Acta Histochemica 123 (2021) 151717

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Acta Histochemica
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Short communication

Nuclear expression of NHERF1/EBP50 in Clear Cell Renal Cell Carcinoma

Baltazar Eduardo Lema a, García Marchiñena Patricio b, Erica Lorena Kreimann c, d, *
a
Private Diagnostic Pathology Laboratory, Anchorena 1510 Capital Federal (1425) C.A.B.A., Buenos Aires, Argentina
b
Servicio de Urología, Hospital Italiano de Buenos Aires, Tte. Gral. Juan Domingo Perón, 4190, (1199). C.A.B.A., Buenos Aires, Argentina
c
Department of Radiobiology, National Atomic Energy Commission of Argentina (CNEA), Argentina
d
National Research Council of Argentina (CONICET), Av. General Paz 1499 (1650), San Martín, Buenos Aires, Argentina

A R T I C L E I N F O A B S T R A C T

Keywords: The Na/H exchange regulatory factor 1 or Ezrin-radixin-moesin-binding phosphoprotein 50 (NHERF1/EBP50) is

NHERF1 an adaptor protein implicated in the stabilization of molecular complexes linking extracellular signals with the
Cancer cytoskeleton machinery. NHERF1 expression at the cell cortex is associated with the maintenance of adherent
Renal
junction integrity in polarized epithelia. The role of NHERF1 in cancer depends on its localization within the cell,
Ovarian
Clear cells
acting, in most cases, as a tumor suppressor when localized at the cell membrane, and as an oncogene, when
Nucleus expressed in the cytoplasm or the nucleus of cancer cells. The distribution of NHERF1 in renal cell carcinoma
(RCC) has not been yet investigated. In this study, NHERF1 expression was examined by immunohistochemistry
in papillary and clear cell RCC. We observed membranous staining in papillary RCC, whereas NHERF1 expression
was nuclear and membranous in clear cell RCC. In comparison, NHERF1 immunohistochemistry in clear cell
carcinomas of the ovary showed mainly nuclear staining. Our finding of the specific NHERF1 nuclear expression
in clear cell carcinomas may help to elucidate the molecular changes that regulate its nuclear accumulation and
to better understand its role in this cell compartment.

1. Introduction %–80 % of all RCCs. The ccRCC histological subtype refers to the
appearance of cells with high concentration of cytoplasmic glycogen and
Renal cell carcinoma (RCC) is responsible for approximately 4.1 % of lipid content. About 80 % of sporadic ccRCC cases are attributed to
adult diseases and represents the most lethal urological cancer (Cairns, somatic von-Hippel Lindau (VHL) gene inactivation (Cho et al., 2011)
2010). It is more frequent in men than in women with a rising incidence and 2–3 % of ccRCC to hereditary disease linked to von-Hippel Lindau
over the last 10 years for both sexes in all races (SEER Stat Fact Sheets: syndrome (VHL disease) (Rini et al., 2008).
Kidney and Renal Pelvis, (http://seer.cancer.gov/statfacts/html/kidrp. Other RCC subtypes include Papillary renal cell carcinoma (PRCC),
html). For tumors confined to the kidney, the standard therapy is the type I and II, responsible for 11–15 % of the cases, Chromophobe (5 %),
partial or radical nephrectomy that results in an 80 % five-year survival and other less frequent subtypes (Escudier et al., 2019; Hsieh et al.,
rate (Hüsch et al., 2012). Unfortunately, by the time of diagnosis, 20–30 2017).
% of the patients already have metastatic dissemination, and 20 % will The Na/H exchange regulatory factor 1 (NHERF1) protein was
eventually progress to metastatic disease during follow up, with 12 % identified for the first time in kidney as a co-factor that required for the
5-year survival rate (Khetani et al., 2020). A greater understanding of regulation of the activity of sodium/hydrogen exchanger type 3 by
RCC biology has led to the development of new therapies for metastatic cAMP. NHERF1 was independent in cell lysates of human placenta in a
RCC. In localized disease, adjuvant treatment of chemotherapy is not successful attempt to identified potential Ezrin-Radixin-Moesin (ERM)
recommended, and targeted therapies are preferred for disseminated protein partnersand called Ezrin-radixin-moesin-binding phosphopro­
kidney cancer (Escudier et al., 2007, 2009; Hudes et al., 2007; Sternberg tein 50 (EBP50) (Weinman et al., 1995; Reczek et al., 1997). NHERF1 is
et al., 2010; Rini et al., 2019). an adaptor protein expressed primarily at the cell cortex on the inner
RCC encompasses a heterogeneous group of tumor subtypes that face of the plasma membrane of polarized epithelia. NHERF1 has 2
include the Clear cell renal cell carcinoma (ccRCC), responsible for 70 tandem PDZ (PSD-95/Disk-large/ZO-1 homology) domains and an

