.1) 1 (2.1) 17 (35.4) 30 (62.5) 33 (68.8) 11 (22.9) 4 (8.3)
Grade 3 10 (20.4) 8 (16.3) 31 (63.3) 0 (0) 5 (10.2) 44 (89.8) 36 (73.5) 12 (24.5) 1 (2)
Total no. 49 (38) 48 (37.2) 32 (24.8) 1 (0.8) 54 (41.9) 74 (57.4) 93 (72.1) 29 (22.5) 7 (5.4)
Po0.05 are in bold.
*Solid pattern versus other patterns (glandular and papillary), Po0.001 (w2 test).
wMarked grade versus other grades (mild and moderate), Po0.001 (w2 test).
zr9/10 versus >9/10 HPF, P = 0.8 (w2 test).
FIGO indicates International Federation of Obstetrics and Gynecology; HPF, high power field (  400).

(P = 0.004) and showed a tendency for longer overall
and disease-free survival, compared with the patients
without endometriosis, but without statistical signifi-
cance in the multivariate analysis (P = 0.381 and
0.208, respectively). The patients with AF component
in CCC showed a tendency for better overall survival
and disease-free survival than the patients without
AF component, but without statistical significance
(P = 0.15 and 0.136, respectively).
Correlation of Emi1, Galectin-3, p53, and Ki-67
Expressions With Clinicopathologic Parameters
The expression of Emi1 was found in 30 (23.3%) of
129 CCCs, and was slightly frequent in tumors in the
older age group (445 yr old) and in tumors with a
solid gross pattern, but its expression was not
correlated with any parameters including FIGO
stage, bilaterality, tumor size, the presence of
necrosis, lymph node metastasis, cell type, histologic

TABLE 4. Relationships between Emi1 expression and clinical data in ovarian clear cell carcinoma
Expression of Emi1
Clinical parameters Case no. Negative (n = 99) (%) Positive (n = 30) (%) P (w2 test)
Age (yr)
45 or younger 48 41 (85.4) 7 (14.6) 0.073
Older than 45 81 58 (71.6) 23 (28.4)
FIGO stage
I and II 96 73 (76) 23 (24) 0.747
III and IV 33 26 (78.8) 7 (21.2)
Laterality
Unilateral 115 87 (75.7) 28 (24.3) 0.519*
Bilateral 14 12 (85.7) 2 (14.3)
Size
r12 cm 80 62 (77.5) 18 (22.5) 0.795
412 cm 49 37 (75.5) 12 (24.5)
Gross pattern
Cystic or mixed 109 85 (78) 24 (22) 0.565*
Solid 20 14 (70) 6 (30)
Necrosis
Absence 58 46 (79.3) 12 (20.7) 0.533
Presence 71 53 (74.6) 18 (25.4)
Lymph node metastasis
None 120 92 (76.7) 28 (23.3) 0.999*
Presence 9 7 (77.8) 2 (22.2)
Endometriosis
Absence 67 49 (73.1) 18 (26.9) 0.313
Presence 62 50 (80.6) 12 (19.4)
Adenofibroma component
Absence 120 92 (76.7) 28 (23.3) 0.999*
Presence 9 7 (77.8) 2 (22.2)
*Fisher exact test.
FIGO indicates International Federation of Obstetrics and Gynecology.

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10 K-W. MIN ET AL.

TABLE 5. Relationships between Emi1 expression and histologic data in ovarian clear cell carcinoma
Expression of Emi1
Histologic parameters Case no. Negative (n = 99) Positive (n = 30) P (w2 test)
Cell type
Clear, cuboidal, or oxyphilic 85 69 (81.2%) 16 (18.8%) 0.098
Hobnail or signet 44 30 (68.2%) 14 (31.8%)
FIGO grade
1 32 29 (90.6%) 3 (9.4%) 0.032
2 and 3 97 70 (72.2%) 27 (27.8%)
Universal grade
1 61 51 (83.6%) 10 (16.4%) 0.081
2 and 3 68 48 (70.6%) 20 (29.4%)
Architecture pattern
Glandular and papillary 97 77 (79.4%) 20 (20.6%) 0.217
Solid 32 22 (68.8%) 10 (31.3%)
Nuclear grade
Mild and moderate 55 47 (85.5%) 8 (14.5%) 0.044
Marked 74 52 (70.3%) 22 (29.7%)
Mitotic count
r9/10 HPF 93 70 (75.3%) 23 (24.7%) 0.524
49/10 HPF 36 29 (80.6%) 7 (19.4%)
Psammoma body
Absence 108 87 (80.6%) 21 (19.4%) 0.044*
Presence 21 12 (57.1%) 9 (42.9%)
Galectin-3 expression (%) 71.1 71 0.988w
p53 expression (%) 17.8 33.7 0.015w
Ki-67 labeling index (%) 30.9 29.3 0.762w
Po0.05 are in bold.
*Fisher exact test.
wStudent t test.
FIGO indicates International Federation of Obstetrics and Gynecology; HPF, high power field (  400).

