Claudin-4 Expression is Associated With Survival in Ovarian Cancer But Not With Chemotherapy Response

Abstract

The tight junction protein claudin-4 has been reported to be overexpressed in advanced ovarian cancer. We investigated the prognostic significance of claudin-4 overexpression and whether claudin-4 expression could predict platinum response in primary ovarian carcinoma (OC). Claudin-4 expression was evaluated by immunohistochemistry in a tissue microarray of 140 OCs. Multivariable Cox-regression models were used to assess the effect of claudin-4 overexpression on progression-free survival and overall survival (OS). Kaplan-Meier survival analyses and the logrank test were performed comparing claudin-4 high and low groups. The association between claudin-4 expression and platinum resistance was assessed using risk ratios and the Pearson χ test. A dataset of >1500 epithelial ovarian cancers was used to study the association between CLDN4 mRNA and survival. Of 140 evaluable cases, 71 (51%) displayed high claudin-4 expression. Claudin-4 overexpression predicted shorter 5-yr progression-free survival and OS in univariable analyses [hazard ratio (HR)=1.6 (1.1-2.5), P=0.020 and HR=1.6 (1.0-2.4), P=0.041, respectively]. Hazard of relapse was similar [HR=1.5 (1.0-2.4)] after adjustment for age, stage, type, and BRCA1/2 status in a multivariable analysis, but the evidence was slightly weaker (P=0.076). Validation in an external cohort confirmed the association between high expression of CLDN4 and poor 10-yr OS [HR=1.3 (1.1-1.5), P<0.001]. However, no confident association between claudin-4 and platinum sensitivity was found in our cohort [risk ratio=1.2 (0.7-2.0), P=0.3]. These findings suggest that high expression of claudin-4 may have a prognostic value in OC. The role of claudin-4 in the development of platinum resistance remains unclear.

Full text

International Journal of Gynecological Pathology
00:1–9, Lippincott Williams & Wilkins, Baltimore
Copyright r2017 by the International Society of Gynecological Pathologists
Original Article
Claudin-4 Expression is Associated With Survival in Ovarian
Cancer But Not With Chemotherapy Response
Laura Martı
´n de la Fuente, M.D., Susanne Malander, M.D., Ph.D., Linda Hartman, Ph.D.,
Jenny-Maria Jo
¨nsson, M.D., Ph.D., Anna Ebbesson, M.Sc., Mef Nilbert, M.D., Ph.D.,
Anna Ma
˚sba
¨ck, M.D., Ph.D., and Ingrid Hedenfalk, Ph.D.
Summary: The tight junction protein claudin-4 has been reported to be overexpressed in
advanced ovarian cancer. We investigated the prognostic significance of claudin-4 over-
expression and whether claudin-4 expression could predict platinum response in primary
ovarian carcinoma (OC). Claudin-4 expression was evaluated by immunohistochemistry in a
tissue microarray of 140 OCs. Multivariable Cox-regression models were used to assess the
effect of claudin-4 overexpression on progression-free survival and overall survival (OS).
Kaplan-Meier survival analyses and the logrank test were performed comparing claudin-4 high
and low groups. The association between claudin-4 expression and platinum resistance was
assessed using risk ratios and the Pearson w
2
test. A dataset of >1500 epithelial ovarian
cancers was used to study the association between CLDN4 mRNA and survival. Of 140
evaluable cases, 71 (51%) displayed high claudin-4 expression. Claudin-4 overexpression
predicted shorter 5-yr progression-free survival and OS in univariable analyses [hazard ratio
(HR) = 1.6 (1.1–2.5), P= 0.020 and HR = 1.6 (1.0–2.4), P= 0.041, respectively]. Hazard of
relapse was similar [HR = 1.5 (1.0–2.4)] after adjustment for age, stage, type, and BRCA1/2
status in a multivariable analysis, but the evidence was slightly weaker (P=0.076).Validation
in an external cohort confirmed the association between high expression of CLDN4 and poor
10-yr OS [HR = 1.3 (1.1–1.5), Po0.001]. However, no confident association between claudin-
4 and platinum sensitivity was found in our cohort [risk ratio = 1.2 (0.7–2.0), P=0.3]. These
findings suggest that high expression of claudin-4 may have a prognostic value in OC. The role
of claudin-4 in the development of platinum resistance remains unclear. Key Words: Claudin-
4—Ovarian carcinoma—Platinum resistance—Prognostic factor.