* Corresponding author at: Department of Radiobiology, National Atomic Energy Commission of Argentina (CNEA), Av. General Paz 1499 (1650), San Martín,
Buenos Aires, Argentina.
E-mail addresses: laboratorioslema@gmail.com (B.E. Lema), patricio.garcia@hospitalitaliano.org.ar (G.M. Patricio), ekreimann@conicet.gov.ar (E.L. Kreimann).

https://doi.org/10.1016/j.acthis.2021.151717
Received 22 October 2020; Received in revised form 24 April 2021; Accepted 26 April 2021
Available online 6 May 2021
0065-1281/© 2021 Elsevier GmbH. All rights reserved.
B.E. Lema et al. Acta Histochemica 123 (2021) 151717

ERM-binding C-terminal region (EB), and they are all essential for its Table 1
functions. NHERF1 stabilizes macromolecular complexes at the cell Renal Cell Carcinoma Cases.
membrane linking extracellular signals with the cytoskeleton machin­ Case Tumor Type Grade NHERF1 Cellular
ery. It is also involve in the regulation of proliferation, apoptosis, cell localization
migration, inflammatory response, tissue injury and metabolic pathways 1 CcRCCa – Nucleus
(Bushau-Sprinkle et al., 2020). 2 CcRCC – Nucleus/Membrane
The cell localization of NHERF1 is altered during cell transformation, 3 CcRCC I Nucleus/Membrane
a shift from the membrane to the cytoplasm and nucleus and had been 4 CcRCC I Nucleus
5 CcRCC II Nucleus/Membrane
described in different types of cancers such as breast, liver, brain, colon, 6 CcRCC II Nucleus/Membrane
prostate cancers among others (Georgescu et al., 2008). Nuclear accu­ 7 CcRCC II Nucleus/Membrane
mulation of NHERF1 had been described in breast cancer (Bellizzi et al., 8 CcRCC I Nucleus/Membrane
2011) gastric and colon cancer (Mangia et al., 2012, 2015; Lin et al., 9 CcRCC. Recurrence. III Nucleus/Membrane
10 CcRCC. Tumor invasion of the renal II Nucleus/Membrane/
2012) and melanoma (Fang et al., 2015). In melanoma cells, NHERF1
capsule. Cytoplasm
interacts with PTEN in the nucleus and its translocation to the cytoplasm 11 CcRCC, partly eosinophilic I Nucleus/Membrane
drives malignant progression (Fang et al., 2015). In advanced colorectal 12 CcRCC, partly eosinophilic Nucleus
cancer (CRC), NHERF1 nuclear expression was associated with poorer 13 CcRCC, partly chromophobe – Nucleus
differentiation grade in the tumor’s invasive front (Malfettone et al., 14 CcRCC, partly chromophobe – Nucleus/Membrane
15 CcRCC, partly chromophobe and II Nucleus/Membrane
2012). Many studies support the principle that NHERF1 acts as a tumor partly eosinophilic.
suppressor when localized at the cell membrane, but as an oncogene 16 PRCCb type I – Apical Membrane*
while expressed in the cytoplasm and cell nucleus (reviewed by Bush­ 17 PRCC type I II Apical Membrane*
au-Sprinkle et al., 2020). In some types of cancer, a different behavior 18 PRCC type I with clear cells foci. I Apical Membrane
/Nucleus of clear cells
was observed, for example, in serous ovarian carcinomas, NHERF1 was
19 PRCC type I with clear cells foci. I Nucleus of clear cells
expressed in the apical membrane of the cells of the tumor papillae that 20 CcRCC, areas of PRCC type II and – Apical Membrane
line the luminal cavities, but was absent in areas of compact tumor chromophobe cells (80 %). /Nucleus of clear cells
(Demacopulo et al., 2016). In gastric cancer, the expression of NHERF1 21 PRCCc type II, relapse in hepatic – Apical Membrane
in the nucleus correlates with clinical response to chemotherapy with parenchyma. Clear cell Foci.

