(American Journal of Pathology. 1999;155:1047-1050.)
© 1999 American Society for Investigative Pathology
Immunohistochemical Analysis of Uroplakins, Urothelial Specific Proteins, in Ovarian Brenner Tumors, Normal Tissues, and Benign and Neoplastic Lesions of the Female Genital Tract
Kumiko Ogawa*
,
Sonny L. Johansson* and
Samuel M. Cohen*
From the Department of Pathology and Microbiology and the Eppley
Institute,*
University of Nebraska Medical Center, Omaha,
Nebraska; and the First Department of Pathology,
Nagoya City University Medical School, Nagoya, Japan
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Abstract
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Uroplakins are the characteristic integral membrane proteins in
terminally differentiated, superficial urothelial asymmetric
unit membrane. Brenner tumors of the ovary and Walthard cell nests of
Fallopian tubes have been considered to represent urothelial
differentiation in the female genital tract, but no definitive
differentiation marker has been demonstrated supporting such a
conclusion. An immunohistochemical analysis was performed to assess the
expression of uroplakins in these lesions as well as in various benign
and neoplastic lesions and normal tissues of the female genital tract.
Focal expression of uroplakins was observed on the luminal surface of
ovarian Brenner tumor cells forming microcysts in all 5 cases examined.
In contrast, uroplakins were slightly expressed in only 1 of 12
cases of Walthard cell nests, even in the presence of microcyst
formation. Uroplakins were not expressed in other benign or malignant
lesions or normal tissues of the female genital tract. These results
support the hypothesis that the Brenner tumor and possibly Walthard
cell nests represent urothelial (transitional cell)
differentiation.
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Introduction
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Brenner tumors comprise 1 to 2% of ovarian neoplasms; the vast
majority are benign and measure less than 2 cm. Microscopically, they
are composed of sharply demarcated, solid to partly cystic epithelial
cell nests surrounded by a stroma composed of tightly packed,
spindle-shaped cells. The epithelial cells are polygonal with pale,
eosinophilic cytoplasm and oval nuclei that display distinct nucleoli
and longitudinal grooving, the so-called coffee-bean appearance.
Walthard cell nests are most commonly found on the peritoneal surface
of the Fallopian tubes and also in the mesovarium and occasionally in
the ovarian hilus. Because of the histological similarity of the
epithelium of Brenner tumors and Walthard cell
nests to the urothelium of the lower
urinary tract, it has been suggested that Brenner tumors and Walthard
cell nests represent urothelial (transitional cell) differentiation.
This has further been supported by electron microscopic analysis
showing similarities between them, including deeply indented nuclei,
sparse granular endoplasmic reticulum, abundant free ribosomes,
presence of glycogen particles, secondary lysosomes, moderate numbers
of desmosomes, markedly tortuous, interdigitating cell membranes, and
large complex intercellular spaces.1-4
Both Brenner
tumors and normal urothelium immunohistochemically stain for
carcinoembryonic antigen5-7
and involucrin.8
However, none of these features are restricted only to urothelium.
The defining characteristic feature of terminal urothelial
differentiation is the presence of the asymmetric unit membrane on the
luminal surface and in intracytoplasmic fusiform
vesicles.9
These structures have not been observed in
Brenner tumors or in Walthard nests by ultrastructural
examination.1-4
The asymmetric unit membrane10,11
is characteristic of
urothelium and is so named because of its highly unusual
ultrastructure. The luminal leaflet of the plasmalemma is almost twice
as thick as the cytoplasmic leaflet (9 versus 4 nm). In the
superficial cells of the urothelium, discoid cytoplasmic vesicles lined
with asymmetric unit membrane are formed in the Golgi
complex.11,12
The luminal plasma membrane of the
superficial cells consists of hexagonal subunits which form
scallop-shaped plaques typically 0.3 to 0.5 nm in diameter when
viewed by transmission and scanning electron microscopy. These plaques
contain four major integral membrane proteins called uroplakins Ia, Ib,
II, and III, which form 16 nm "twisted-ribbon"-shaped particles
arranged in a well-ordered hexagonal lattice with p6
symmetry.13
The existence of asymmetric unit membrane
plaques and their protein constituents, uroplakins, is
urothelium-specific and differentiation-dependent. Possible functions
of the asymmetric unit membrane include serving as a permeability
barrier, as a means of stabilizing the apical bladder surface during
expansion and contraction, and as a molecular device to change the
surface area of the plasma membrane.