Ovarian cancer is one of the leading causes of
cancer death among women due to difficulties in both
diagnosis and therapy. Because of the insidious onset
of the disease and the lack of reliable screening
methods, two thirds of patients present with ad-
vanced stage disease upon diagnosis (1). The 5-yr
From the Department of Clinical Sciences, Division of Oncology and Pathology, Lund University and Ska
˚ne University Hospital
(L.M.d.l.F., S.M., L.H., J.-M.J., A.E., M.N., I.H.); Regional Cancer Center South Sweden (L.H., M.N.); Department of Surgical Pathology,
Division of Laboratory Medicine, Ska
˚ne University Hospital (A.M.); and CREATE Health Strategic Center for Translational Cancer
Research, Lund University (I.H.), Lund, Sweden.
I.H.: received grant support from the Swedish Cancer Society, the G Nilsson Cancer Foundation, the B Kamprad Foundation, the Cancer
and Allergy Foundation, King Gustaf V’s Jubilee Foundation, the Lund University Hospital Research Foundation, and governmental
funding of clinical research within the National Health Services (ALF). The remaining authors declare no conflict of interest.
Address correspondence and reprint requests to Laura Martı
´n de la Fuente, MD, Department of Clinical Sciences, Division of Oncology
and Pathology, Lund University Cancer Center/Medicon Village, Lund SE-223 81, Sweden. E-mail: laura.martin_de_la_fuente@med.lu.se.
Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML
and PDF versions of this article on the journal’s Website, www.intjgynpathology.com.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0
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1DOI: 10.1097/PGP.0000000000000394

relative survival rate is 27% for patients with
advanced disease (stage III-IV) (2). Despite efforts
aimed at early detection and new therapeutic
approaches, the poor survival persists and may be
explained by the heterogeneity and poorly under-
stood pathogenesis of ovarian carcinoma (OC).
Platinum compounds comprise the most active
chemotherapy available and constitute standard treat-
ment after cytoreductive surgery for the majority of
women diagnosed with ovarian cancer (3). First-line
chemotherapy with platinum-based chemotherapy yields
response rates of >80%. However, nearly all patients
relapse and eventually develop platinum resistance (4). A
biomarker capable of predicting platinum resistance at
primary surgery could make it possible to identify
patients who may not be suitable for platinum-based
therapy, and thus be spared its side effects, or who could
be eligible for other treatment options.
Claudins belong to a multigene family, with B24
members, and are considered one of the main tight
junction (TJ) forming proteins (5,6). A TJ acts as a
cellular barrier and is involved in paracellular perme-
ability and cell polarity (7,8). Since the discovery of
claudins in 1998, many studies have aimed to character-
ize the claudin family, revealing that the specific
combination and coexpression of different claudin
species determines the TJ barrier characteristics in a
tissue-specific manner (9,10). TJ structure and function
are often altered in human carcinomas, where TJ loss
can contribute to cancer progression (11). For example,
claudin-1 has been found to be downregulated in breast
cancer, and claudin-2 is downregulated in breast and
prostate cancer (12,13). In contrast, claudin-4 has been
reported to be upregulated in pancreatic, colorectal,
gastric, breast, prostate, and ovarian cancers (13–20).
Previous studies have reported increased expression of
claudins 3 and 4 in OC compared with normal ovarian
surface epithelium and benign ovarian tumors (20–22).
Claudin-4 has been proposed as a possible diagnostic
and prognostic biomarker in OC (3,23). However, there
are limited and contradictory data regarding the involve-
ment of claudins in chemotherapy resistance (24–26).
