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[CANCER RESEARCH 61, 7268 –7276, October 1, 2001]
Multipoint Imprinting Analysis Indicates a Common Precursor Cell for Gonadal
and Nongonadal Pediatric Germ Cell Tumors1
Dominik T. Schneider, Amy E. Schuster, Michael K. Fritsch, Jie Hu, Thomas Olson, Stephen Lauer, Ulrich Göbel,
Elizabeth J. Perlman,2 the Pediatric Oncology Group, and German Pediatric Germ Cell Tumor Study Group
Division of Pediatric Pathology, Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287 [D. T. S., A. E. S., M. K. F., J. H., E. J. P.];
Department of Pediatric Hematology and Oncology, Children’s Hospital, Heinrich-Heine-University, Medical Center, Düsseldorf, Germany D-40225 [D. T. S., U. G.]; Department
of Pathology, University of Wisconsin-Madison, Madison, Wisconsin [M. K. F.]; Department of Genetics, University of Pittsburgh, Pennsylvania [J. H.]; and Pediatric
Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia 30322 [T. O., S. L.]
ABSTRACT
Pediatric germ cell tumors (GCTs) commonly arise at extragonadal
sites. It has been proposed that nongonadal GCTs arise from ectopic
primordial germ cells that have aberrantly migrated during embryogenesis. During a time between their migration and development to mature
gametes, primordial germ cells are characterized by their lack of imprinting, which can be assessed by the evaluation of allelic gene expression and
DNA methylation in differentially methylated control regions. To elucidate the cellular origin of nongonadal GCTs, we evaluated the imprinting
status of 21 gonadal and 21 nongonadal pediatric GCTs. Allele-specific
H19 and IGF-2 expression was assessed with reverse transcription-PCR
followed by digestion at polymorphic restriction sites. DNA methylation
was evaluated after bisulfite modification, PCR amplification, and restriction digestion at a consistently methylated CpG dinucleotide within the 5ⴕ
flanking region of the SNRPN gene. These results were compared with
genetic gains and losses determined by comparative genomic hybridization. Seven of 15 informative tumors showed biallelic H19 expression, and
8 of 17 informative tumors showed biallelic IGF-2 expression. The frequency of biallelic gene expression was comparable in gonadal and nongonadal GCTs. Sixteen of 19 gonadal GCTs and 17 of 21 nongonadal
GCTs showed absence of methylation of SNRPN consistent with loss of
imprinting. One testicular GCT and three nongonadal GCTs showed a
somatic methylation pattern. Two ovarian teratomas and one mediastinal
teratoma showed only methylated SNRPN, consistent with entry into
meiosis. Twenty-one of 22 non-GCT control samples showed a somatic
methylation pattern. Gonadal and nongonadal germ cell tumors are derived from primordial germ cells that have consistently lost the imprinting
of SNRPN and partly lost imprinting of H19 and IGF-2. Because the
imprinting pattern of the latter genes differs from that found in testicular
GCTs of adult patients, our data suggest that pediatric GCTs arise from
a different stage of germ cell development.
INTRODUCTION
GCTs3 can develop at any age and site including the gonads as well
as extragonadal midline sites such as the coccyx, the mediastinum,
and the brain. They show a marked heterogeneity in their histologic
appearance, which may vary from mature organoid differentiation
patterns in teratoma to highly malignant and clinically aggressive
tumors such as seminoma, YST (also known as endodermal sinus
tumor), embryonal carcinoma, and choriocarcinoma. However, gonadal and nongonadal GCTs of all ages are grouped together and
termed “germ cell tumors” in keeping with the theoretical concept of
Received 4/10/01; accepted 7/26/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by the American Cancer Society (RPG-97-113-01-CCE). D. T. S. receives
a Dr. Mildred Scheel Grant from the German Cancer Aid (Deutsche Krebshilfe e.V.).
2
To whom requests for reprints should be addressed, at Division of Pediatric Pathology, Johns-Hopkins Medical Institutions, 600 N. Wolfe Street, Baltimore, MD 21287.
Phone: (410) 614-1988; Fax: (410) 614-9386; E-mail: [email protected].
3
The abbreviations used are: GCT, germ cell tumor; YST, yolk sac tumor; PGC,
primordial germ cell; LOI, loss of imprinting; ICR, imprinting control region; SNRPN,
small nuclear ribonucleoprotein polypeptide N gene; CGH, comparative genomic hybridization; RT-PCR, reverse transcription-PCR.
Teilum (1) that all subtypes develop from a common precursor cell,
the PGC.
There are at least two lines of evidence that support this hypothesis.
First, malignant nongonadal GCTs show genetic aberrations that
resemble those of gonadal GCTs of the corresponding age group
(2–5). Only the association between Klinefelter syndrome and GCTs
appears to be unique to nongonadal GCTs (particularly mediastinal
GCTs) of adults (6, 7). It is remarkable, however, that GCTs arising
in infancy and those arising after the onset of puberty show profound
differences in their molecular biology despite identical histology.
Postpubertal malignant testicular, ovarian, and nongonadal GCTs are
characterized by an isochromosome 12p in about 80% of cases and
amplification of segments of 12p in most of the remaining cases (5, 8,
9). In contrast, nongonadal GCTs arising in infants and toddlers
display different genetic aberrations such as imbalances of chromosome 1 and loss of 6q, and they lack amplification of 12p in most
cases (3–5, 10, 11). In summary, genetic analysis distinguishes subgroups of GCT by age, rather than by tumor site or specific histological subentities (8). This raises the possibility that pre- and postpubertal GCTs may develop from germ cells at different stages of their
development, or alternatively, some subsets may arise from embryonic stem cells.
