(American Journal of Pathology. 2001;159:51-55.)
© 2001 American Society for Investigative Pathology
Glial Implants in Gliomatosis Peritonei Arise from Normal Tissue, Not from the Associated Teratoma
Amy W. Ferguson*,
Hidetaka Katabuchi
,
Brigitte M. Ronnett
and
Kathleen R. Cho*
From the Department of Pathology,*
University of
Michigan Hospital, Ann Arbor, Michigan; the Department of Obstetrics
and Gynecology,
Kumamoto University School of
Medicine, Kumamoto, Japan; and the Department of
Pathology,
The Johns Hopkins Hospital,
Baltimore, Maryland
 |
Abstract
|
|---|
Metaplasia of subcoelomic mesenchyme has been implicated,
but not proven, in the pathogenesis of common gynecological
diseases such as endometriosis and rarer entities such as
leiomyomatosis peritonealis disseminata and gliomatosis peritonei (GP).
GP is associated with ovarian teratomas and is characterized by
numerous peritoneal and omental implants composed of glial tissue. Two
theories to explain the origin of GP have been proposed. In
one, glial implants arise from the teratoma, whereas in
the other, pluripotent Müllerian stem cells in the
peritoneum or subjacent mesenchyme undergo glial metaplasia. To address
the origin of GP, we exploited a unique characteristic of many
ovarian teratomas: they often contain a duplicated set of maternal
chromosomes and are thus homozygous at polymorphic microsatellite (MS)
loci. In contrast, DNA from matched normal or metaplastic
tissue (containing genetic material of both maternal and paternal
origin) is expected to show heterozygosity at many of these same MS
loci. DNA samples extracted from paraffin-embedded normal
tissue, ovarian teratoma and three individual laser-dissected
glial implants were studied in two cases of GP. In one case,
all three implants and normal tissue showed heterozygosity at each of
three MS loci on different chromosomes, whereas the teratoma
showed homozygosity at the same MS loci. Similar results were observed
in the second case. Our findings indicate that glial implants in GP
often arise from cells within the peritoneum, presumably
pluripotent Müllerian stem cells, and not from the
associated ovarian teratoma. This finding has important implications
for more common gynecological entities with debatable
pathogenesis, such as endometriosis, by definitively
demonstrating the metaplastic potential of stem cells within the
peritoneal cavity.
|
Introduction
|
|---|
Gliomatosis peritonei (GP),
characterized by numerous peritoneal and omental implants composed of
glial tissue, is a rare benign condition associated with both mature
and immature teratomas of the ovary.1-8
GP has also been
reported in association with a gastric teratoma in a male
infant9
and an immature endometrial
teratoma.10
Two major theories regarding the pathogenesis
of GP have been proposed. The first suggests that glial foci arise from
the primary teratoma either through rupture of the capsule with
subsequent implantation of tissue within the peritoneum, or via
angiolymphatic spread as in carcinoma metastasis.7
The
second suggests that glial foci arise independently from pluripotent
Müllerian stem cells in response to favorable intraperitoneal
conditions, as a so-called "field effect."11
To address whether glial implants are genetically related to the
associated ovarian teratoma or whether they arise independently, we
exploited the unique genetic make-up of many ovarian teratomas.
Approximately 65% of teratomas are derived from a single germ cell
after the first meiotic division with subsequent failure of meiosis 2
or endoreduplication of a haploid ovum.12
Teratomas
arising through these mechanisms show homozygosity at most, if not all,
polymorphic microsatellite (MS) loci.13,14
In this study,
we used MS loci demonstrating a heterozygous pattern in normal tissue
and a homozygous pattern in the ovarian teratoma from the same patient
to determine the origin of glial implants in GP. We presumed that if
the glial implants showed a homozygous pattern similar to the teratoma,
they were most likely related to the teratoma and arose via capsular
rupture or angiolymphatic dissemination. If they demonstrated a
heterozygous pattern, similar to normal tissue, they most likely arose
via metaplasia of normal cells within the peritoneum.