epirubicin / oxaliplatin / capecitabine (EOX) (Mangia et al., 2015). a

Clear-cell renal-cell carcinoma.
The regulatory mechanisms of NHERF1 cell expression is still a b
Papillary renal-cell carcinoma type I.
c
matter of extensive research and it may depend on variables specific to Papillary renal-cell carcinoma type II.
each tumor including tumor architecture, cell type, cell cycle, gene
mutations, phosphorylation, protein interactions and activation of preserve patients confidentiality expressed in the Argentinean privacy
different signaling pathways within each cell. For instance, the phos­ ethical rules (ANMAT 5330/97). This study adheres to the internationals
phorylation of human NHERF1 in Ser162/339/340 induces a shift from standards such as the Nuremberg code, the Helsinki declaration and its
its localization from the cell periphery to the cytosol (Vaquero et al., modifications.
2017). In hepatocellular carcinoma the nuclear localization of NHERF1
was mediated by the direct binding of NHERF1 to β-Catenin, placing
NHERF1 as an activator of the Wnt signaling pathway (Shibata et al., 2.2. Histology and immunohistochemistry
2003). In colon cancer, the expression of cytoplasmic NHERF1 during
the adenoma-carcinoma stage progression activates genes regulated by The tissue specimens were fixed in 10 % formalin in phosphate
β-catenin, with a consequent increase in cell migration and invasion buffered saline (PBS) for 24 h, and then transferred to an 70 % ethanol
(Hayashi et al., 2010). solution and storage at 4 ⁰ C until processed for paraffin embedding,
Up to date, there are no reports on the expression of NHERF1 in within the week upon received. Tissue sections (5− 8 μm) were placed in
RCCs. Molecular biology studies will not only allow a better pathological 3-aminopropyltrietoxysilane (Sigma-Aldrich, St. Louis, MO, USA)
classification of RCCs, but also help to detect new therapeutic targets coated slides and stained with hematoxylin-eosin for histopathological
and diagnostic and prognostic biomarkers, thus contributing to better studies. The immunohistochemistry analysis was performed within in
management of patients with RCC. The aim of this study is to describe unstained sections (from 0 to 3 months old paraffin blocks) using two
the cellular expression of NHERF1 in two tumor types’ ccRCC and PRCC commercially available kits. The Vectastain Universal Elite ABC KIT,
that account for at least 85–90 % of all RCCs. previous blocking of the samples with Avidin/Biotin blocking reagent
(Vector Laboratories, Inc., Burlingame, CA, USA) and the polymer based
2. Materials and methods detection system NovoLink™ Min Polymer Detection System (cat.
RE7290-K, Novocastra, Leica 157 Biosystems Inc., Buffalo Grove, IL,
2.1. Tumor samples USA, 60089). The protocols were performed according to manufac­
turer’s instructions and the experiment was repeated two times with
The study was carried out in tissue sections from paraffin blocks in 21 each kit. Primary antibodies: 1) NHERF1 polyclonal rabbit antibody
RCC samples collected from pathology service laboratories in the Buenos (catalog No. PA1-090, Invitrogen-Thermo Fisher Scientific 168 Third
Aires area between the years 2014− 2017. Cancer subtypes include Avenue Waltham, MA, USA 02451; 1:500). 2) Proliferating cell nuclear
ccRCC and PRCC type I and II according to the WHO classification of antigen (PCNA) clone PC10 (catalog No sc-56, Santa Cruz Biotech­
tumors of the urinary system and male genital organs 4th Edition, Lyon, nology, Inc. 10410 Finnell Street, Dallas, TX, USA 75220; 1:500). The
France, International Agency for Research on Cancer (Moch et al., 2016) tissue sections were incubated with the antibodies overnight at 4 ◦ C, in
(Table 1). The study was approved by the Ethical Committee of The humidity chambers and in the dark. Negative controls were processed
Huésped Foundation. Three specimens of ovarian adenocarcinoma OC excluding the primary antibody as our previous studies demonstrated
mostly serous with foci of clear cells were included in the analysis, the the specificity of the NHERF1 antibody using non-specific mouse IgG1
samples were collected from the Institute of Oncology Angel H Roffo in antibody and purified rabbit pre-immune serum (DAKO, Kingsgrove,
Buenos Aires, and the study was approved by the ethics committee of the NSW, Australia) as isotype controls (Kreimann and Cabrini, 2013; Cuello
same institution. The OC disease was classified according to the WHO Carrion et al., 2010).
classification (Kurman et al., 2014). All the samples were coded to The photographs of the slides were taken with a 100x objective, and