Therefore, in this study we investigated the role of
claudin-4 as a potential prognostic marker in 140 patients
with primary OC. The relationship between platinum
resistance and claudin-4 expression was also investigated.
MATERIALS AND METHODS
Patients
A total of 128 patients with OC were recruited
consecutively in the southern Swedish health care
region between 1998 and 2000 (27). In addition, 18
patients with OC were recruited at the oncogenetic
counseling at Lund University Hospital (Sweden)
between 1981 and 1997. Six patients with unknown
primary tumor or missing follow-up information
were excluded. Thus, a cohort of 140 OC patients was
assessed for claudin-4 protein expression.
The study was approved by the Ethics Committee
at Lund University, Sweden, waiving the requirement
for informed consent. The histologic subtype and
grade were determined according to WHO 2014 (28),
and all tumors were staged according to the Interna-
tional Federation of Gynecology and Obstetrics
(FIGO) criteria (29). Tumors were also classified into
types I and II on the basis of both histology and
grade (30). Platinum resistance was defined as
primary progressive disease or recurrence within
6 mo of completing platinum-based therapy, the
definition used in clinical practice today.
The majority of the tumors (70%) were of serous
histology, followed by endometrioid, mucinous, and
clear cell histologies. Approximately 75% of the
patients were diagnosed with advanced stage (III/IV)
disease, 64% were classified as type II, and 31/140
(22%) were carriers of a germline BRCA1 or BRCA2
mutation. Platinum-based chemotherapy was admin-
istered postoperatively to 132 patients: Carboplatin
(AUC5) combined with paclitaxel (175 mg/m
2
)to70
patients, Carboplatin (AUC5) combined with cyclo-
phosphamide (500 mg/m
2
) to 25 patients, and un-
specified platinum-based chemotherapy to 36
patients. Thirty-nine of the 132 patients who received
chemotherapy (30%) were platinum resistant. Seven
patients with early-stage (IA-B) disease and type I
tumors were excluded from the study of claudin-4
expression as a treatment predictive marker for
platinum response, as they had not received
platinum-based therapy, in accordance with current
guidelines. One patient was excluded from the same
analysis because she received non–platinum-based
chemotherapy postoperatively.
Data from a cohort of 1582 OC patients available
through the online tool Kaplan-Meier Plotter (31)
were used for validation.
Tissue Microarray (TMA) Construction and
Immunohistochemistry
We used a TMA with 0.6 mm triplicate core needle
biopsies from viable tumor areas (27). Sections, 3–4 mm
in thickness, were deparaffinized, rehydrated, and
stained. Antigen retrieval was performed using Dako
2 L. MARTI´N DE LA FUENTE ET AL.
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

Target retrieval solution of pH 6 (Dako A/S, Glostrup,
Denmark) in a pressure cooker 2100 Retriever
(Histolab Products AB, Gothenburg, Sweden). The
sections were incubated with a claudin-4 mouse
monoclonal antibody (Cat.No. 32-9400; Invitrogen
Corporation, Camarillo) at a 1:100 dilution for 30 min.
Immunohistochemical reactions were performed using
the Autostainer Plus with the Dako REAL EnVision
Detection system (Dako). Normal colon tissue was
included as a positive control. The negative controls
were ovarian stroma and follicle cells as well as smooth
muscle and adipose tissue, all reported as negative for
claudin-4 expression in The Human Protein Atlas (32).
We also stained whole-tissue sections with benign
ovary, fallopian tube, and endometrium, as well as 3
cases of serous cystadenomas.
A semiquantitative analysis of claudin-4 was
performed by 2 blinded investigators following
recommendations from a senior gynecologic pathol-
ogist (A.M.) and on the basis of the similar type of
expression pattern observed for HER2. Claudin-4
staining was distinct and predominantly confined to
the cell membrane. Two staining patterns were
observed: (i) punctate and partial staining around
the membrane; and (ii) complete membrane staining,
covering the circumference of the membrane surface.