|
Materials and Methods
|
|---|
Case Selection
Five cases of GP associated with ovarian teratomas were retrieved
from the pathology archives of The Johns Hopkins Hospital, Baltimore,
MD (two cases), The University of Michigan Hospital, Ann Arbor, MI (one
case), and the Kumamoto University Hospital, Kumamoto, Japan (two
cases). Hematoxylin and eosin (H&E)-stained tissue sections were
reviewed for confirmation of diagnosis. Three cases were excluded from
further analysis, one because DNA from the teratoma and matched normal
tissue demonstrated identical alleles at all polymorphic MS loci
tested, and two because amplifiable DNA could not be extracted from the
available tissues. The two cases from Baltimore proved suitable for
additional study. In one case GP was associated with a mature teratoma
and in the second case, GP was associated with an immature teratoma
(grade 2). This neoplasm contained a minor component of immature
neuroepithelial tissue (2 of 19 slides). Four paraffin blocks were
selected from each of these two cases: one containing ovarian teratoma,
one containing nonneoplastic tissue (cervix or fallopian tube), and two
containing either omentum or peritoneal surface with numerous (>50)
glial implants.
DNA Extraction
Neoplastic (teratoma) and nonneoplastic (cervix or fallopian tube)
tissue was isolated with a razor blade using an H&E-stained section as
a dissection guide. DNA was extracted as previously described with
slight modifications.15
Briefly, 4-µm-thick wax sections
were incubated in 300 µl of lysis buffer (200 µg/ml proteinase K,
50 mmol/L Tris, pH 8.3, 0.5% Tween-20, 100 µg/ml glycogen) for 48
hours at 60°C with the addition of 200 µg/ml proteinase K after the
first 24 hours. The lysate was then extracted twice with
phenol/chloroform (1:1) and allowed to incubate for 2 minutes after the
addition of 36 µl of ethidium bromide (5 mg/ml). Phenol/chloroform
(X1) and chloroform (X1) extractions were performed after the addition
of one-half volume of 7.5 mol/L ammonium acetate; followed by the
addition of one drop of Chelex-100 (Bio-Rad, Bethesda, MD) bead slurry.
After a 5-minute incubation, DNA was ethanol-precipitated, washed with
70% ethanol, and resuspended in 10 to 50 µl of water. The same
technique was used to extract DNA from glial implants stained and
microdissected per the manufacturers’ protocol using an Arcturus
laser-capture microscope (Mountain View, CA).
Genetic Analysis
DNA samples were subjected to polymerase chain reaction (PCR)
amplification of multiple MS markers including D5S592 (5pter-5qter),
D16S2624 (16q), and D17S1987 (17q). Primer pairs and annealing
temperatures are shown in Table 1
. Each
10-µl PCR reaction contained 1x PCR buffer (10 mmol/L Tris-HCl, pH
9.2, 1.5 mmol/L MgCl2, 75 mmol/L KCl), 200
µmol/L dATP, 200 µmol/L dGTP, 200 µmol/L dTTP, 25 µmol/L dCTP,
2 µCi (3000 Ci/mmol) dCTP-32P, 0.1 µmol/L of
each primer, and 1.0 U Taq polymerase. Input DNA varied from
1 to 4.8 µl as DNA obtained from the laser-captured glial implants
required a higher input volume than DNA obtained from normal tissue and
teratoma. After an initial denaturation step of 95°C for 5 minutes,
DNA templates were amplified using 40 cycles of 95°C for 30 seconds,
55°C (D5S592, D16S2624), or 57°C (D17S1987) for 1 minute and 72°C
for 1 minute, followed by a final extension step at 72°C for 7
minutes. PCR reactions were diluted with 5 to 10 µl stop buffer (20
mmol/L Tris-HCl, 10 mmol/L ethylenediaminetetraacetic acid, 0.1 mmol/L
dCTP) and PCR products were resolved by electrophoresis on a 6%
acrylamide-8 mol/L urea gel that was dried and subjected to
autoradiography.
