(American Journal of Pathology. 1999;154:987-991.)
© 1999 American Society for Investigative Pathology
Microdissection-Based Analysis of Mature Ovarian Teratoma
Alexander O. Vortmeyer*
,
Mojgan Devouassoux-Shisheboran
,
Guang Li*
,
Victoria Mohr*
,
Fattaneh Tavassoli
and
Zhengping Zhuang*
From the Laboratory of Pathology,*
National Cancer
Institute, Bethesda, Maryland, and the Armed Forces Institute of
Pathology,
Washington, DC
 |
Abstract
|
|---|
The genotypic features of mature ovarian teratomas (MOTs) are
controversial. Early studies detected a homozygous genotype in MOTs
suggesting that these tumors are composed of germ cells that have
undergone meiosis I. Other studies, however, revealed a
heterozygous genotype in a substantial proportion of MOTs suggesting an
origin either from premeiotic germ cells or from a somatic cell line.
In view of the complex morphology of MOTs and to increase the
sensitivity of teratoma genotyping, we applied tissue
microdissection before genetic analysis of teratomatous tissue. This
approach allowed selective analysis of different heterotopic tissue
elements as well as the lymphoid tissues within MOTs the origin of
which is unknown. After DNA extraction, the tissue samples were
polymerase chain reaction amplified using a random panel of highly
informative genetic markers for different chromosomes to evaluate
heterozygosity versus homozygosity. In all seven cases that
were analyzed, heterotopic tissues consistently revealed a
homozygous genotype with several markers; in two cases,
heterozygosity was detected with a single marker, indicating a
meiotic recombination event. Lymphoid aggregates within MOTs were
heterozygous and derived from host tissue rather than from teratomatous
growth. However, well differentiated thymic tissue was
consistently homozygous, suggesting lymphoid differentiation
capability of MOTs. We conclude that potential pitfalls in genotyping
of teratomas including meiotic recombination and host cell
participation can be avoided by a microdissection-based approach in
combination with a panel of genetic markers.
|
Introduction
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Teratomas are tumors that are
composed of a variety of tissue elements derived from two or more of
the embryonic germ layers.1,2
Classic theories of the
origin of teratomas include incomplete twinning, neoplastic
proliferation of sequestered totipotent blastomeres or primordial germ
cells, derepression of totipotent genetic information in the nuclei of
somatic cells, and parthenogenetic development of germ
cells.1,2
Due to the frequent occurrence of teratomas in
the ovary, a germ cell origin was postulated decades ago. Utilizing
enzyme polymorphisms and chromosome banding studies, Linder and
co-workers demonstrated that teratomas are homozygous for chromosomal
polymorphisms whereas nonteratomatous host tissue is
heterozygous.3-6
These findings not only proved a
fundamentally different genetic composition of the teratomatous tissue
as compared with normal host tissue but also strongly suggested that
the teratomatous genotype was acquired secondary to meiotic cell
division. The data were subsequently confirmed by larger
studies7
and experimental mouse models8
that
continued to support a germ cell origin of ovarian teratomas.
Subsequent studies, however, failed to consistently detect a homozygous
genetic composition of teratomas.9-13
Instead,
heterozygous centromeric markers and other chromosomal heteromorphisms
were reported in a subset of tumors, raising the possibility of either
postmeiotic or premeiotic origin of these tumors.
Compared with other tumors, teratomas exhibit unique histological
features being composed of a variety of architecturally and
cytologically mature tissues rather than a proliferating pool of
neoplastic cells. Histologically, teratomas are composed of heterotopic
tissues, including tissues such as epidermis, central nervous system
tissue, or mature cartilage.1,2
However, teratomas also
contain nonspecific tissue types, eg, lymphoid tissue or fibrous
stroma. Whereas the teratomatous nature of specific heterotopic tissues
is evident and even mandatory for diagnosis, it is unknown to what
extent immunological cell elements within heterotopic parenchyma
represent either pluripotent differentiation capability of teratomatous
tissue or host cell reactivity. Better clarification of this question
may reveal the full differentiation potential of teratomatous growth
that may include not only heterotopic parenchymal tissue but also
lymphoid cells. As development of specific tissue is dependent on
specific signaling pathways, more selective analysis of the
histopathological features of teratomas may explain the enormous
intratumor and intertumor diversity of these neoplasms. In addition, we
may obtain closer insight into host-tumor interactions that are
represented by different cellular systems with fundamental genetic
diversity, yet full immunological compatibility.