Results from the core with the highest/strongest
positivity were recorded into 5 subgroups on the basis
of staining pattern and fraction of stained cancer cells
(0: no staining, 1: <50% of cells with punctate/
partial membrane staining, 2: 50%–75% of cells with
punctate/partial membrane staining, 3: 50%–75% of
cells with complete membrane staining, 4: >75% of
cells with complete membrane staining).
As claudin-4 was expressed in the majority of OCs
examined and no consensus for cutoff was available
in the literature, an exploratory cutoff was used in the
present study. Tumors with a Z3+ score were
considered to have high expression and the rest had
low expression. This cutoff was established on the
basis of the percentage of patients with primary
platinum-resistant disease and the 2 staining patterns
observed, before evaluating the effect on prognosis or
platinum resistance.
Statistical Analyses
The prognostic value of claudin-4 was investigated
using 5-yr progression-free survival (PFS) time and 5-yr
overall survival (OS) time as endpoints. PFS time was
defined as the time interval between date of diagnosis
and the first sign of disease recurrence (clinical and/or
radiologic) or death, whichever occurred first. OS time
was defined as the time interval between date of
diagnosis and death. Of the patients who died within
5 yr after diagnosis, all but 2 died of ovarian cancer.
Statistical analyses were performed with SPSS for
Windows version 22. To analyze the probability of a
patient being platinum resistant, we calculated the
relative risk (risk ratio) of high versus low claudin-4
expression with 95% confidence interval (95% CI).
Associations between claudin-4 expression and plat-
inum resistance and other clinical parameters were
assessed using the Pearson w
2
test or the Fisher exact
test, except for the ordinal variable stage, which was
compared using the Mann-Whitney Utest. Survival
analyses for PFS and OS were performed using the
Kaplan-Meier method, and differences between
groups were tested using the logrank test. The effect
of claudin-4 expression on survival was expressed
using hazard ratios (HR) with 95% CI, estimated
using univariable (crude effect) and multivariable
(adjusting for clinical factors known to influence
ovarian cancer survival) Cox regression. These
factors included age at diagnosis (Z70 y vs. <70),
tumor type (II vs. I), stage (III/IV vs. I/II), and
BRCA1/2 mutation status (wild-type vs. muta-
tion) (30,33–35) and were analyzed as binary factors.
The cutoff of 70 yr was chosen on the basis of
previous studies reporting an inferior outcome
among elderly women (33). The classification into
type I and II is made on the basis of both histology
and grade, and defines 2 fundamentally different
groups regarding tumorigenesis and prognosis (30). A
large meta-analysis showed better OS and PFS for
BRCA1 and BRCA2 mutation carriers compared
with noncarriers (35). The prognostic factor residual
disease following cytoreductive surgery was not
included in the analyses, as this information was
not documented. Cox regression was performed for
the type II tumors alone as a stability analysis. All P-
values are 2-sided.
RESULTS
Immunohistochemical Staining of Claudin-4
Seven of the 140 OCs displayed no staining, and
the distribution in subgroups 1–4 was as follows:
1: n = 32, 2: n = 30, 3: n = 18, 4: n = 53. Thus, 71 cases
(51%) displayed high claudin-4 expression. Figure 1
shows representative examples of high and low claudin-
4expressioninOC.Theexpressionofclaudin-4(lowvs.
high) in the different histologic subtypes, stages, and
types, as well as in relation to age, BRCA1/2 mutation
3CLAUDIN-4 EXPRESSION IN OVARIAN CARCINOMA
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

status, and platinum sensitivity, is shown in Table 1.
High expression of claudin-4 correlated with high-grade
serous histology compared with low-grade serous
histology (w
2
test, P= 0.037) and high age at diagnosis
(w
2
test, P= 0.049). A correlation between type II
tumors and high claudin-4 expression was also
observed, but this did not reach statistical significance
(w
2
test, P= 0.071). No correlation was found with
BRCA1/2 status (wild-type vs. mutant, w
2
test, P=0.6)
or stage (Mann-Whitney Utest, P=0.1) (Table 1).