|
Results
|
|---|
In case 1, the teratoma was classified as a benign cystic teratoma
that contained mature tissue derived from all three germ layers (Figure 1A)
. The teratoma from case 2 displayed a
similar histological picture but also included a minor component of
immature neural tissue, leading to its classification as a grade 2
immature teratoma per the criteria of Thurlbeck and
Scully.16
All glial implants from both cases were composed
of mature glial tissue (Figure 1B)
. DNA was extracted from the
teratoma, nonteratomatous normal tissue and three individual
laser-captured microdissected glial implants for each of the two cases.
A representative histological section stained with a modified H&E stain
demonstrates a glial implant before microdissection (Figure 1C)
, the
same implant after microdissection (Figure 1D)
and the cap onto which
the tissue was captured (Figure 1E)
.
View larger version (90K):
[in this window]
[in a new window]
|
Figure 1. A: H&E-stained section of a mature teratoma containing
keratinizing squamous epithelium and hair follicle
(bottom left),
mature glial tissue
(center), and
cerebellar tissue (top
right) (original
magnification, x40). B: H&E-stained
tissue section of omentum. Five individual foci of mature glial tissue
are pink and round to ovoid in shape (original
magnification, x100). C: Modified
H&E-stained section showing a glial implant
(left) and
adjacent blood vessels
(right) before
laser-capture microdissection (original
magnification, x200). D: Same
section as shown in C after laser-capture microdissection
(original magnification,
x200). E: Captured glial implant.
The dark bubbles overlying the implant are an artifact of the capture
membrane (original magnification,
x200).
|
|
Polymorphic MS loci on three chromosomes (D5S592, D16S2624, and
D17S1987) were amplified using these DNA samples as PCR templates. In
both cases, DNA from normal tissue and individually microdissected
glial implants demonstrated a heterozygous pattern at the
polymorphic loci evaluated, whereas DNA from the matched teratoma
showed a homozygous pattern at the same loci. Representative data from
both cases are shown in Figure 2
.
View larger version (41K):
[in this window]
[in a new window]
|
Figure 2. Representative data from analysis of polymorphic MS loci D5S592,
D16S2624, and D17S1987 in two cases of GP. At all loci, nonteratomatous
normal tissue (N)
demonstrates a heterozygous pattern; teratoma
(T) demonstrates a
homozygous pattern and three individual glial implants
(I-1, I-2, and I-3)
demonstrate a heterozygous pattern, similar to normal.
|
|
Notably, although some of the PCR reaction products from implant DNA
favor the allele present in the teratoma DNA, others show either allele
amplification similar to that seen in the matched normal tissue, or
even slight bias toward the allele absent in the teratoma DNA.
Preferential amplification of one MS allele over the other is a
well-recognized problem when amplifying very small quantities of
template DNA, as would be expected from DNA extracted from small glial
implants in GP.17-19
In some cases preferential allele
amplification can be so severe as to cause total allele drop out. The
problem is further compounded by the availability for this study of
only formalin-fixed, paraffin-embedded tissue, which is known to yield
DNA more prone to PCR artifacts than DNA from frozen
tissue.20
Recognizing that preferential allele
amplification might be encountered in PCR reactions using very small
quantities of template DNA, we tailored our experimental approach to
minimize the likelihood of being misled by PCR artifacts. First, we
used meticulous laser capture microdissection rather than manual
microdissection to minimize contamination of the implant samples by
nonglial cells. We estimate that nonglial cells comprised <5% of the
cells harvested for DNA extraction. Second, for several implants
yielding sufficient quantities of DNA, results were verified with at
least one and often two additional independent PCR reactions at a given
locus. Third, we analyzed multiple implants from each case with as many
markers as the small quantities of DNA extracted from individual
implants would allow. Our findings strongly support the interpretation
that the glial implants are derived from cells in the peritoneum and
not from teratoma tissue contaminated by nonneoplastic cells.