The second line of evidence supporting a germ cell origin of
extragonadal GCTs comes from the analysis of early germ cell development and migration. Embryologic studies (12, 13) reveal that
PGCs first appear in the extraembryonic mesoderm and actively
migrate along the mesentery to the posterior hindgut, in very close
proximity to the coccyx, before they enter the gonadal ridges. This
migration is directed by the c-kit and stem cell factor receptor-ligand
pair among others and requires the interaction with extracellular
matrix [for review, see Wylie (14)]. In light of these findings, it has
been proposed that nongonadal GCTs develop from PGCs that for
some reason failed to enter the gonadal ridge. Fetal mice often contain
extragonadal germ cells; however, these have never been seen in the
mediastinum or brain and instead are more commonly found in such
sites as the adrenal gland, sites where GCTs do not arise. Extragonadal germ cells have not been observed in human fetuses. It has been
suggested that they may undergo apoptosis because of lack of growth
factor support (12, 15–17). However, the possibility that extragonadal
GCTs may instead arise from embryonal stem cells has also been
raised and is supported by the midline location almost exclusively
seen in these tumors.
The above observations have led to yet another hypothesis regarding the origin of nongonadal GCTs that also takes into account their
genetic similarity to gonadal tumors. Chaganti et al. (2, 8) proposed a
model that mediastinal GCTs develop from early gonadal lesions,
giving rise to cells that “recapitulate embryonal memory and reverse
migrate to thymus and pineal where receptive environments permit
their establishment as primary tumors.” In support of this hypothesis,
the authors refer to the thymus as an organ that is well appreciated as
an intermediate migratory site of other cell types such as T lymphocytes. In addition, the abundance of germ cells in the testis compared
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
with the ovary might explain the fact that nongonadal GCTs predominantly develop in males. However, this is not true for nongonadal
GCT in children, which show a female predominance (18).
In summary, there is no evidence that nongonadal GCTs indeed
derive from PGCs, and molecular markers are needed to evaluate the
presumably common origin of gonadal and nongonadal GCTs. Immunological and genetic markers have been informative in tracing
back the origin of some childhood neoplasms to the fetal period. For
instance, in childhood acute lymphoblastic leukemia, monoclonal
antigen receptor rearrangements and leukemia-specific chromosomal
rearrangements were detected in neonatal blood samples, and the
molecular genetic evaluation of identical twins with leukemia suggested a fetal origin of the leukemia (19, 20). Unfortunately, these
methods cannot be applied to the analysis of solid neoplasms such as
GCTs, because corresponding prenatal tissue samples are not available apart from rare fetal teratoma.
Therefore, we used an alternative approach to study the cellular
origin and time of origin of extragonadal GCTs. We examined GCTs
for unique molecular features of their presumed cell of origin, the
PGC. Others (21–25) have already proposed that LOI may serve as
such a molecular marker of a defined stage of PGC development.
However, insufficient data are available for nongonadal and pediatric
GCTs to arrive at meaningful conclusions.
The term imprinting refers to the phenomenon that in somatic cells
the two alleles of some genes are expressed in an asymmetric pattern
depending on the inheritance of the allele from either father or mother
(26 –28). DNA methylation of 5⬘-CG-3⬘ (CpG) dinucleotides represents the most significant biochemical marker of imprinting and
allows the distinction between the paternal and the maternal alleles.
Among all of the imprinted genes, H19 and IGF-2 have been studied
most extensively (22, 24, 29). They are part of an imprinted gene
cluster on chromosome 11p15, and they share a common enhancer
downstream of the H19 gene. Differential methylation of the ICR
upstream of the H19 promoter controls imprinting of IGF-2 by interfering with the binding of the vertebrate enhancer-blocking protein
CTCF, which prevents the activation of the IGF-2 promoter by its
enhancer (boundary model; Fig. 1; Refs. 30, 31). In addition, imprinting of IGF-2 is controlled independently of H19 by a differentially
methylated silencer element (32).
The imprinted gene for the small nuclear ribonucleoprotein
polypeptide N gene (SNRPN) is located within a region on chromosome 15q11–13, which is microdeleted in Prader-Willi syndrome or
Angelman syndrome (33, 34). Evaluation of the SNRPN methylation
pattern has been proposed as a diagnostic test for both syndromes,
because the methylated maternal allele is lost in Angelman syndrome,
whereas the unmethylated paternal allele is deleted in Prader-Willi
syndrome (34, 35). Loci on both 11p15 and 15q11–13 have been
reported to be deleted only rarely in pediatric and adult GCTs (10, 36).
Therefore, it is unlikely that cytogenetic events will substantially alter
the imprinting pattern of GCTs.
During their development, PGCs erase their inherited imprint and
establish a new sex-specific imprinting pattern. If one hypothesizes
that GCTs preserve the original imprinting status of their cell of
origin, one may assume that GCTs arising from PGCs before entry
into the gonadal ridge and GCTs arising from premeiotic germ cells
will show erased imprinting (biallelic expression of H19 and IGF-2;
no methylation of the H19 ICR or the SNRPN gene). On the other
hand, GCTs developing from PGCs that have entered meiosis should
display a sex-specific gametic imprinting pattern.
This study summarizes our analysis of the IGF-2, H19, and SNRPN
imprinting status of 42 gonadal and nongonadal GCTs in children and
adolescents.