Despite the ongoing controversy about the genotype of ovarian teratoma,
and our ignorance about reactivity versus neoplasticity of
various teratomatous elements, selective analysis of different
histological components has never been attempted. In the present study,
we performed selective genetic analysis of seven teratomas by
microdissection of a wide diversity of histological features.
|
Materials and Methods
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Patients and Tumors
Seven solitary mature ovarian teratomas were retrieved from the
files of the Armed Forces Institute of Pathology. The patients' ages
at surgery were between 10 and 20 years. All cases showed a variety of
areas with different histological differentiation (Table 1
and Figure 1
). From each case, between 8 and
18 different histological areas were selectively dissected as described
previously.14,15
The following tissues were selectively
dissected from the seven cases (Table 1)
: 1) areas of specific
histological differentiation, eg, respiratory epithelium and salivary
gland; contamination with cells that are not mandatory constituents of
the targeted area, eg, cells from other tissue types or blood-borne
cells, was avoided or minimized; 2) areas in which a few lymphocytes
were admixed with various tissue elements; and 3) pure lymphoid
aggregates that were observed in association with different types of
histological differentiation; the lymphoid aggregates consisted of
clusters of lymphoid cells that did not contain germinal centers.
Whenever feasible, tissues were removed in duplicate or triplicate.
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Figure 1. Representative histological areas from different mature ovarian
teratomas that were selectively procured and genetically analyzed; the
microdissected areas are surrounded by arrowheads.
a–c: Areas of specific histological differentiation:
a, sebaceous glands; b, cartilage; c, squamous
epithelium. d–f: Areas with lymphoid tissue: d,
lymphoid aggregate in gastrointestinal epithelium; e, lymphoid
aggregate associated with respiratory epithelium; f, thymic
tissue. g–i: Areas in which a few lymphocytes were admixed
with various tissue elements: g, lymphocytes in squamous
epithelium: h, lymphocytes associated with sebaceous gland;
i, lymphocytes in neuroglial tissue.
|
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Microdissection
Unstained 5-µm sections on glass slides were deparaffinized with
xylene, rinsed in ethanol from 100% to 80%, briefly stained with
hematoxylin and eosin, and rinsed in 10% glycerol in TE buffer.
Tissue microdissection was performed as described
previously.14,15
DNA Extraction
Produced cells were immediately resuspended in 30 µl of buffer
containing Tris/HCl, pH 8.0, 10 mmol/L ethylenediamine tetraacetic
acid, pH 8.0, 1% Tween 20, and 0.5 mg/ml proteinase K and were
incubated at 37°C overnight. The mixture was boiled for 10 minutes to
inactivate the proteinase K, and 1.5 µl of this solution was
used for polymerase chain reaction (PCR) amplification of the DNA.
Genetic Analysis
Tissue samples were analyzed with different microsatellite
markers, including D1S1646 (1p), Int-2 (11q13), Ank-1 (8p), D9S303
(9q), D9S171 (9p), IFNA (9p), D5S346 (5q), and D3S2452 (3p14–21). Each
PCR sample contained 1.5 µl of template DNA as described above, 10
pmol of each primer, 20 nmol each of dATP, dCTP, dGTP, and dTTP, 15
mmol/L MgCl2, 0.1 U of Taq DNA polymerase, 0.05
ml of [32P]dCTP (6000 Ci/mmol), and 1 µl of 10X buffer
in a total volume of 10 µl. PCR was performed with 35 cycles:
denaturing at 95°C for 1 minute, annealing at 55°C to 60°C for 1
minute, and extending at 72°C for 90 seconds. The final extension was
continued for 10 minutes. Labeled amplified DNA was mixed with an equal
volume of formamide loading dye (95% formamide, 20 mmol/L EDTA, 0.05%
bromophenol blue, and 0.05% xylene cyanol). Samples were then
denatured for 5 minutes at 95%, loaded onto a gel consisting of 6%
acrylamide (acrylamide:bisacrylamide 49:1), and electrophoresed at 1800
V for 90 minutes. After electrophoresis, the gels were transferred to
3-mm Whatman paper and dried. Autoradiography was performed with Kodak
X-OMAT film (Eastman Kodak, Rochester, NY).