Two whole-tissue sections from a benign ovary with
mesothelial hyperplasia revealed negative staining in
the mesothelium, including the hyperplastic areas,
whereas cortical serous inclusion cysts were strongly
positive (Fig. 1). Epithelial cells in the 3 cases of serous
cystadenomas also displayed strong claudin-4 expres-
sion. Other cell types in the ovary, including stromal
and smooth muscle cells, showed no claudin-4
expression in all evaluated cases, in line with the
Human Protein Atlas (32). Epithelium from normal
fallopian tube and normal endometrium displayed
high claudin-4 expression, also in line with the Human
Protein Atlas (5 and 10 cases, respectively) (32).
Claudin-4 Expression and Platinum Sensitivity
Among the 132 patients who had received
platinum-based chemotherapy, a patient with high
claudin-4 expression had a 1.2 (95% CI = 0.7–2.0,
P= 0.3) times higher risk (risk ratio) of being
platinum resistant compared with a patient with
low claudin-4 expression (Table 1). No associations
with platinum responsiveness were found in separate
analyses of type II tumors (n = 86, P= 0.6), type I
tumors (n = 42, Fisher Exact test, P= 0.4), the
serous subtype (n = 97, P= 0.7), late-stage (III-IV)
disease (n = 96, P= 0.8), early-stage (I-II) disease
(n = 36, Fisher Exact test, P= 0.2), or BRCA1/2
wild-type status (n = 100, P= 0.2).
Claudin-4 Expression and Prognosis of OC
Univariable analyses revealed an association be-
tween PFS and claudin-4 expression [HR = 1.7
(1.1–2.6), P= 0.020] (Table 2), with patients whose
tumors expressed high levels of claudin-4 displaying
an inferior outcome (Kaplan-Meier survival
analysis, Fig. 2). Hazard of relapse was similar
[HR = 1.5 (1.0–2.4)] after adjustment for age, stage,
type, and BRCA1/2 status in a multivariable analysis,
but the evidence was slightly weaker (P= 0.076)
(Table 2). A comparable association was observed for
OS in the univariable and multivariable analyses
[HR = 1.6 (1.0–2.4), P= 0.041 and HR = 1.5
(0.9–2.3), P= 0.1] (Table 3). Analyses of type II
tumors (n = 86) showed a similar HR in multivariable
analysis compared with all OCs [1.6 (0.9–2.8),
FIG. 1. Immunohistochemical evaluation of claudin-4 in ovarian tissues, including claudin-4 high (A) and low (B) serous ovarian cancer, and
claudin-4 high (D) and low (E) endometrioid ovarian cancer. Negative ovarian mesothelium and positive cortical inclusion cyst (C). Positive
control, healthy colon (F). Magnification: 40 .
4 L. MARTI´N DE LA FUENTE ET AL.
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

P= 0.078 for PFS and 1.6 (0.9–2.7), P= 0.086 for
OS].
To extend our findings, we investigated the role of
CLDN4 mRNA levels in a cohort of 1582 OC
patients available through the online tool Kaplan-
Meier Plotter (31). The online tool comprises gene
expression data and survival information from 13
independent public OC data sets (Gene Expression
Omnibus and The Cancer Genome Atlas, n = 28-565,
2015 version). The best performing threshold was
used as cutoff, where 1105 patients (70%) were
classified as CLDN4 low and 477 (30%) as CLDN4
high. These results were in line with our findings, as
CLDN4-high patients displayed worse 5-yr OS in a
univariable analysis [HR = 1.3 (1.1–1.5), Po0.001]
compared with CLDN4-low patients. Interestingly,
the results were consistent after 10 yr of follow-up [see
Fig. Supplemental Digital Content 1, http://link-
s.lww.com/IJGP/A57, HR = 1.3 (1. 2–1.5), Po0.001
for 10-yr OS]. However, a similar difference in 10-yr
PFS was not observed [HR = 1.2 (1.0–1.3),
P= 0.064].