pattern, mitotic count, and universal grade. However,
Emi1 expression was more frequently observed in
FIGO grades 2 and 3 than grade 1 (P = 0.032) and at
a high nuclear grade than at mild and moderate
nuclear grades (P = 0.044). The presence of psammoma
body was associated with Emi1 expression (P = 0.044).
Emi1 expression was associated with p53 expression,
but not with Galectin-3 expression and Ki-67 LI
(Tables 4 and 5).
High Galectin-3 expression (Z80%) was seen in
77 (59.7%) of 129 CCCs, and was associated with
FIGO stages III and IV than with stages I and II
(P = 0.029), but not with other clinicopathologic
parameters. The expression of p53 was seen in 82
(63.6%) of 129 CCCs. High expression of p53
(Z50%) was seen in 24 cases (18.6%), including
51% to 70% in 13 and >70% in 11 cases. The
expression of p53 did not show any significant
association with the clinicopathologic parameters.
High Ki-67 LI (Z50%) was seen in 35 (27.1%)
CCCs, and was frequently found in high mitotic
counts (49/10 HPF), but without statistical signifi-
cance (P = 0.062).
High expressions of Galectin-3 and p53 were
associated with high Ki-67 LI (P = 0.025 and 0.001,
respectively). There were no differences in Galectin-3,
p53, and Ki-67 expression between CCCs with and
without endometriosis. CCCs with AF component
showed lower Ki-67 LI than those without AF
component, although there was no statistical signifi-
cance (P = 0.112).
Effect of Histologic Findings and Emi1, Galectin-3,
p53, and Ki-67 Expression on the Patient’s Overall
and Disease-free Survivals
A solid pattern and a high nuclear grade showed a
tendency to correlate with poor overall (P = 0.611 and
0.765, respectively) and disease-free survivals (P = 0.824
and 0.51, respectively), whereas a clear cell type tended
to have good overall and disease-free survivals (all
P = 0.562), but without statistical significance. The
FIGO stage and lymph node metastasis were associated
with poor overall and disease-free survivals in the
univariate analysis, but only the FIGO stage was
correlated with survival in the multivariate analysis.
The patients with Emi1 expression in tumor cells
were associated with a poor overall survival com-
pared with the patients with low Emi1 expression
(P = 0.027), whereas it showed a tendency to
correlate with disease-free survival in univariate
analysis (P = 0.063) (Fig. 6). After adjustment of
confounders such as lymph node metastasis, the

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 EMI1 EXPRESSION IN OVARIAN CLEAR CELL CARCINOMA 11

FIG. 6. Overall survival and disease-free survival for the patients with ovarian clear cell carcinoma according to Emi1-positive groups versus
Emi1-negative groups (overall survival and disease-free survival: P = 0.027 and 0.063, respectively).

FIGO and universal grades, and the FIGO stage,
Emi1 expression was an independent prognostic
factor for overall survival (P = 0.027) (Table 6).
However, Galectin-3, p53, and Ki-67 expressions
were not correlated with overall and disease-free
survival in both univariate and multivariate analyses.
DISCUSSION
CCC of the ovary is a relatively rare neoplasm
accounting about 5% in North America and Europe
(5.8% in Korea, unpublished data), although its
prevalence is increasing and accounting 15% to 25%
in Japan. Ovarian CCC usually presents as a pelvic
mass at low clinical stage, and its low-stage outcome
is better than stage-matched HGSCA. However, it
has been well known that some CCCs are associated
with an aggressive clinical course with a worse
prognosis and poor response to standard carboplatin
paclitaxel chemotherapy. The histologic cell type, the
universal histologic grade, the clinical stage, and
residual tumor are considered to be prognostic
factors influencing the patient’s survival (18–20).
However, the clinicopathologic characteristics of
CCCs are not well defined so far. In this study, we
collected cases from 18 hospitals in Korea and
clarified the histologic diagnosis with consensus and
IHC stains to define the prognostic markers. Because