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B.E. Lema et al. Acta Histochemica 123 (2021) 151717

were saved in uncompressed TIFF format (resolution of 2040 × 1530 nuclear, membrane and cytoplasmic expression (Table 1, case 10)
pixels). For quantification of NHERF1 staining, an average of 30–50 cells (Fig. 1e and f, and Table 1). Positive staining of PCNA was observed in
was analyzed on each slide. Images were grayscale transformed using the same tumor and it was used as positive control for nuclear staining
the free Image J software, Version 1.42 (National Institutes of Health, (Fig. 1c).
USA), and intensity was measured in the apical membrane and clear cell Fig. 2 shows the analysis in two samples of PRCC type I tumors,
nucleus, using the same rectangular area. The intensity of the different where we did not detect NHERF1 nuclear labeling. Positive staining was
slides was normalized by the background measured in the stroma. The only observed in the apical and the upper region of the basolateral
mean of each group was calculated as the mean of the means of each membrane of the cells that line the tumor papillae, as we previously
area of the different slides (n = 3 cases). Statistical comparisons were described in the serous OC tumors (Fig. 2b and d) (Demacopulo et al.,
made using the t-test. 2016).
Clear cell tumors occur in other organs such as the female genital
3. Results tract, they were describe in the ovary, endometrium and cervix.
Although these tumors are rare, special attention is paid to them for their
We analyzed the expression of NHERF1 in 21 samples of RCC resistance to conventional treatments (Kar et al., 2020). Our group has
(Table 1) and three cases of OC, with foci of clear cells. It was previously previously described the expression of NHERF1 in human serous OCs
described that in normal kidney, NHERF1 was expressed in the brush (Demacopulo et al., 2016). In this report, we present the distribution of
border membranes of the proximal tubule’s epithelial cells, in human NHERF1 in clear cell ovarian tumors with a mixed, serous and clear cell
and rodents (Weinman et al., 2002; Morales et al., 2004, Murray et al., phenotype, and compare them with the distribution of NHERF1 in RCC.
2013). In Fig. 3, we show the expression and distribution of NHERF1 in RCC
In this report, we observed the same pattern in kidney healthy tissue and OC tumors. The panels on the left show a case of PRCC type I with
margins of RCC samples, as NHERF1 expression was positive in the apical staining of NHERF1 in tumor cells of the papillae (a, b), in
apical membrane and cytoplasm of proximal tubule’s epithelial cells, contrast to the nuclear accumulation in clear cells in the same tumor (c,
but negative in the glomerulus (Fig. 1b). In contrast, in ccRCC tumor arrow). The right panel of (d–f) corresponds to a bilateral ovarian cys­
cells, NHERF1 was expressed in the nucleus and at the cell membrane in tadenocarcinoma, mostly serous, with foci of clear cells. The tumor is
13 cases, only in the nucleus in 5 cases, and one tumor presented characterized by an atypical epithelial proliferation of cells with marked

Fig. 1. NHERF1 nuclear expression in ccRCC.

(a) Tissue section of an area of normal kidney
area adjacent to the tumor showing the prox­
imal tubule and glomerulus (H&E). (b) NHERF1
is expressed in the apical membrane and cyto­
plasm of renal proximal tubules (arrow), in
contrast to the negative staining observed in the
glomerulus (head arrow). (c) PCNA expression
in ccRCC, as positive control of nuclear stain­
ing. (d) CcRCC tumor (H&E), inset showing the
typical clear cytoplasm characteristic of clear
cells (H&E). (e-f) The panel shows the positive
expression of NHERF1 in the nucleus and
membrane of clear cancer cells. Pictures
magnification: 200x (a-b, d-e) and 1000x (c and
f and inset).