DISCUSSION
A previous study has reported low or absent
claudin-4 expression in normal ovarian surface
epithelium and ovarian cystadenomas, in contrast
to the high expression reported in OC, which
according to the authors supports the involvement
of claudin-4 in malignant transformation (20). We
also found absence of expression in ovarian surface
mesothelium, and interestingly also in the hyper-
plastic changes. In contrast to the former study, we
found strong positive expression in cortical inclusion
cysts (CIC) and serous cystadenomas. Furthermore,
we also found strong positive expression in normal
fallopian tube epithelium, consistent with data
reported in the Human Protein Atlas (32).
TABLE 1. Claudin-4 expression and clinicopathologic parameters
N (%)
N (%) Claudin-4 low Claudin-4 high P*
All ECs 140 69 (49) 71 (51)
Age (yr)
Mean (range) 59 (26–83) 58 (26–83) 61 (26–83)
o70 110 (79) 59 (54) 51 (46) 0.049
Z70 30 (21) 10 (33) 20 (67)
BRCA1/2 status
Mutant 31(22) 14 (45) 17 (55) 0.6
Wild-type 109 (78) 55 (51) 54 (49)
Histology
High-grade serous 80 (70) 36 (45) 44 (55) 0.037w
Low-grade serous 18 (13) 13 (72) 5 (28)
Endometrioid FIGO 3 5 (3.6) 1 4
Endometrioid FIGO 1/2 16 (11.4) 7 9
Mucinous 10 (7) 4 6
Clear cell 5 (3.6) 5 0
Carcinosarcoma 1 0 1
Mixed and missing 5
Tumor type
I 49 (36) 29 (59) 20 (41) 0.071
II 86 (64) 37 (43) 49 (57)
Missing 5
Stage
I 27 (12) 17 (65) 10 (37) 0.1z
II 17 (13) 9 (53) 8 (47)
III 77 (60) 35 (45) 42 (55)
IV 19 (15) 8 (42) 11 (58)
Platinum responsiveness
Sensitive 91 (70) 43 (50) 44 (50) 0.3
Resistant 39 (30) 17 (44) 22 (56)
Missing 2
Bold values are statistically significant Pvalue o0.05.
*w
2
test between claudin-4 low and high groups.
ww
2
test comparing the high-grade and low-grade serous ovarian carcinomas.
zMann-Whitney Utest between claudin-4 low and high groups.
5CLAUDIN-4 EXPRESSION IN OVARIAN CARCINOMA
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

A new paradigm for the pathogenesis of ovarian
serous carcinoma, which questions the traditional
model of ovarian mesothelium as the cell of origin for
serous neoplasia, has been proposed. Cogent argu-
ments support the role of fallopian tube epithelium in
the genesis of both low and high-grade serous
OCs (36–43). Serous tubal intraepithelial carcinomas
found in prophylactic salpingo-oophorectomies in
BRCA mutation carriers and sporadic cases of pelvic
high-grade serous carcinoma are this far the best
evidence of this extraovarian hypothesis (40,42,43).
Furthermore, a new model of serous tumorigenesis
proposes that normal tubal epithelium may shed and
implant on the ovarian surface, thereby forming
CICs. CICs and serous cystadenomas follow a benign
course in most cases, but in some cases they can
eventually lead to low and sometimes high-grade
serous tumors (42). Thus, no conclusion regarding
the involvement of claudin-4 in malignant trans-
formation should be made on the basis of the
comparison with ovarian mesothelium (40).
In our study, fallopian tube epithelium, as well as
CICs and serous cystadenomas, displayed high
claudin-4 expression, arguing against the upregula-
tion of claudin-4 in malignant transformation.
Furthermore, we examined endometrial epithelium,
as endometrioid and clear cell tumors have been
associated with endometriosis and because their gene
expression profiles resemble those of endometrial
epithelium (40,43). High endometrial claudin-4 ex-
pression was observed, also consistent with the
Human Protein Atlas (32).
Decreased expression of claudins in cancer is in
agreement with the hypothesis that tumorigenesis is
accompanied by TJ disruption and loss of cell-cell
adhesion, leading to disease dissemination (11). In
contrast, overexpression of claudin-4 has been reported
in several cancer types, including OC (20–22,44).