PATIENTS AND METHODS
Tumor Samples, DNA Extraction, and RNA Extraction. Thirty-five
fresh-frozen and seven archival germ cell tumor samples from children and
adolescents were collected at the Pediatric Oncology Group- and MAKEI(German Maligne Keimzelltumoren Study Group) affiliated institutions. The
histology of all of the tumors was reviewed centrally. Control specimens
included 12 non-neoplastic tissue specimens and 12 pediatric tumors in which
loss of imprinting has been reported previously (eight renal tumors and four
hepatoblastomas) (28, 37). Before DNA and RNA extraction, specimens were
analyzed histologically to confirm ⬎80% viable tumor cell content. Tissue
sections were digested with proteinase K in SDS, and DNA was extracted with
phenol/chloroform followed by ethanol precipitation. For RNA extraction,
0.1– 0.4 g of frozen tissue was minced, homogenized, and dissolved in 1 ml of
TRIzol (Life Technologies, Inc., Rockville, MD). RNA was extracted with
chloroform and precipitated with isopropanol. The extraction was repeated
Fig. 1. Boundary model of the IGF-2 and H19
cluster on chromosome 11p15. IGF-2 and H19
share a common enhancer downstream of H19. On
the maternal allele, the ICR upstream of H19 is
unmethylated and binds the vertebrate enhancerblocking protein CTCF, which inhibits the activation of the IGF-2 promoter by the enhancer. On the
paternal allele, the H19 promoter and ICR are
methylated, thus silencing H19 and interfering with
CTCF binding. The IGF-2 promoter is activated by
its enhancer (30, 31).
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
once. After incubation with 1 unit/␮l of DNase (Roche Molecular Biochemicals, Mannheim, Germany) for 20 min at 37°C, phenol/chloroform extraction,
and ethanol precipitation, the amount of RNA was quantified by UV spectroscopy. RNA quality was assessed on an ethidium bromide-stained 1% agarose
gel, which should reveal the 28 S and 18 S bands of rRNA.
CGH. Nick translation, comparative hybridization, and image analysis
were performed as described in detail previously (3). Briefly, DNA (1 ␮g)
from tumor and normal male control tissue was labeled with Spectrum-Green
and Spectrum-Red (Vysis Inc., Downers Grove, IL) by nick translation. Equal
amounts of labeled tumor and control DNA were cohybridized with Cot-1
DNA against normal male metaphase spreads. After posthybridization washes,
slides were counterstained with 0.1 ␮g/ml 4⬘,6-diamidino-2-phenylindole-2
HCl in antifade. Gray level images were acquired for each fluorescent dye
using the dedicated Cytovision software and hardware of the Applied Imaging
Corporation (Santa Clara, CA). Chromosomes were identified using reverse
4⬘,6-diamidino-2-phenylindole banding. At least 15 representative chromosomes were analyzed to generate a green:red ratio profile with mean and SE for
each chromosome. A deviation above 1.2 and below 0.8 was considered gain
or loss of chromosomal material, respectively.
Allelic Expression of IGF-2 and H19. The polymorphisms at the ApaI
restriction site in exon 9 of IGF-2 and at the AluI restriction site in exon 5 of
H19 were analyzed (38). In a first step, the genomic polymorphism of each
patient was evaluated for both sites (39). Tumors from heterozygous patients
were further analyzed by RT-PCR for allele-specific gene expression. RNA
was reverse-transcribed with Superscript reverse transcriptase (Life Technologies, Inc.) and random hexamers (Perkin-Elmer, Branchburg, NJ). PCR
amplification was performed on a Perkin-Elmer 2400 PCR thermocycler using
2.5 ␮M of each primer (Table 1), buffer 3 (IGF-2) or 9 (H19; Stratagene, La
Jolla, CA), 2 mM of each deoxynucleotide triphosphate, and AmpliTaq (H19)
or AmpliTaq Gold (IGF-2; Perkin-Elmer).
PCR conditions for IGF-2 were 94°C for 10 min, 30 cycles of 94°C for 30 s,
60°C for 1 min, and 72°C for 30 s, followed by final extension at 72°C for 10
min. PCR and RT-PCR of IGF-2 both resulted in a 292-bp product. PCR
included water blanks as negative controls. For each sample analyzed by
RT-PCR, a negative control without reverse transcriptase was included to
monitor for contamination with genomic DNA. PCR products were ethanol
precipitated, dissolved in distilled water, and digested overnight at 25°C with
20 units of ApaI, according to the manufacturer’s instructions (New England
Biolabs, Beverly, MA). Digestion products were separated on a 2% ethidiumbromide-stained agarose gel. Samples showing both a 292-bp and a digested
227-bp DNA fragment were considered heterozygous for DNA or biallelic
RNA expression, respectively. The 65-bp fragment was not reliably detected
on the agarose gels.
H19 was amplified with nested PCR under the following conditions: 94°C
for 5 min, 20 cycles of 94°C for 1.5 min, 56°C for 1 min, and 72°C for 1.5 min.
One ␮l of the first PCR was transferred to a new tube with fresh reagents, and
nested PCR was performed under identical conditions for another 20 cycles.
The amplification of genomic DNA resulted in a 833-bp product. Because of
introns between the exons 3 and 4, as well as 4 and 5, RT-PCR resulted in a
672-bp product, and the absence of the 833-bp product served as an additional
control for contamination with genomic DNA. Amplification products were
ethanol precipitated and digested overnight with 20 units of AluI, according to
the manufacturer’s instructions (Life Technologies, Inc.). The 833-bp PCR
product amplified from genomic DNA was digested with AluI into 261-, 153-,
147-, 91-, 76-, 49-, 41-, 10-, and 5-bp fragments. The restriction site at
nucleotide 10380 (GenBank accession no. AF087017) is polymorphic, and as
a consequence, the digestion may result in a 300-bp fragment instead of the
two 153-bp and 147-bp fragments. RT-PCR products were digested into 261-,
147-, 76-, 72-, 60-, 41-, 10-, and 5-bp fragments. Polymorphism at the AluI
restriction site results in a 219-bp fragment. Therefore, biallelic gene expression results in 261-bp, 219-bp, and 147-bp bands. Monoallelic expression,
however, results either in 261-bp and 219-bp bands or in 261-bp and 147-bp
bands.