|
Results and Discussion
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In all seven cases, host tissue genotype and teratoma were
analyzed by microdissecting and analyzing normal ovarian/Fallopian tube
tissue and different areas of teratomatous tissue with a series of
markers, including D1S1646 (1p), Int-2 (11q), Ank-1 (8p), D9S303 (9q),
D9S171, (9p), IFNA (9p), D5S346 (5q), and D3S2452 (3p). In each
individual case, the majority of markers revealed a heterozygous and
therefore informative genotype in normal ovarian tissue. With the same
informative markers, selectively procured teratomatous tissue was
genetically homozygous whenever the dissected tissue was strictly
parenchymal, ie, selectively dissected without potential contamination
with nonconstitutive cells (Figure 2A)
.
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Figure 2. Representative findings after genetic analysis of various histological
components from different mature ovarian teratomas. After PCR, the
amplification products were separated using 8% polyacrylamide gels;
each lane depicted below represents a separately procured
tissue sample. A: Case 1, various histological components being
consistently homozygous with markers Int-2, D1S1646, and D3S2452;
normal host ovarian stroma is heterozygous. lane 1, normal
ovarian stroma; lane 2, squamous epithelium; lane 3,
neuroglial tissue; lane 4, sebaceous gland; lane 5,
striated muscle; lane 6, neuroglial tissue; lane 7,
sebaceous gland; lane 8, neuroglial tissue; lane 9
, sebaceous gland; lane 10, squamous epithelium;
lane 11, squamous epithelium; lane 12, neuroglial
tissue; lane 13, squamous epithelium)
B: Case 7, evidence of genetic recombination. Lane 1,
Normal ovarian tissue; lane 2, normal ovarian tissue;
lane 3, squamous epithelium; lane 4, lymphoid aggregate
in squamous epithelium. Teratomatous squamous epithelium is homozygous
with Ank-1 but heterozygous with D9S303; in contrast, normal ovarian
tissue and lymphoid aggregate are heterozygous with both markers.
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In two cases, tumor areas were homozygous with all markers except one
marker that indicated heterozygosity (Figure 2B
, marker D9S303). This
finding was interpreted as a recombination event during meiosis I
involving the respective marker locus resulting in preservation of
heterozygosity.
Vice versa, aggregates of lymphoid tissue that were usually
associated with different epithelial structures were consistently
heterozygous (Figure 2B
, lane 4; Figure 3A
, lane 7; Figure 3B
, lanes 14 and 15).
Although these findings could indicate heterogeneity of different tumor
compartments, we interpret these findings in most cases as a
contaminating effect caused by infiltration of host lymphoid cells.
This interpretation is based on our observations on teratomatous
tissues in which the number of lymphoid cells did not exceed that of
strictly ectopic parenchymal cells (Figure 3B
, lanes 6 to 8). These
samples demonstrated variable intensities of the second allele with
different markers; eg, in case 2, teratoma tissues containing
lymphocytes showed similar allelic intensities with D9S303 and Ank-1
and variable intensities with Int-2, whereas no second allele was
observed with IFNA (Figure 3B
, lanes 6 to 8). This observation
indicates allelic imbalance secondary to the presence of heterozygous
cells that contribute a second allele of variable intensity; it also
points out that different genetic markers may show different degrees of
amplification sensitivity of heterogeneous cell populations. The
interpretation of cellular contamination rather than true heterogeneity
is further corroborated by the observation that selective procurement
of lymphoid aggregates demonstrated two alleles of equal intensity
(Figure 2B
, lane 4; Figure 3A
, lane 7; Figure 3B
, lanes 13 and 14). In
contrast, thymic tissue from case 3 was genetically homozygous (Figure 3C)
. It therefore appears that the differentiation potential of mature
ovarian teratoma does include lymphoid tissue.