Upregulation of CLDN3 and CLDN4 has been shown
to increase cell invasion and motility in OC cell lines,
and, conversely, knockdown caused the opposite
effects, suggesting that claudin-3 and 4 may indeed
promote ovarian tumorigenesis (45). In the present
study, we found that high levels of claudin-4 were
associated with decreased PFS and OS. The hazards of
relapse or death were similar but not significant when
TABLE 2. Univariable and multivariable analyses of progression-free survival
5-yr PFS univariable Cox 5-yr PFS multivariable Cox
N (events) HR (95% CI) PHR (95% CI) P
Claudin-4 expression
Low 64 (38) 1
High 70 (55) 1.7 (1.1–2.6) 0.020 1.5 (1.0–2.3) 0.076
Age at diagnosis (yr)
o70 107 (71) 1
Z70 27 (22) 1.5 (0.9–2.4) 0.1 1.1 (0.6–1.8) 0.7
BRCA1/2 status
Mutant 29 (18) 1
Wild-type 105 (75) 1.7 (1.0–3.1) 0.052 2.6 (1.5–4.6) 0.001
Stage
I/II 40 (11) 1
III/IV 94 (82) 6.2 (3.3–11.7) o0.001 4.4 (2.2–8.8) o0.001
Type
I 47 (20) 1
II 82 (68) 2.9 (1.8–4.8) o0.001 2.2 (1.2–3.8) 0.023
Bold values are statistically significant Pvalue o0.05.
CI indicates confidence interval; HR, hazard ratio; PFS, progression-free survival.
FIG. 2. Kaplan-Meier survival analysis. Association between
progression-free survival (PFS) and the level of claudin-4
expression. Patients with high claudin-4 expression (solid line,
n = 70) had shorter PFS than those with low expression (dotted
line, n = 64). Median PFS 18 versus 37 mo. Logrank P= 0.018.
6 L. MARTI´N DE LA FUENTE ET AL.
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

adjusting for known prognostic factors, probably due
to lack of power. The group with low claudin-4
expression had better prognosis and moreover dis-
played lower claudin-4 expression than the normal
tissue levels used for comparison. Thus, there is a
possibility that tumors that downregulate claudin-4
expression during malignant transformation become
less aggressive tumors with better prognosis. Our
findings also suggest that claudin-4 may have prog-
nostic value.
Currently, the development of resistance can only
be determined retrospectively, after patients have
experienced the burden and toxicity of ineffective
therapy (3). Clinical recurrences that occur within
6 mo after cessation of primary platinum-based
chemotherapy are considered platinum resistant. This
definition of platinum resistance is a strong predictor
of response to second-line therapy, with very low
response rates for patients defined as platinum
resistant (46,47). A biomarker capable of predicting
platinum resistance at primary surgery could enable
the identification of patients not suitable for
platinum-based therapy and who may thus be spared
its side effects. Furthermore, patients with over-
expression of claudin-4 and platinum-resistant dis-
ease may be candidates for clinical trials, including
claudin-4-targeted therapy, as claudin-4 is potentially
druggable using the C-terminal fragment of Clostri-
dium perfringens enterotoxin (24–26). Such a bio-
marker could therefore provide the basis for
individualization of therapy and improved outcome.
In the current study we attempted to establish
whether claudin-4 might be predictive of platinum
sensitivity. Only a few reports are available on the
association between platinum resistance and claudin-
4 expression, and the results are contradictory. A
proteomic study found 7-fold increased levels of
claudin-4 protein in a platinum-resistant cell line
compared with the sensitive parental line (3). Two
studies have reported associations between high levels
of claudin-4 protein and platinum resistance (24,26),
whereas a third study failed to detect any association
with platinum resistance or survival (25). These
studies are, however, small (n = 12–43) and may
not be representative of the whole spectrum of OC. In
fact, one of the studies reporting significantly higher
claudin-4 expression in chemoresistant compared
with chemosensitive cases had an overrepresentation
of clear cell histology, known to be inherently more
chemoresistant (10/43 cases, 23%), compared with
our cohort (4/130 cases, 3%) (26). The association
between platinum resistance and claudin-4 may hence
be confounded by the histologic type, emphasizing
the importance of stratification of histologic and
probably also molecular subtypes when investigating
potential treatment predictive biomarkers in OC. The
other study compared CLDN4 mRNA levels at
primary surgery from 6 patients with chemotherapy
naive serous OC with chemotherapy resistant tumors
from 6 patients with recurrent disease, showing
CLDN4 upregulation at recurrence (24). In contrast,
we investigated only tumor tissue from primary
surgery, as we were interested in investigating the
potential predictive value at first-line chemotherapy.