Methylation of the SNRPN Gene. Rather than expression analysis, methylation analysis was performed to determine the imprinting status of the
SNRPN gene, because this approach does not depend on genetic polymorphism
and therefore allows examination of all of the available tumor samples. The
restriction site for CfoI in the 5⬘ flanking region of the SNRPN gene is
methylated on the maternal chromosome in more than 96% of normal individuals and unmethylated on the paternal chromosome (33, 35). DNA modification was performed by overnight incubation with sodium bisulfite and
hydrochinone according to the manufacturer’s instructions (Intergen, Purchase,
NY). This modification was followed by a DNA extraction step with glass
beads. Bisulfite-modified DNA (250 ng) was added to a 50-␮l PCR reaction
mix, which consisted of 100 mM Tris-HCl (pH 9.2), 35 mM MgCl2, 250 mM
KCl, 0.2 mM of each nucleotide, 1 ␮M of each primer (Table 1), and 2.5 units
of Taq DNA polymerase (Taq Gold; Stratagene). The primers bind to bisulfitemodified DNA only, and the primer-binding sites include no potentially
methylated CpG dinucleotides, thus allowing amplification of the imprinted
and nonimprinted allele. A negative control with unmodified DNA was included in each experiment. PCR conditions were 94°C for 10 min (one cycle),
1 min at 94°C, 1 min at 51°C, 1 min at 72°C for 35 cycles, and 25 cycles for
nested PCR, respectively.
The resulting 340-bp PCR product was ethanol precipitated and digested
overnight with 20 units of CfoI (Promega, Madison, WI). The reaction products were separated in an ethidium bromide-stained 2% agarose gel. To
monitor for complete restriction digestion, the H19-PCR product from
genomic DNA was added to each reaction. CfoI digests this 833-bp product
into 386-bp, 260-bp, 130-bp, and 57-bp fragments. Samples, which showed
incomplete digestion of H19, were excluded from the analysis of the SNRPN
methylation status.
PCR products from methylated and therefore unmodified SNRPN alleles are
digested into two 224-bp and 116-bp fragments, whereas PCR products from
unmethylated (modified) alleles are not digested (340-bp band only). In cases
showing both bands, the relative band intensities of the ethidium bromide
staining was accessed with the Eagle Eyes system (Stratagene). ⌽X174 DNA
cut with HaeIII was run in parallel to ensure that the measurement was in a
linear range. As a result of the size difference between the two SNRPN bands,
a 1.5:1 ratio was consistent with identical molar concentrations of both bands.
In contrast, a ratio above 4.5:1 was considered predominance of the unmethylated allele, and a ratio below 0.5:1 was considered predominance of the
methylated allele.
RESULTS
Clinical and Histological Data. We analyzed 21 gonadal and 21
nongonadal GCTs. Among the gonadal tumors, there were five testicular and 16 ovarian GCTs (Table 2). Of the latter, one tumor
(dysgerminoma with a gonadoblastoma component) arose in a phe-
Table 1 PCR-primersa
Gene
IGF-2
H19
SNRPN (after bisulfite treatment)
a
Primer
Sequence
Forward
Reverse
Forward
Reverse
Forward–nested
Reverse–nested
Forward
Reverse
Forward–nested
Reverse–nested
5⬘-CTTGGACTTTGAGTCAAATTGG-3⬘
5⬘-GGTCGTGCCAATTACATTTCA-3⬘
5⬘-AACACCTTAGGCTGGTGG-3⬘
5⬘-TGCTGAAGCCCTGGTGGG-3⬘
5⬘-CACTATGGCTGCCCTCTG-3⬘
5⬘-TCGGAGCTTCCAGACTAG-3⬘
5⬘-GGTTTTTTTTTATTGTAATAGTGTTGTGGGG-3⬘
5⬘-CTCCAAAACAAAAAACTTTAAAACCCAAATTC-3⬘
5⬘-GGTTTTAGGGGTTTAGTAGTTTTTTTTTTTTAGG-3⬘
5⬘-CAATACTCCAAATCCTAAAAACTTAAAATATC-3⬘
Refs. 34, 35, 38, and 39.
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
Table 2 Age, histology, CGH profiles, and SNRPN methylation of 21 gonadal germ cell tumorsa
1
2
3
4
5
6
7ab
7bb
8
9
10
11c
12
13
14
15
16
17
18
19
20
21
Age
Sex
Site
Histology
Chromosomal gains and losses
3 mo
5 mo
6 mo
7 mo
11 mo
9 yr
12 yr
12 yr
14 yr
14 yr
16 yr
17 yr
18 yr
22 yr
4 yr
8 yr
12 yr
15 yr
17 yr
24 yr
4 yr
21 yr
M
M
M
M
M
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
T
T
T
T
T
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
YST
YST
YST
YST
YST
DYS
DYS
DYS
DYS
DYS
DYS
DYS/GO
DYS
DYS
MGCT
MGCT
YST
MGCT
MGCT
YST
IT
IT
⫹17p, ⫹20q
⫹3, ⫺4p, ⫺6q, ⫹8q, ⫹13, ⫺14, ⫺20p, ⫹20q, ⫹22
⫺4, ⫺16, ⫹17q, ⫹22, ⫹X
⫺1p, ⫺13q, ⫺16
⫹1q, ⫹3p, ⫺4p, ⫺5q, ⫺6, ⫹7p, ⫹9q, ⫺8
⫹20q
⫹6p, ⫹12p, ⫹21
⫹7, ⫹12p, ⫹21
⫹1q, ⫺4, ⫺7p, ⫺12
⫺1p, ⫹1q, ⫹4q, ⫹7, ⫹8, ⫹12, ⫺13, ⫹15, ⫹21
⫹8, ⫹12p, ⫹15, ⫹21
⫺4, ⫹8, ⫹12p, ⫹X, ⫺Y
⫹3, ⫹12p, ⫺13q
⫹1q, ⫹8, ⫹21
⫹8, ⫹12p
⫹12p, ⫹21
⫺1p, ⫹1q, ⫺5, ⫹12p, ⫺13, ⫺18q, ⫹20
⫺4, ⫹7, ⫹8, ⫺10, ⫹12p, ⫺18
⫹1q, ⫹10, ⫹12
⫺1p, ⫹7p, ⫹8, ⫺9, ⫺11, ⫹12p, ⫺13, ⫺15, ⫺20, ⫹21, ⫺22
Normal, XX
⫹14
H19
IGF-2
SNRPN
Monoallelic
U
M⫹U
U
Monoallelic
Monoallelic
Monoallelic
Monoallelic
Biallelic
Monoallelic
Monoallelic
Biallelic
Biallelic
Biallelic
Biallelic
Monoallelic
Biallelic
Biallelic
Monoallelic
Biallelic
Biallelic
Monoallelic
Monoallelic
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
M
M
a
CGH profiles of patient numbers 1 to 5 and 6 to 21 have been reported by Perlman et al. (Ref. 3) and Riopel et al. (Ref. 9) respectively. T, testis; O, ovary; DYS, dysgerminoma;
GO, gonadoblastoma; MGCT, mixed malignant GCT; IT, immature teratoma; M, methylated allele; U, unmethylated allele.