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B.E. Lema et al. Acta Histochemica 123 (2021) 151717

Fig. 2. Apical staining of NHERF1 in PRCC

tumors. (a) H&E showing the structure of the
tumor papilla, the arrow indicates the apical
membrane of a tumor cell. (b) NHERF1
expression if mainly located in the apical
membrane of the tumor papilla (arrow) and
negative in the basal membrane. It resembles
the results observed in ovarian carcinomas
(Demacopulo B et al., 2016). (c) A second PRCC
tumor displaying internal luminal spaces, H&E.
(d) NHERF1 is strongly expressed in the border
of tumor papillary structures, but also in tumor
cells coating the intratumoral lumens. Interest­
ingly, we notice some staining in the upper half
of the basolateral membrane of cells facing
luminal spaces. Pictures magnification: 400 × .

anaplasia, nuclear atypia and numerous mitotic figures, which form well cause uncertainty at the time of diagnosis (Kar et al., 2020).
differentiated papillary structures. In this tumor, we detected the same Although migration of NHERF1 from the apical membrane to the
pattern as in RCC with strong nuclear expression of NHERF1 in clear nucleus has been described in several types of cancers, including car­
cells, but apical staining in the papillary areas, with exception of some cinomas of the liver, breast, and colon, this pattern has never been
cell protrusions which need to be further investigated (e–f). associated with a specific cell type until now (Shibata et al., 2003;
The semiquantitative analysis of the immunohistochemical reaction Paradiso col., 2017; Mangia et al., 2012). Studying the nuclear role of
showed some significant differences between both tumors. For example, NHERF1 in clear cells will allow us to characterize one more aspect of
the intensity of NHERF1 expression between the nucleus and the the biology of these tumor cells and the mechanisms that allow them to
membrane of the tumor cells of the RCC was similar. In contrast, nuclear proliferate, migrate and colonize other organs. From a clinical point of
expression in OCs was much stronger than that detected at the cell view, clear cells are characterized by their resistance to chemotherapy
membrane, (p ≤ 0.01) (Fig. 4). Furthermore, a greater intensity was and traditional radiotherapy, which is why they constitute an important
observed in the nuclear staining of NHERF1 in OC cells compared to the research topic. (Ljungberg et al., 2010; Itamochi et al., 2008; Sugiyama
nuclear expression of NHERF1 in RCC (p ≤ 0.05) (Fig. 4). et al., 2000).
The NHERF1 protein can undergo various post-translational modi­
4. Discussion fications that regulate its activity and interactions. For example, the
cleavage of its C-terminal region and the expression of isoforms have
The study of biomarkers has had a great impact on the detection and been described (Morales et al., 2004). These post-translational modifi­
diagnosis of various types of cancers, helping to identify the tissue of cations affect the affinity of the NHERF1 protein for its ligands and
origin, stage and tumor subtypes. The NHERF1 protein is an adapter determine the formation of different macromolecular complexes. Pre­
molecule that participates in maintaining the structure of polarized vious work described the nuclear exclusion of NHERF1 after deletion of
epithelia. Due to its apical location, it is a marker of normal epithelial the PDZ2 domain. On the contrary, the loss of the EB or PDZ1 domains
cell polarization. In transformed cells, NHERF1 is detected mainly in the caused the delocalization of NHERF1 in the nucleus (Voltz et al., 2007).
nucleus and cytoplasm where it participates in oncogenic pathways such Unfortunately, our study does not allow us to discriminate whether we
as those involved in epithelial-mesenchymal transformation or EMT are detecting the full-length protein or a processed form, as we use a
(Kreimann et al., 2007; Georgescu et al., 2008). polyclonal antibody targeting the recombinant human NHERF1 protein.
Here we describe the expression of NHERF1 in the nucleus of clear Future studies should determine if the form that is located in the nucleus
cancer cells of renal and ovarian carcinomas. Our study shows the is the full-length protein, an isoform, a cleavage product or a modified
expression pattern of NHERF1 in 21 cases of human RCC, where form due to phosphorylation that can modify NHERF1’s protein
NHERF1 is expressed with a membranous and nuclear pattern, while in interactions.
PRCC-type carcinomas the protein remains in the apical region as it had Biomarkers are used for the selection of treatment and the follow-up
been described in the ovarian tumors (Demacopulo et al., 2016). A of the evolution of the disease, acting in some cases at a predictive level
different staining was observed in clear OC cells where the staining was before the detection of tumor recurrence. In the case of NHERF1 there
mainly nuclear. If confirmed with a larger number of samples, the dif­ are some studies evaluating its potential as a biomarker. For example in
ferential expression of NHERF1 observed in RCC and OC could hepatocellular carcinoma (HCC), higher levels of NHERF1 mRNA were
contribute to the identification of clear cells of renal and ovarian origin detected in tumors and cell lines compared to their normal counterpart.
in distant organs. It also opens the possibility of studying the behavior of In both cases, NHERF1 was located in the cell nucleus co-localized with
NHERF1 in other types of cancers where the presence of clear cells can β-catenin (Centonze et al., 2018). In colorectal carcinogenesis,