However, pairwise comparisons between primary
surgery and relapse may be interesting and could
potentially inform treatment decisions in recurrent
disease, as there may be a group of patients in whose
TABLE 3. Univariable and multivariable analyses of overall survival
5-yr OS univariable Cox 5-yr OS multivariable Cox
N (events) HR (95% CI) PHR (95% CI) P
Claudin-4 expression
Low 69 (36) 1
High 71 (48) 1.6 (1.0–2.4) 0.041 1.5 (0.9–2.3) 0.1
Age at diagnosis (yr)
o70 110 (60) 1
Z70 30 (24) 1.9 (1.2–2.9) 0.011 1.7 (1.0–2.9) 0.037
BRCA1/2 status
Mutant 31 (16) 1
Wild-type 109 (68) 1.4 (0.8–2.4) 0.2 2.1 (1.2–3.7) 0.013
Stage
I/II 44 (13) 1
III/IV 96 (71) 4.2 (2.3–7.6) o0.001 2.8 (1.5–5.3) 0.002
Type
I 49 (17) 1
II 86 (64) 3.0 (1.7–5.1) o0.001 2.1 (1.1–3.8) 0.020
Bold values are statistically significant Pvalue o0.05.
CI indicates confidence interval; HR, hazard ratio.
7CLAUDIN-4 EXPRESSION IN OVARIAN CARCINOMA
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2017

tumors claudin-4 is upregulated upon development of
platinum resistance. This line of research warrants
further attention in larger cohorts with matched primary
tumors and relapses. Nevertheless, we did not find a
confident association between claudin-4 expression and
platinum sensitivity. Our findings are, however, con-
sistent with a previous report showing that claudin-4
expression, determined by qRT-PCR and IHC, did not
differ between platinum-sensitive and platinum-resistant
tumors in a cohort of 36 patients with high-grade,
advanced-stage serous ovarian cancer (25). Further-
more, the definition of platinum resistance differed in
the previously reported studies. Taken together, we did
not find claudin-4 to be a useful predictive marker for
platinum sensitivity in the clinical situation, either in an
unselected cohort or within any other subgroup.
Of interest, claudin-4 has been suggested as a
potential therapeutic target. Suppression of claudin-4
expression in human serous ovarian cancer cell lines
by siRNA led to cellular accumulation of cisplatin
and a significant increase in cisplatin sensitivity (26),
suggesting that upregulation of claudin-4 expression
may contribute to platinum resistance by decreasing
drug uptake. Additional mechanisms of drug resist-
ance may nevertheless also be present (48), and it
remains to be confirmed whether claudin-4 over-
expression is involved. We hypothesize that the
heterogeneity and complexity of resistance mecha-
nisms may explain why no differences between
resistant and sensitive tumors were observed in the
present study.
In conclusion, we identified claudin-4 as a potential
prognostic biomarker in OC. Although previous studies
have suggested a relationship between claudin-4 and
platinum resistance, we were not able to validate this in
our larger cohort. The druggable nature of claudin-4
nevertheless makes it appealing to pursue further as a
target for circumventing platinum resistance. Further
investigations aimed at studying the relationship be-
tween claudin-4 and platinum resistance are needed, in
addition to validation studies to find application into
clinical practice.
Acknowledgment: The authors would like to acknowl-
edge Kristina Lo
¨vgren for assistance with TMAs and
stainings.
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