b
Patient 7a/7b, (a) tumor right ovary, (b) tumor left ovary.
c
Patient 11, gonadal dysgenesis with male pseudohermaphroditism.
notypic female with a constitutional 46, XY karyotype and streak
gonads (number 11).
Among the nongonadal tumors, 11 developed at the sacrococcygeal
region, eight in the mediastinum, and two in the central nervous
system (Table 3). Fifteen nongonadal tumors developed in infants or
children, and five developed in adolescents. Six patients were female,
and 15 patients were male.
In infants and prepubertal children, the only histological subtypes
identified in both gonadal and nongonadal GCTs were YST and
teratoma. After the onset of puberty, we also observed dysgerminoma,
YST, immature teratoma, and mixed malignant GCTs, which showed
a composite histology including germinomatous and nongerminomatous elements such as embryonal carcinoma, YST, choriocarcinoma,
or teratoma (Tables 2 and 3).
CGH. This analysis included 10 pure teratomas (six mature and
four immature teratomas). All of the mature teratomas and three
immature teratomas showed normal CGH profiles (Tables 2 and 3).
One immature ovarian teratoma showed gain of chromosome 14. All
of the malignant GCTs showed chromosomal imbalances on CGH
analysis (Tables 2 and 3). Specific CGH profiles did not correlate with
histological differentiation or tumor site. However, two distinct CGH
patterns could be distinguished by age. Regardless of site, malignant
GCTs in infants and children younger than eight years (n ⫽ 16) most
commonly showed imbalances at chromosome 1 (loss 1p and/or gain
1q), gain of 3p, loss of 4 and 6q, and gain of 20q. Only one ovarian
malignant GCT in a four-year-old girl showed gain of 12p. Conversely, gain of 12p was the most consistent aberration found in GCTs
arising after eight years of age and was found in 11 of 16 tumors.
Other recurrent aberrations in adolescents were gain of 1q, gain of
chromosome 8 and 21, and loss of chromosome 13. One tumor
(number 19) showed loss of chromosome 11 and 15. Because this
tumor showed biallelic expression of IGF-2, the tumor is most probably polyploid. Four other tumors showed gain of chromosome 15.
Otherwise, no chromosomal imbalances of the chromosomes 11 and
15 were detected with CGH.
Methylation Pattern of the 5ⴕ Flanking Region of the SNRPN
Gene. In 40 patients, DNA was available for analysis of the methylation of the 5⬘ flanking region of the SNRPN gene. In 16 of 19
Table 3 Age, histology, CGH analysis, and SNRPN methylation of 21 nongonadal germ cell tumorsa
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Age
Sex
Site
Histology
Chromosomal gains and losses
1d
1 wk
1 mo
2 yr
14 yr
14 yr
15 yr
16 yr
3 yr
14 yr
11 mo
1 yr
2 yr
2 yr
2 yr
3 yr
3 yr
4d
10 d
Infant
1 yr
M
M
M
M
M
F
F
M
M
M
F
M
M
M
M
M
M
F
M
F
F
Med
Med
Med
Med
Med
Med
Med
Med
CNS
CNS
Co
Co
Co
Co
Co
Co
Co
Co
Co
Co
Co
IT
IT
MT
YST, IT
MGCT
MT
MT
YST
MGCT
GER
YST
YST
YST
YST
YST
YST
YST
IT, YST
MT
MT
MT
Normal, XY
Normal, XY
Normal, XY
⫺1p, ⫹2p, ⫹3, ⫺4, ⫺5q, ⫺13, ⫹18q, ⫹21
⫹8, ⫹12p, ⫺18, ⫹X
Normal, XX
Normal, XX
⫹2p, ⫹3p, ⫹8q, ⫹12p, ⫺13, ⫹14q, ⫹17q, ⫹20, ⫹21
⫹1q, ⫹2p, ⫹3p, ⫺20q
⫹17q
⫹1q, ⫺4q, ⫹15, ⫹20q
⫺1p, ⫹1q, ⫹3p, ⫺4q, ⫺6q, ⫹10, ⫹14q, ⫹17q, ⫹20q
⫺1p, ⫹3p, ⫹6p, ⫹13, ⫹14q
⫺1p, ⫹1q, ⫹3q, ⫺6q, ⫺8q, ⫹11q, ⫹12q, ⫹19p, ⫹20q
⫹3p, ⫺4, ⫹6p, ⫹10, ⫹15, ⫹20
⫺1p, ⫹2p
⫺5q, ⫺6q, ⫹20
⫹1q
Normal, XY
Normal, XY
Normal, XX
H19
IGF-2
Biallelic
Monoallelic
Monoallelic
Biallelic
Monoallelic
Monoallelic
Biallelic
Biallelic
Monoallelic
Biallelic
Monoallelic
Biallelic
Biallelic
SNRPN
M
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U⫹M
U
U⫹M
U
U
U⫹M
a
CGH profiles of patient numbers 34 and 35 have been reported by Perlman et al. (Ref. 3); Med, mediastinum; Co, coccyx; IT, immature teratoma; MT, mature teratoma; MGCT,
mixed malignant GCT; GER, germinoma; M, methylated allele; U, unmethylated allele; CNS, central nervous system.