|
Discussion
|
|---|
The differentiation capability of pluripotent Müllerian stem
cells and their role in the pathogenesis of various gynecological
diseases has been debated for decades. These cells have been implicated
in the development of endometriotic foci, noninvasive implants of
papillary serous tumors of low malignant potential, neoplastic foci in
women with multifocal primary peritoneal carcinoma, smooth muscle foci
in disseminated peritoneal leiomyomatosis, and glial implants in
GP.11
The unusual chromosomal composition of ovarian
teratomas allowed us to use molecular tools to definitively evaluate
the differentiation potential of normal cells within the peritoneum and
subjacent mesenchyme in GP.
The origin of glial implants in GP and the factors responsible for the
development of GP in association with selected teratomas has been the
subject of much debate. One theory regarding the origin of glial tissue
suggests that it is genetically related to the associated teratoma,
being either extruded from the primary neoplasm through capsular
defects or disseminated via angiolymphatic channels. Capsular defects
have been described in resected teratomas and in some instances,
teratomatous material has been observed protruding through these
defects.3-7
In addition, capsular rupture at the time of
primary surgery has been described in cases where GP was only apparent
at the time of second-look laparotomy.1
In support of
lymphatic dissemination, mature glial tissue has been found in
mesenteric, para-aortic, and retroperitoneal lymph nodes in association
with immature teratomas in the presence or absence of
GP.3,7,8,21,22
Moreover, cystic peritoneal masses composed
of tissue from all three germ layers (including immature neural
elements) have been described in addition to small glial foci in some
cases of GP, suggesting that the former are true
metastases.2,3,6,7
It remains unclear whether these
metastases arise through the same or different mechanism as the foci of
mature glial tissue observed in typical GP.
An alternate theory, supported by our study, suggests that glial foci
are genetically unrelated to the associated teratoma and arise from
normal cells in response to favorable environmental conditions. The
most likely candidate normal cells are pluripotent Müllerian
stem cells on the peritoneal surface or in the subcoelomic
mesenchyme. This hypothesis is based on the observation that many
gynecological diseases seem to have a multifocal intraperitoneal origin
and is further supported by reports of glial implants admixed with
endometrial tissue.2,3,4,23
Endometrial tissue is
Müllerian in origin and is uncommon in teratomas. Given that we
analyzed only two cases of GP and a limited number of implants, it is
possible that some glial foci result from true implantation, whereas
others arise via metaplasia of normal stem cells within the peritoneum.
Why some intraperitoneal stem cells (or tissues) differentiate along a
glial pathway whereas others do not requires some speculation. The
remarkable ability of stem cells derived from various organs to
differentiate along divergent pathways has been the subject of multiple
recent articles, including reports of studies demonstrating that bone
marrow-derived stem cells can undergo glial
differentiation.24,25
It has been suggested that a stem
cell’s microenvironment can induce a specific differentiation pathway
or pathways, and it is possible that some teratomas with an abundant
glial component secrete factors that induce glial differentiation in
the peritoneum. Protein secretion by teratomas is a well-recognized
phenomenon. For example, teratomas with a prominent thyroid component,
such as struma ovarii and struma-carcinoid, have been shown to secrete
thyroid hormone and calcitonin, respectively.26,27
Notably, murine astrocyte cells and teratocarcinoma cell lines have
been shown to secrete ß-nerve growth factor in
vitro.28-30
Moreover, GP has been described in
children without teratomas who have had ventriculoperitoneal shunts
placed early in infancy.31
Neural growth factors normally
present in cerebrospinal fluid may enter the peritoneum through the
shunt and induce glial differentiation in the same manner.