The expression patterns of uroplakins changed in carcinogen-treated rat
bladder epithelium, even before tumor appearance, during the
hyperplastic phase of the process.14
In addition to
superficial cells, intermediate cells of
N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced
simple hyperplastic urothelium occasionally stained positive for
uroplakins as well as occasional cells in papillomas and carcinomas.
Furthermore, staining for uroplakins was occasionally decreased to
absent in the luminal-lining cells of FANFT-induced papillary
and nodular hyperplasia and of uracil-induced papillomas. Cells lining
the bladder lumen in these lesions generally are not terminally
differentiated urothelial superficial cells, but rather resemble
intermediate and basal cells. The changes in uroplakin expression in
experimental bladder carcinogenesis demonstrate the altered
differentiation associated with the process.
In rat and human transitional cell carcinomas, the number of cells that
express uroplakins is markedly reduced or absent.14,15
However, expression is still present in human metastatic transitional
carcinoma.15
The aim of the present investigation was to demonstrate evidence of
urothelial differentiation in Brenner tumors and Walthard nests by
immunohistochemical analysis for uroplakins. Other benign or malignant
lesions as well as normal tissues of the female genital tract were also
studied by the same methods.
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Materials and Methods
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The material included five typical Brenner tumors,16
twelve Walthard cell nests, and two of each of the following lesions:
serous cyst adenoma, serous cyst adenocarcinoma, mucinous cyst adenoma,
mucinous cyst adenocarcinoma, and granulosa cell tumors of the ovary,
hyperplastic endometrium, endometrial adenocarcinoma, squamous cell
carcinoma and adenocarcinoma of the cervix, and mesothelial inclusion
cysts of the Fallopian tube. Normal urinary bladder, cervical,
endometrial, myometrial, placental, Fallopian tube, and ovarian tissues
were also examined. Specimens came from patients treated between 1993
and 1995 at the University of Nebraska Medical Center or between 1997
and 1998 at the Nagoya Midori Municipal Hospital.
These surgically excised tissues were fixed in 4% phosphate-buffered
formaldehyde and processed routinely for paraffin embedding. The
avidin-biotin-peroxidase complex method17
was used for
immunohistochemical analysis of uroplakin. After deparaffinization,
unstained slides were treated with microwave heating in antigen
retrieval solution (BioGenex; San Ramon, CA) for 10 minutes.
Anti-asymmetric unit membrane (AUM) antibody that reacts with all four
uroplakins (generously provided by Dr. Tung-Tien Sun) was made against
highly purified bovine AUM18
and applied at a 1:104
dilution. Anti-rabbit IgG secondary antibody and Vectastain ABC kit was
purchased from Vector Laboratories, Inc. (Burlingame, CA).
Diaminobenzidine was used as a substrate and then counter stained with
hematoxylin.
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Results
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Figure 1
shows typical uroplakin
staining of normal human urinary bladder. The luminal surface of the
superficial cells has distinct and consistent expression of uroplakins.
In general, the cytoplasm did not show uroplakin expression.
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Figure 1. Immunohistochemical stain for uroplakins in normal human urinary
bladder. Note the positive continuous expression on the luminal surface
of the superficial cells.
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All five Brenner tumors also showed positive staining for uroplakins.
However, it was mostly present focally on the luminal surface of the
small cysts (Figure 2a)
present in four
of the tumors. Tumor cells lining the lumens had positive staining.
There was no apparent histological difference between
uroplakin-positive microcystic areas compared to those that were
negative. Neither was there a relationship with positivity and
thickness of the tumor cell layers around the cystic areas. One of the
Brenner tumors had diffusely positive staining on the cell membrane as
well as lumens (Figure 2
b). In addition, the cytoplasm of some
tumor cells stained weakly positive.
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Figure 2. Immunohistochemical stain for uroplakins in a Brenner tumor. a:
Expression is observed at the luminal surface of the microcyst. The
staining is focally discontinuous. b: Diffuse expression of
uroplakins on the cell membrane is seen in one case.
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Expression of uroplakins was not observed in 11 cases of Walthard nests
despite the histological appearance of multiple cell layers around
microcysts similar to transitional cells and similar to the microcysts
seen in the Brenner tumors. Only one case of Walthard nest showed
slight uroplakin expression on the luminal surface of the cysts (Figure 3,a and b)
.
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Figure 3. Immunohistochemical stain for uroplakins in a Walthard nest showing
only faint luminal uroplakin expression. a: Low power field of
Walthard cell nests. Arrowhead shows positive lumen. b:
High power field of uroplakin-positive lumen.