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
Fig. 2. Analysis of the methylation status of the
5⬘ flanking region of SNRPN. One control sample,
five gonadal GCTs, and five nongonadal GCTs.
Agarose gel electrophoresis after bisulfite DNA
modification, PCR amplification, and restriction
digestion at a monoallelically methylated CpG
dinucleotide. The 833-bp PCR product for H19 is
included in the reaction to monitor for complete
digestion. DYS, dysgerminoma; MGCT, mixed malignant GCT; IT, immature teratoma; MT, mature
teratoma; Test., testicular; Ov., ovarian; Med., mediastinal; Cocc., coccygeal.
gonadal GCTs, only the unmethylated allele was present in the tumor,
as demonstrated by the presence of the 340-bp band and absence of
the 224-bp band (Table 2; Fig. 2). One testicular YST (number 3)
showed both bands at the exact 1.5:1 ratio for ethidium bromide
fluorescence, consistent with a somatic imprinting pattern in this
tumor. Contamination with non-neoplastic tissue was excluded by
histological evaluation of an adjacent section, and CGH analysis of
the same DNA vial showed characteristic aberrations. Both ovarian
immature teratomas showed the 224-bp but no 340-bp band, indicating the presence of a methylated SNRPN and absence of unmethylated
SNRPN. Both tumors also showed monoallelic expression of IGF-2.
This constellation is consistent with a maternal pattern of methylation.
In 15 of 21 nongonadal GCTs, the analysis yielded only the 340-bp
band of the unmethylated SNRPN allele. Among these, there were six
teratoma and nine malignant GCTs. In two additional sacrococcygeal
YSTs (Table 3; numbers 33 and 36), the 340-bp band was significantly more intense than the 224-bp band. The assessment of the
relative ethidium bromide fluorescence intensities yielded a 4.9:1 ratio
and a 13.6:1 ratio, respectively, indicating that more than half of the
cells showed loss of SNRPN methylation. All of the tumors showing
gain of chromosome 15 on CGH analysis demonstrated only the
unmethylated SNRPN allele. Another sacrococcygeal YSTs (Table 3;
number 37) showed a 3.9:1 ratio, consistent with loss of SNRPN
methylation in 44% of cells. Lastly, one malignant GCT with a
predominant immature teratoma component and about 25% YST
component (number 39) showed a 2.4:1 ratio, consistent with loss of
the methylated SNRPN in approximately 22% of cells. In this tumor,
expression of both H19 and IGF-2 was biallelic. One teratoma (numbers 42 and 43) showed both bands but with a predominance of the
unmethylated allele (ratio, 2.9:1). In contrast, one immature mediastinal teratoma of a neonate (Table 3; number 33) exclusively showed
the 224-bp band, indicating that the unmethylated allele is absent in
the tumor or that both alleles are methylated.
The analysis of 12 non-neoplastic tissue samples and of 11 of 12
pediatric tumors yielded both bands at the expected 1.5:1 ratio (range,
1.3 to 1, 2.7 to 1). Only one hepatoblastoma showed no 224-bp band,
indicating loss of SNRPN methylation (no chromosomal loss was
detected with CGH).
Allele-specific Expression of IGF-2 and H19. In 39 patients,
fresh frozen tumor samples were available for the evaluation of IGF-2
and H19 expression through RT-PCR. Eighteen of 39 patients were
heterozygous for IGF-2. Nine tumors showed monoallelic expression
of IGF-2, and nine tumors showed biallelic IGF-2 expression (Table
4; Fig. 3).
Twenty of 39 patients were heterozygous for H19, but in five
patients, poor RNA quality did not allow for amplification. Seven of
the remaining 15 tumors showed biallelic expression, and eight tu-
mors showed monoallelic expression of H19 (Table 4; Fig. 4). The
frequency of biallelic expression of H19 and IGF-2 was comparable
among gonadal and nongonadal tumors and did not correlate with
histological differentiation, sex, or age of the patients or with distinct
CGH patterns (Table 4). Furthermore, there was no sex-dependent
difference in the frequency of biallelic expression of IGF-2 or H19,
respectively. Five patients were heterozygous for both H19 and
IGF-2. One ovarian GCT showed biallelic expression of both genes,
three tumors of female patients showed biallelic expression of H19
but monoallelic expression of IGF-2, and one tumor in a male showed
monoallelic H19 but biallelic IGF-2 expression.
DISCUSSION
PGCs and Imprinting. The discussion of the biological significance of imprinting in mammals has focused on the impact of imprinted genes on the development of the offspring generation [parent
offspring conflict hypothesis; for review, see Tilghman (26)]. Considered from a teleological perspective, it has been suggested that
imprinting control mechanisms of fathers will support growth of their
offspring, whereas mothers will try to limit and equally distribute their
resources during pregnancy. This discussion very much reflects the
ability of many imprinted genes such as H19 and IGF-2 to effectively
alter cell growth and proliferation. On the other hand, the biological
and evolutionary impact of other genes such as SNRPN is not as
readily assessable, because these most probably control neurological
maturation (26).
Nevertheless, imprinted genes share common features. They are
localized within several chromosomal clusters that include GC-rich
sequences with differentially methylated CpG dinucleotides (DNA
methylation sites). The allele-specific expression of imprinted genes is
regulated by methylation of their promoters (40), silencer elements
(32), or a shared imprinting control region, which interferes with
Table 4 Allele-specific expression of H19 and IGF-2 with regard to tumor site,
histology, and sex, and age
Fifteen patients were informative for H19, and 18 patients were informative for IGF-2.