TABLE 6. Survival analyses of Emi1 (negative: r5% versus positive: >5%) and clinicopathologic parameters in ovarian clear
cell carcinoma
Variables Significance univariate* Significance multivariatew Hazard ratio 95% CI
Overall survival
Emi1 expression (r5%. vs.45%) 0.027 0.027 2.788 1.126–6.905
Lymph node metastasis (negative vs. positive) 0.001 0.547 1.398 0.47–4.158
FIGO grade (grade 1 and 2 vs. grade 3) 0.8 0.615 1.268 0.503–3.201
Universal grade (grade 1 vs. grade 2 and 3) 0.753 0.912 1.055 0.413–2.694
FIGO stage (I and II vs. III and IV) 0.001 0.001 12.815 4.792–34.271
Disease-free survival
Emi1 expression (r5%. vs.45%) 0.063 0.055 2.36 0.983–5.665
Lymph node metastasis (negative vs. positive) 0.001 0.684 1.249 0.429–3.634
FIGO grade (grade 1 and 2 vs. grade 3) 0.855 0.528 1.331 0.548–3.228
Universal grade (grade 1 vs. grade 2 and 3) 0.753 0.912 1.052 0.43–2.573
FIGO stage (I and II vs. III and IV) 0.001 0.001 11.838 4.68–29.944
*Breslow test.
wCox proportional hazard model.
CI indicates confidence interval; FIGO, International Federation of Obstetrics and Gynecology.