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Uroplakins were not expressed in any of the normal tissues or any of
the other benign or malignant lesions examined including the
mesothelial inclusion cyst of the Fallopian tube. The squamous
epithelium of the cervix was likewise negative for uroplakin staining.
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Discussion
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The luminal plasma membrane of cells surrounding microcysts in
Brenner tumors focally expressed uroplakins, and similar distribution
of expression was observed in one Walthard nest. In contrast, staining
for uroplakins was negative in normal tissue from the cervix,
endometrium, myometrium, tubes, ovaries, and placenta sites. Likewise,
no staining for uroplakins was detected in ovarian serous and mucinous
cystadenomas, ovarian serous and mucinous adenocarcinomas, ovarian
granulosa cell tumor, hyperplastic endometrium, endometrial
adenocarcinoma, cervical squamous cell and adenocarcinoma or
mesothelial inclusion cysts of the Fallopian tube.
Based on light microscopic and ultrastructural studies, Brenner tumors
and Walthard cell nests have been considered to show urothelial
differentiation, but asymmetric unit membranes have not been observed.
An electron microscopic study by Roth3
showed that the
Walthard nests had a rather simple fine structure resembling the deeper
layers of the urinary bladder epithelium, as evidenced by prominent
intercellular spaces containing a profusion of cytoplasmic processes.
However, oval or fusiform vesicles were not observed nor were luminal
plaques. The Brenner tumors and bladder urothelium share similar
immunohistochemical characteristics. Thus, both express chromogranin,
neuron specific enolase, serotonin, carcinoembryonic antigen,
epithelial membrane antigen and keratin,7
but Brenner
tumors and Walthard nests tended to be cytokeratin-7 positive whereas
bladder urothelium was cytokeratin-20 positive.19
Furthermore, involucrin, a protein precursor present in human stratum
corneum, which has been found only in squamous epithelium and
urothelium, was also observed in Brenner tumor and Walthard
nests.8
However, these ultrastructural and
immunohistochemical changes are not specific to urothelium.
The presence of uroplakins, which are urothelial-specific
differentiation proteins, as demonstrated in the present study,
provides the first definitive evidence that the cells of the Brenner
tumor show urothelial differentiation, not just morphologically
resembling urothelial cells. The staining appeared specific for the
Brenner tumors, as we did not observe it in any of the other normal
tissues or benign and malignant lesions of the female genital tract,
including several types of cystic lesions. The focal and variable
expression of uroplakins in Brenner tumors suggests that terminal
differentiation does not occur in all cells. The urothelial
differentiation may be a result of urothelial metaplasia of ovarian
celomic epithelium or mesothelium.7
Because of the
resemblance of the Walthard nests to urothelial cells, we expected
positive staining for uroplakins in these cells, also. However, we
observed it in only one case, possibly because these cells resemble
more the deeper layers of the urothelium. The uroplakins are terminally
differentiated proteins and are mainly expressed in the superficial
cells. It has been suggested that Walthard nests are precursor lesions
for Brenner tumors.7
This seems unlikely, since the
majority of Walthard nests are identified in extra-ovarian tissue and
the cells seldom express uroplakins.
The histogenesis of Brenner tumors has been suggested to be most likely
from differentiation of pluripotential celomic
epithelium,5,20,21
occasionally occurring with features of
mucinous differentiation. However, similar tumors, with or without
mucinous differentiation, have been reported in ovarian
teratomas,20,21
suggesting another possible histogenesis.
It is likely that either of these pathways can occur as they are not
mutually exclusive. The present study, although demonstrating the
urothelial differentiation of Brenner tumors, does not resolve the
question of histogenesis.
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Acknowledgements
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We gratefully acknowledge the advice and constructive comments of
Dr. Eva Uzvolgyi (University of Nebraska Medical Center) and Dr.
Tomoyuki Shirai (Nagoya City University), and the assistance of
Michelle Moore with the preparation of this manuscript. We are
especially grateful to Dr. Tung-Tien Sun (New York University) for
providing the antibody used for the studies.
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Footnotes
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Address reprint requests to Samuel M. Cohen, Havlik-Wall Professor of Oncology, Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135. E-mail:scohen{at}unmc.edu
Supported in part by grants CA32513 and CA36727 from the National Cancer Institute and DK39753 and DK47529 from the National Institute of Diabetes and Digestive and Kidney Diseases.
Accepted for publication June 24, 1999.
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Abstract
Introduction