H19
Site
Histology
Age
Sex
Total
Gonadal
Nongonadal
Teratoma
Malignant
Infant
Adolescent/adult
Female
Male
IGF-2
Biallelic
Monoallelic
Biallelic
Monoallelic
5
2
1
6
3
4
6
1
7
4
4
2
6
5
3
4
4
8
4
5
1
8
5
4
6
3
9
7
2
2
7
5
4
4
5
9
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
Fig. 3. Allele-specific expression of IGF-2 of
five gonadal and six nongonadal GCTs. Each lane
of these agarose gels represents a RT-PCR product
before (⫺) and after (⫹) restriction digestion of a
polymorphic ApaI restriction site. Presence of both
292-bp and 227-bp bands proves biallelic expression. Abbreviations as above.
promoter activation through an enhancer (Fig. 1; Refs. 30, 31). On the
basis of the present knowledge, it appears that imprinting is controlled
within these clusters, and different clusters are controlled independently.
The presence of imprinted genes in mammals poses specific tasks
to the developing germ cells. In contrast to somatic cells, which
maintain the parent-specific imprinting pattern, germ cells have at
some point to erase the imprint and establish a new, sex-specific
imprinting pattern. At present, it is unclear whether the parental
imprint is completely erased and by which biochemical mechanisms
imprinting is erased and reestablished (26). Furthermore, the exact
timing of erasure and establishment in germ cells has yet to be
determined in more detail. Szabo and Mann (41) found biallelic
expression of several imprinted genes (including IGF-2, H19, and
SNRPN) in mouse PGCs that have already entered the gonads. In this
study, biallelic expression was observed throughout gonadal germ cell
development. In contrast, the comparison of allele-specific gene expression at the tip and the base of the allantois, where PGCs can first
be appreciated as a distinct population, suggested that at least SNRPN
may be expressed monoallelically in premigratory PGCs. However,
one has to keep in mind that even somatic cells may show biallelic
expression of imprinted genes at the single-cell level, suggesting that
some post-transcriptional control mechanism may be of additional
importance (42). Furthermore, biallelic expression of imprinted genes
may be seen despite imprinting-specific DNA methylation (43, 44).
However, the hypothesis that PGCs have erased their imprint by the
time they enter into the gonadal ridge is supported by a recent study
evaluating the methylation imprint of the imprinting control region of
the human H19 (Fig. 1). The authors (25) found no methylation in
fetal postmigratory PGCs but imprinting-specific methylation in adult
male germ cells that enter into meiosis.
In summary, these studies delimit a time frame in males ranging
from at least the entry of PGCs into the gonadal ridge, maybe earlier,
until meiosis entry, during which PGCs are void of imprinting. Evidence suggests the same boundaries for germ cells of females, al-
though the actual time during which the PGCs are void of imprinting
is considerably shortened because of entry into meiosis late in fetal
life. Conversely, somatic cells and premigratory PGCs show somatic
methylation patterns and monoallelic expression of imprinted genes.
This suggests that imprinting is erased during PGC migration to the
gonadal ridge. However, there are no data concerning the timely order
of imprint erasure of different imprinted gene clusters during germ
cell migration.
LOI and Cancer. The role of imprinted genes and epigenetic
changes in cancer biology was not elucidated until 1993, when LOI
was reported in approximately 70% of Wilms’ tumors (45– 47). Other
studies followed that reported LOI in a broad variety of carcinomas
and pediatric embryonal cancers (29). In addition, LOI has been
detected in normal tissue such as colonic mucosa and blood cells of
patients with colon cancer (48). Therefore, LOI has to be considered
not only a frequent event in tumorigenesis, but it may in some patients
also constitute a predisposing factor for cancer development (49 –51).
Its occurrence in somatic tumors may not be surprising, when one
considers that many of the imprinted genes interfere with the regulation of cell growth and proliferation.
In contrast, several studies of the SNRPN gene in cancer have not
revealed a single case with LOI (29, 52, 53). These data are further
supported by our analysis of 12 non-neoplastic and 12 neoplastic
control samples, of which only one hepatoblastoma showed LOI.
These data suggest that SNRPN is not involved in tumor development
(26). This feature makes SNRPN an ideal candidate for the evaluation
of the “native” imprinting pattern of the cell of origin of GCTs.
Imprinting and Germ Cell Tumors. Van Gurp et al. (39) were
the first to study the allele-specific expression of H19 and IGF-2 in
adolescent testicular GCTs. They found biallelic expression of IGF-2
in 10 of 11 tumors and biallelic H19 expression in 12 of 14 informative tumors. They concluded that testicular GCTs develop from germ
cells that have erased their imprint. LOI of IGF-2 and H19 appears to
contribute to an increased expression of these genes in GCTs, as has
Fig. 4. Allele-specific expression of H19;
672-bp RT-PCR product before restriction digestion, seven gonadal and five nongonadal GCTs, and
one control. Agarose gel electrophoresis after RTPCR and restriction digestion of a polymorphic
AluI restriction site. Presence of 261-bp, 219-bp,
and 147-bp bands proves biallelic expression.
Monoallelic expression results in either 261-bp and
219-bp bands or 261-bp and 147-bp bands.
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
been demonstrated with Northern blotting, RT-PCR, and in situ hybridization experiments (54, 55).
A recent study of 11 GCTs in children, adolescents, and young
adults included two testicular GCTs, which both showed LOI of H19
and IGF-2 (24). Three of five ovarian GCTs showed biallelic expression of both genes, and one showed monoallelic H19 but biallelic
IGF-2 expression. Among four nongonadal GCTs, only one tumor
showed biallelic IGF-2 expression, and three showed biallelic H19
expression. In summary, virtually all of the postpubertal testicular
GCTs showed LOI, and a majority of ovarian GCTs also showed LOI.