Int J Gynecol Pathol Vol. 32, No. 1, January 2013

12 K-W. MIN ET AL.
of the histologic diversity, CCCs sometimes pose
diagnostic difficulty. A recent review study showed
that 23% of low-stage ovarian cancers were not
diagnosed correctly (21). In this study, 14 of 155 cases
(9.0%) were not CCC or were mixed with other
epithelial surface epithelial tumors such as SCA or
ECA. IHC stains are used in the differential diagnosis
from ECA and HGSCA. CCC is positive for HNF-
1b, but negative for ER and WT-1, whereas HGSCA
shows negative stain for HNF-1b, but positive stains
for ER and WT-1 (15). ECA is positive for ER, but
negative for HNF-1b and WT-1. In this study, cases
that were negative for HNF-1b and positive for WT-1
or ER in >10% of tumor cells with histologic
ambiguity were excluded from pure CCC. Two cases
with a positive reaction for WT-1 and 1 case in which
>70% of the tumor cells were positive for ER and
associated with endometriosis were included in pure
CCCs. Six of 129 CCCs (4.7%) were negative for
HNF-1b. Mutant p53 has been found to be the most
common genetic defect in human malignancies (22),
and high expression of p53 has been known to be
an indicator of HGSCA among ovarian cancers.
However, because 18.6% of the cases showed p53
expression in Z50% of the tumor cells, and 11 of 129
cases (8.5%) showed p53-positive cells in >70% of
tumor cells in this study, we considered that p53
expression could not be used in the differential
diagnosis from HGSCA, but suggest that its alter-
ation may be involved in the malignant transforma-
tion of endometriosis-associated ECA or CCC
(23,24). Psammoma body is considered to be
characteristic of SCA, but it was also found in
16.3% of the present cases. Psammoma body was not
limited to the papillary pattern, but was also seen in
glandular and solid patterns.
On analysis of the clinicopathologic parameters
with the patient’s survival, the clear cell type, a solid
histologic pattern, and a high nuclear grade were
associated with a high FIGO grade, but there was no
correlation with the patient’s survival. The FIGO
stage was the only prognostic factor predicting the
patient’s overall and disease-free survivals.
Recent advances in understanding the molecular
pathogenesis of ovarian epithelial cancer showed
that HGSCA is characterized by p53 mutation and
BRCA1 and/or BRCA2 dysfunction, whereas low-
grade SCA and mucinous carcinoma are related with
KRAS and BRAF mutation. ECA is often associated
with KRAS and PTEN dysfunction. The molecular
changes of CCC remain largely unknown. It has been
described that ovarian CCCs are pathogenetically
Int J Gynecol Pathol Vol. 32, No. 1, January 2013
related with endometriosis or clear cell adenofibro-
ma (14,25). Genetic instability or damage caused by
iron-dependent oxidative stress in the ectopic endo-
metrium leads to downregulation or upregulation of
tumor suppressor genes or oncogenes. Upregulation
of HNF-1b, Emi1, and mTOR is considered to be a
critical factor in the development of atypical endo-
metriosis-CCC sequence, whereas APC and PTEN
gene alteration has been speculated as an early event
of clear cell adenofibroma-CCC sequence in the
ovarian carcinogenic pathway (25).
An interesting recent meta-analysis study (16)
reported that 54 genes were highly upregulated in
CCC, 47 of these being redox-related genes and 22
involved in the HNF-1b pathway. One substance,
Galectin-3, is an adhesion molecule that is involved in
various processes such as induction of oxidative
stress, proinflammatory cytokines and reactive oxy-
gen species (ROS) production, ischemic-reperfusion
injury, apoptosis, neutrophilic adhesion, and mast
cell migration and degranulation (8,26). Upregula-
tion of Galectin-3 is known to enhance proliferation
and inhibit apoptosis. It is related to aggressive
metastatic events by binding with MUC1 on the
cancer cell surface, thus promoting cancer cell
adhesion to the vascular endothelium (27,28). The
expression of Galectin-3 was upregulated in malig-
nancies of the thyroid, the stomach, and the
liver (29–31). One study described that Galectin-3
expression was observed in 88.7% epithelial ovarian
cancers, but not in normal ovarian tissues, and a high
expression was correlated with a shorter progression-
free survival of the patients (32) Another study (33)
showed that Galectin-3 expression was seen most
frequently in CCCs (12/13). The association of
Galectin-3 with CCC was suggested as the factor
responsible for chemoresistance in CCC patients (3,7).
The responders to chemotherapy among CCC
patients had a higher proliferation activity than the
nonresponders, and the S-phase fraction was in-
creased by knocking down Galectin-3 in cell lines.
Galectin-3 expression might exert suppressive influ-
ences on the cell proliferation in CCC cells, and thus
the expression of Galectin-3 might contribute to its
lower cell proliferation and resistance to chemo-
therapy (7). In this study, high expression of
Galectin-3 was seen in 59.7% of CCCs. However,
its expression did not show any relationship with the
patient’s survival, even though high expression was
associated with high Ki-67 LI and an advanced
FIGO stage. The role of Galectin-3 in CCC remains
to be determined.
 EMI1 EXPRESSION IN OVARIAN CLEAR CELL CARCINOMA
Another substance known to be increased in
endometriosis and CCC is the polo-like kinase, which
is activated by reactive oxygen species or hydrogen
peroxide; it promotes genome stability and phosphor-
ylates Emi1. Emi1 drives cell cycle progression from G1/
S through early mitosis by inhibiting APC/C ubiquitin
ligase activity, thus facilitating the accumulation of
APC/C substrates. Emi1 overexpression leads to pro-
liferation, tetraploidy, and genome instability of cells in
the absence of p53 (9). High expression of Emi1 was
reported in ovarian CCCs and correlated with cyclin E
expression, but not with Ki-67 LI (11,34). In this study,
Emi1 expression was observed in 23.3% of 129 ovarian
CCC cases. High Emi1 expression showed correlation
with a poor patient survival and high FIGO and nuclear
grades. However, its expression was not correlated with
the presence of endometriosis and Ki-67 LI.
In conclusion, these results obtained from a
relatively large-scale, multi-institutional, retrospec-
tive study provide a new point of view that Emi1
expression may be significantly correlated with a high
histologic grade, and can be utilized to predict the
survival in patients with ovarian CCC. Therefore, the
inhibitor that targets Emi1 can be considered as a
therapeutic approach in patients with CCC.
Acknowledgments: The authors are grateful to Dr
Silverberg SG for his assistance in the diagnostic review
of ovarian clear cell carcinoma. They also thank Park HH,
PhD, for technical assistance.
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