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Figure 3. A: Case 4, analysis of lymphoid aggregate (lane 7,
marked by asterisk) reveals two
(heterozygous) alleles
with markers D1S1646, D9S303, and Ank-1, whereas microdissected
teratomatous tissues of variable heterotopic differentiation are
consistently homozygous.lane 1, normal ovarian stroma;
lane 2, normal ovarian stroma; lane 3, cerebellum;
lane 4, cerebellum; lane 5, squamous epithelium;
lane 6, neuroglial tissue; lane 7, lymphoid aggregate
in squamous epithelium; lane 8, sebaceous glands;
lane 9, pilar follicles. B: Case 2, various
histological components being consistently homozygous with markers
D9S303, Ank-1, Int-2, and IFNA. Normal host ovarian stroma is
heterozygous; however, a second allele of variable intensity can be
observed in samples 6, 7, and 8 (marked by
asterisks) that represent different
teratomatous tissues with lymphoid cells. Samples 14 and 15
(marked by
asterisks) represent selectively
procured lymphoid aggregates with two alleles of equal intensity.
Lane 1, normal ovarian stroma; lane 2, normal ovarian
stroma; lane 3, sebaceous gland; lane 4; sebaceous
gland; lane 5, squamous epithelium; lane 6, neuroglial
tissue; lane 7, squamous epithelium with lymphoid cells;
lane 8, sebaceous glands with lymphoid cells; lane 9,
neuroglial tissue; lane 10, cartilage; lane 11,
sebaceous gland; lane 12, respiratory epithelium;
lane 13, sebaceous glands; lane 14, lymphocytes
associated with respiratory epithelium; lane 15, lymphocytes
associated with respiratory epithelium; lane 16, sebaceous
glands; lane 17, respiratory epithelium; lane 18,
respiratory epithelium. C: Case 3. Thymic tissue obtained from
case 3 reveals homozygosity with markers Ank-1 and Int-2. Normal host
ovarian stroma is heterozygous T, thymus; N, normal ovarian tissue.
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As postulated more than 100 years ago, the germ cell line
differentiates during early embryonic life and is responsible for
species preservation by developing along the Keimbahn or germ
track.2
In contrast to the somatic cell line, germ cells
physiologically undergo a series of unique and fundamental genetic
modifications. In particular, it is the unique event of meiotic
division that distinguishes germ cells from all other somatic cells by
producing an offspring of cells with fundamentally modified genetic
composition. In contrast to regular mitotic division, division I of
meiosis (meiosis I) creates cells in which both of the two DNA copies
of each chromosome are derived from only one of the two homologous
chromosomes present in the original cell.16,17
In other
words, during meiosis I, the heterozygous premeiotic germ cell
(oogonium) divides into two homozygous postmeiotic cells. In addition,
parts of homologous chromosomes are exchanged by genetic recombination
during the long prophase of meiosis I. On average, one or two crossover
events occur on each pair of homologs,16
allowing for
occasional detection of allelic heterozygosity in postmeiotic cells. In
two of the analyzed cases, heterozygosity was retained with one and two
markers, respectively. However, as the same samples were consistently
homozygous with other chromosomal markers, we interpret these findings
as evidence for genetic recombination rather than intratumor
heterogeneity.
Analysis of heterozygosity versus homozygosity has been of
fundamental importance to establish germ cell derivation of ovarian
teratomas. Original studies with enzyme polymorphisms3
were
subsequently confirmed cytogenetically.5,7
However, several
studies identified a subgroup of teratomas that was genetically
heterozygous,9-13
challenging the concept of a
consistently postmeiotic germ cell origin of these tumors. Analysis of
purely heterotopic teratomatous areas in our series of seven cases
revealed consistent homozygosity of the tumor tissue with all or the
majority of genetic markers.
For analysis of genetic polymorphoisms, it is desirable or even
mandatory to separate and selectively procure the neoplastic
cells.14,15
Vice versa, PCR-based identification
of genetic monomorphism in tissue DNA extracted from a heterozygous
host may indicate that the genetic changes affect all cellular
constituents. With this study, we performed a tissue-specific genetic
analysis of seven ovarian teratomas using tissue microdissection and
PCR analysis with a panel of polymorphic markers. Analyzed tissues were
selectively procured, guided by histopathological appearance.