The results presented in this study demonstrate the ability of
intraperitoneal cells, presumably pluripotent Müllerian stem
cells, to undergo glial differentiation. This finding provides
important insight into the pathogenesis of gynecological diseases
characterized by intraperitoneal multifocality. Identifying the factors
responsible for the intraperitoneal field effect in GP will be an
important area of future investigation and may further advance our
understanding of more common gynecological diseases such as
endometriosis.
|
Acknowledgements
|
|---|
We thank Professor Hitoshi Okamura of the Kumamoto University
School of Medicine for generously providing two cases of ovarian
teratomas associated with gliomatosis peritonei; and the Laser Capture
Microdissection Core, supported by the University of Michigan Cancer
Center, for excellent technical assistance.
|
Footnotes
|
|---|
Address reprint requests to Kathleen R. Cho, M.D., Department of Pathology, University of Michigan, 4301 MSRB-III, Box 0638, 1150 West Medical Center Dr., Ann Arbor, MI 48109. E-mail: kathcho{at}umich.edu
Accepted for publication March 29, 2001.
|
References
|
|---|
-
Gocht A, Lohler J, Scheidel P, Stegner H-E, Saeger W: Gliomatosis peritonei combined with mature ovarian teratoma: immunohistochemical observations. Path Res Pract 1995, 191:1029-1035
-
Calder CJ, Light AM, Rollason TP: Immature ovarian teratoma with mature peritoneal metastatic deposits showing glial, epithelial and endometrioid differentiation: a case report and review of the literature. Int J Gynecol Path 1994, 13:279-282
-
Harms D, Janig U, Gobel U: Gliomatosis peritonei in childhood and adolescence: clinicopathological study of 13 cases including immunohistochemical findings. Path Res Pract 1989, 184:422-430
-
Bassler R, Theele CH, Labach H: Nodular and tumorlike gliomatosis peritonei with endometriosis caused by a mature ovarian teratoma. Path Res Pract 1982, 175:392-403
-
Truong L, Juro S, III, McGavran HH: Gliomatosis peritonei, report of two cases and review of the literature. Am J Surg Path 1982, 6:433-449
-
Neilsen SNJ, Scheithauer BW, Gaffey TA: Gliomatosis peritonei. Cancer 1985, 56:2499-2503[Medline]
-
Robboy SJ, Scully RE: Ovarian teratoma with glial implants on the peritoneum: an analysis of 12 cases. Hum Pathol 1970, 1:643-653[Medline]
-
Norris HJ, Zirkin HJ, Benson WL: Immature (malignant) teratoma of the ovary: a clinical and pathologic study of 58 cases. Cancer 1976, 37:2359-2372[Medline]
-
Coulson WF: Peritoneal gliomatosis from a gastric teratoma. Am J Clin Pathol 1990, 94:87-89[Medline]
-
Ansah-Boateng Y, Wells M, Poole DR: Coexistent immature teratoma of the uterus and endometrial adenocarcinoma complicated by gliomatosis peritonei. Gynecol Oncol 1985, 21:106-110[Medline]
-
Dallenbach-Hellweg G: Critical commentary to "Gliomatosis peritonei combined with mature ovarian teratoma." Pathol Res Pract 1995, 191:1037[Medline]
-
Surti U, Hoffner L, Chakravarti A, Ferrell RE: Genetics and biology of human ovarian teratomas. I. Cytogenetic analysis and mechanism of origin. Am J Hum Genet 1990, 47:635-643[Medline]
-
Vortmeyer AO, Devouassoux-Shisheboran M, Li G, Mohr V, Tavassoli F, Zhuang Z: Microdissection-based analysis of mature ovarian teratoma. Am J Pathol 1999, 154:987-991[Abstract/Free Full Text]