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B.E. Lema et al. Acta Histochemica 123 (2021) 151717

Fig. 3. Nuclear expression of NHERF1 in clear

cells vs. apical expression at the border of
papillary structures, in RCC and OC. (a-b) PRCC
type I tumor showing the apical expression of
NHERF1 in tumor cells at the border of the
papillae, in contrast with the absence of label in
the basal area of the cells (arrow). (c) Nuclear
and membrane expression of NHERF1 in a
group of clear cells (arrow) as it was showed in
ccRCC tumors in picture 1. (d) NHERF1 staining
in a tumor slide from a bilateral cys­
tadenocarcinoma of the ovaries with mixed
histology, mostly serous, with clear cell foci
(arrow). The tumor is composed by an atypical
epithelial proliferation of cells with marked
anaplasia and nuclear atypia and numerous
figures of which form well differentiated
papillary structures (e), with apical expression
of NHERF1 as it was previously described
(Demacopulo et al., 2016). (f) The picture
shows a linear group of clear cells with nuclear
labeling of NHERF1 to the staining in RCC tu­
mors in picture (c). Pictures magnification: 20x
(a, d) and 1000x (b, c-e, f).

up-regulation of NHERF1 was associated with the transition from

normal colonic mucosa to carcinoma, and nuclear expression of NHERF1
was correlated with a more aggressive phenotype. A significant associ­
ation was also found between NHERF1 levels, disease progression and
the appearance of liver metastases (Mangia et al., 2012; Leiphrakpam
et al., 2020). In patients with breast cancer, long-term follow-up of a
clinical cohort demonstrated the prognostic value of nuclear expression
of NHERF1 (nNHERF1). Patients with positive expression of nNHERF1
showed greater disease-free survival (DFS) compared to patients with
negative nuclear expression, with no differences in OS between the two
groups (Paradiso et al., 2013).
Although these results, together with the findings reported in
different types of cancer, show the usefulness of NHERF1 as a biomarker,
its specific application cannot be generalized. There is a general
consensus towards a delocalization from the apical region towards the
cytoplasm and the nucleus during cell transformation, but the function
Fig. 4. Semiquantitative analysis of the immunohistochemical reaction of of NHERF1 in each compartment should be studied according to the type
NHERF1 in clear cells (nucleus and membrane) in RCC and OC tumor cells. The of cancer. It would be important in the future to achieve a consensus
results are represented as the mean (± SD) of the NHERF1 staining intensities (n between the studies carried out in different laboratories so that NHERF1
= 3 cases; 30-50 cells / sample) measured as described in material and
expression criteria could be incorporated into clinical practice for the
methods. A similar expression of NHERF1 was quantified in the nucleus and
benefit of patients.
membrane of RCC tumors. In contrast, a higher intensity of nuclear NHERF1
was detected in clear cells of the OC tumors compared to the membrane la­
beling (p ≤ 0.01). Furthermore, the amounts of NHERF1 in the nucleus of OC 5. Conclusion
clear cells were significantly higher than the measured in RCC tumors (p
≤ 0.05). In RRC, current risk stratification methods are largely based on

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Kurman, R.J., Carcangiu, M.L., Herrington, C.S., Young, R.H., 2014. WHO Classification
Orrea, and to the tumor bank of the Institute of Oncology "Angel H. of Tumours of Female Reproductive Organs, 4th ed. WHO Press, 1211 Geneva 27,
Roffo", University of Buenos Aires, Av. San Martín 5481, (1417), Buenos Switzerland.
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