In conclusion, the results of this study indicate that 1) ovarian
teratomas reveal genetic homozygosity in the procured tissues strongly
supporting Linder's original hypothesis of a germ cell origin; with a
few markers, heterozygosity was observed in some tumors indicative of
genetic recombination. 2) Within teratomas with homozygous genotype,
lymphoid aggregates reveal allelic heterozygosity indicative of host
cell contamination. In one case, well differentiated thymic tissue
revealed a homozygous genotype emphasizing the pluripotent capability
of teratoma cells to differentiate along hematopoetic cell lines. 3)
Tissue microdissection may be the most sensitive method to evaluate the
genotype of individual teratomas.
|
Footnotes
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Address reprint requests to Dr. Zhengping Zhuang, Laboratory of Pathology, Building 10 Room 2A33, National Cancer Institute/NIH, 9000 Rockville Pike, Bethesda, MD 20892.
A.O.V. and M.D.-S. contributed equally to this study.
Accepted for publication December 22, 1998.
|
References
|
|---|
-
Scully RE: Tumors of the ovary and maldeveloped gonads. Atlas of Tumor Pathology. 1979, Armed Forces Institute of Pathology, Washington, DC,
-
Gonzales-Crussi F: Extragonadal teratomas. Atlas of Tumor Pathology. 1982, Armed Forces Institute of Pathology, Washington, DC,
-
Linder D: Gene loss in human teratomas. Proc Natl Acad Sci USA 1969, 63:699-704[Abstract/Free Full Text]
-
Linder D, Power J: Further evidence for post-meiotic origin of teratomas in the human female. Ann Hum Genet 1970, 34:21-30[Medline]
-
Linder D, Hecht F, McCaw BK, Campbell JR: Origin of extragonadal teratomas and endodermal sinus tumors. Nature 1975, 254:597-598[Medline]
-
Kaiser-McCaw BK, Hecht F, Linder D, Lovrien EW, Wyandt H, Bacon D, Clark B, Lea N: Ovarian teratomas: cytologic data. Cytogenet Cell Genet 1976, 16:391-395[Medline]
-
Patil SR, Kaiser-McCaw B, Hecht F, Linder D, Lovrien EW: Human benign ovarian teratomas: chromosomal and electrophoretic enzyme studies. Birth Defects Original Article Ser 1978, 14:297-301
-
Eppig JJ, Kozak LP, Eicher EM, Stevens LC: Ovarian teratomas in mice are derived from oocytes that have completed the first meiotic division. Nature 1977, 269:517-518[Medline]
-
Carritt B, Parrington JM, Welch HM, Povey S: Diverse origins of multiple ovarian teratoma in a single individual. Proc Natl Acad Sci USA 1982, 79:7400-7404[Abstract/Free Full Text]
-
Parrington JM, West LF, Povey S: The origin of ovarian teratomas. J Med Genet 1984, 21:4-12[Abstract/Free Full Text]
-
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]
-
Deka R, Chakravarti A, Surti U, Hauselman E, Reefer J, P. MP, Ferrell RE: Genetics and biology of human ovarian teratomas. II. Molecular analysis of origin of nondisjunction and gene-centromere mapping of chromosome I markers. Am J Hum Genet 1990, 47:644–655
-
Dahl N, H. GK, Rune C, Gustavsson I, Pettersson U: Benign ovarian teratomas: an analysis of their cellular origin. Cancer Genet Cytogenet 1990, 46:115–123
-
Zhuang Z, Bertheau P, Emmert-Buck MR, Liotta LA, Gnarra J, Linehan WM, Lubensky IA: A microdissection technique for archival DNA analysis of specific cell populations in lesions <1 mm in size. Am J Pathol 1995, 146:620-625[Abstract]
-
Zhuang Z, Vortmeyer AO: Applications of tissue microdissection in cancer genetics. Cell Vis 1998, 5:43-48[Medline]
-
Kleckner N: Meiosis. How could it work? Proc Natl Acad Sci USA 1996, 93:8167-8174[Abstract/Free Full Text]
-
Bascom-Slack CA, Ross LO, Dawson DS: Chiasmata, crossovers, and meiotic chromosome segregation. Adv Genet 1997, 35:253-284[Medline]
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Top
Abstract
Introduction