-
Devoussoux-Shisheboran M, Vortmeyer AO, Silver SA, Zhuang Z, Tavassoli FA: Teratomatous genotype detected in malignancies of a non-germ cell phenotype. Lab Invest 2000, 80:81-86[Medline]
-
Mutter GL, Chaponot ML, Fletcher JA: A polymerase chain reaction assay for non-random X-chromosome inactivation identifies monoclonal endometrial cancers and precancers. Am J Pathol 1995, 146:501-508[Abstract]
-
Thurlbeck WM, Scully RE: Solid teratoma of the ovary: a clinicopathologic analysis of 9 cases. Cancer 1960, 1:804-811
-
Kimpton CP, Oldroyd NJ, Watson SK, Frazier RR, Johnson PE, Millican ES, Urquhart A, Sparkes BL, Gill P: Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification. Electrophoresis 1996, 17:1283-1293[Medline]
-
Garvin AM, Holzgreve W, Hahn S: Highly accurate analysis of heterozygous loci by single cell PCR. Nucleic Acids Res 1998, 26:3468-3472[Abstract/Free Full Text]
-
Bluteau O, Legoix P, Laurent-Puig P, Zucman-Rossi J: PCR-based genotyping can generate artifacts in LOH analyses. Biotechniques 1999, 27:1100-1102[Medline]
-
Sieben NL, ter Haar NT, Cornelisse CJ, Fleuren GJ, Cleton-Jansen AM: PCR artifacts in LOH and MSI analysis of microdissected tumor cells. Hum Pathol 2000, 31:1414-1419[Medline]
-
El Shafie M, Furay RW, Chablani LV: Ovarian teratoma with peritoneal and lymph node metastases of mature glial tissue: a benign condition. J Surg Oncol 1984, 27:18-22[Medline]
-
Perrone T, Steiner M, Dehner LP: Nodal gliomatosis and
-fetoprotein production: two unusual facets of grade I ovarian teratoma. Arch Pathol Lab Med 1986, 110:975-977[Medline]
-
Dworak O, Knopfle G, Varchmin-Schultheiss K, Meyer G: Gliomatosis peritonei with endometriosis externa. Gynecol Oncol 1988, 29:263-266[Medline]
-
Vogel G: Stem cells: new excitement, persistent questions. Science 2000, 290:1672-1674[Free Full Text]
-
Kopen GC, Prockop DJ, Phinney DG: Marrow stromal cells migrate throughout the forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999, 96:10711-10716[Abstract/Free Full Text]
-
Simkin PH, Ramirez LA, Zweizig SL, Afonso SA, Fraire AE, Khan A, Dunn AD, Dunn JT, Braverman LE: Monomorphic teratoma of the ovary: a rare cause of triiodothyronine toxicosis. Thyroid 1999, 9:949-954[Medline]
-
Blaustein A: Calcitonin secreting struma-carcinoid tumor of the ovary. Hum Pathol 1979, 10:222-228[Medline]
-
Dicou E, Houlgatte R, Brachet P: Synthesis and secretion of ß-nerve growth factor by mouse teratocarcinoma cell lines. Exp Cell Res 1986, 167:287-294[Medline]
-
Furukawa S, Furukawa Y, Satoyoshi E, Hayashi K: Synthesis and secretion of nerve growth factor by mouse astrocytes in culture. Biochem Biophys Res Commun 1986, 136:57-63[Medline]
-
Tarris RH, Weichsel ME, Jr, Fisher DA: Synthesis and secretion of nerve growth stimulating factor by neonatal mouse astrocyte cells in vitro. Pediatr Res 1986, 20:367-372[Medline]
-
Hill DA, Dehner LP, White FV, Langer JC: Gliomatosis peritonei as a complication of a ventriculoperitoneal shunt: a case report and review of the literature. J Pediatr Surg 2000, 35:497-499[Medline]
This article has been cited by other articles:
|
|
|
|
 
N. Gurfield and K. Benirschke
Equine Placental Teratoma
Vet. Pathol.,
September 1, 2003;
40(5):
586 - 588.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