However, the data for nongonadal GCTs are heterogeneous and do not
allow determining the cellular origin of nongonadal GCTs, which
would be by far more significant than in gonadal GCTs, in which the
germ cell origin is more obvious. This is the focus of our study.
H19 and IGF2 Allelic Expression in Gonadal and Extragonadal
Germ Cell Tumors. Three conclusions can be drawn from our data
on the allelic expression of H19 and IGF-2. First, compared with
testicular GCTs in adults studied with the same methodology, the
frequency of LOI is lower in infantile testicular GCTs, adolescent
ovarian GCTs, and pediatric nongonadal GCTs. Second, the frequency of LOI is comparable in pediatric gonadal and nongonadal
GCTs. Third, these data alone do not provide reliable information
regarding the germ cell origin of either gonadal or nongonadal pediatric GCTs.
It is remarkable, however, that we observed three female patients
with monoallelic IGF-2 and biallelic H19 expression. Ross et al. (24)
found two additional female patients with nongonadal GCTs with the
same imprinting pattern. This constellation is unique to female patients and different from somatic tumors such as Wilms’ tumors,
which show LOI of IGF-2 but epigenetic silencing of the maternally
expressed H19 allele by DNA hypermethylation. The latter pattern is
consistent with the boundary model of an imprinting control region
that in its methylated state silences H19 and allows activation of the
IGF-2 promoter by its enhancer (Fig. 1; Refs. 30, 31). This hypothesis
has been substantiated recently (56) by methylation analysis of the
H19 imprinting control region. Conversely, the above-mentioned constellation observed in this report and by Ross et al. (24) would rather
be consistent with an unmethylated state of the imprinting control
region and of the H19 promoter on both alleles, thus allowing biallelic
expression of H19 and silencing of IGF-2 by binding of CTCF (Fig.
1; Refs. 30, 31). This pattern has to be established during development
of female gametes.
SNRPN Methylation Analysis in Gonadal and Extragonadal
GCTs. In this context, the SNRPN data provide important additional
information. This analysis shows loss of methylation, consistent with
LOI, in the vast majority of both gonadal and nongonadal GCTs. The
frequency of LOI was comparable for gonadal and nongonadal GCTs,
indicating that both arise from PGCs that have erased the SNRPN
imprint.
Although this has to be addressed critically, we consider it unlikely
that different methodologies may account for the discrepancy between
the SNRPN and the IGF-2/H19 data. The assay to detect allelespecific expression of H19 and IGF-2 is well validated and has been
broadly used. In addition to a DNase digestion step, this assay includes control experiments such as exclusion of RT-PCR for monitoring for contamination with genomic DNA. In addition, because the
primers span two introns, the H19 RT-PCR product can be distinguished from the PCR product of genomic DNA by its shorter size.
Our experimental procedure to determine SNRPN methylation is
based on an assay that relies on the monoallelic methylation of one
CpG dinucleotide (35). Because this site is methylated in ⬎96% of
normal individuals, it is unlikely that constitutional lack of methylation substantially contributed to our results (33). In addition, our assay
has been optimized by the inclusion of an internal control to monitor
for incomplete restriction digestion. The reliability of our approach is
further demonstrated by the analysis of control samples and by performing repeated examinations, which always yielded identical results.
Bussey et al. (57) have recently presented preliminary data of
SNRPN methylation of pediatric GCTs, most of which were assessed
after short-term cell cultures. This analysis was based on a different
experimental approach that uses methylation-dependent restriction
digestion and Southern blotting. This study also found loss of methylation at the SNRPN locus, but at a somewhat lower frequency,
particularly in nongonadal GCTs. In our opinion, such data must be
interpreted with caution. GCTs usually grow poorly in cell culture,
often resulting in contamination or overgrowth with non-neoplastic
somatic cells such as fibroblasts. This is of particular concern, because
the SNRPN analysis was performed on samples that were also analyzed cytogenetically, and these demonstrated a high rate of karyotypically normal infantile malignant GCTs (13 of 20 tumors; Ref. 10).
These data stand in contrast to all of the other studies of malignant
GCTs in infants, which show virtually all of the malignant GCTs to
have cytogenetic abnormalities by conventional cytogenetics, CGH,
or fluorescence in situ hybridization analysis (3, 4, 11, 58). In addition, different cell culture conditions may significantly interfere with
DNA methylation (59).
Summary and Conclusion. The results of the current study provide evidence that both gonadal and nongonadal GCTs share a common cell of origin, which is a PGC that has already erased the SNRPN
imprint. IGF-2 and H19, which are localized in a separate chromosomal cluster than SNRPN, are regulated by a different imprinting
control mechanism (30, 31, 40). Therefore, one may assume that the
erasure of SNRPN methylation may occur earlier during PGC migration than the erasure of imprinting of IGF-2 and H19.
The incomplete erasure of H19 and IGF-2 reported here and by
Ross et al. (24) suggests that the initiation of some childhood GCTs
may occur during germ cell migration, whereas the initiation of
testicular GCTs in adults occurs at a later stage of PGC development.
This may be one possible explanation for the profound biological
differences between prepubertal and postpubertal GCTs as summarized in the introduction. Alternatively, the failure to erase the imprint
of IGF-2 and H19 in a physiological pattern during PGC migration
may reflect an early step of tumor development.
Lastly, ovarian teratoma must be considered a distinct biological
entity, because these commonly develop from an error in meiosis II
resulting in isodisomy (10). Hence, it is not surprising that both
ovarian teratomas showed only the methylated allele of SNRPN and
monoallelic IGF-2 expression, mimicking the imprinting status of
female gametes (Table 2). The data are supported by Miura et al. (22),
who also reported that in ovarian teratoma, the methylation imprint of
H19 and SNRPN changes as the cellular origin of the tumor advances
in its development. Remarkably, we also observed one male neonate
with an immature mediastinal teratoma who showed an identical
methylation pattern of SNRPN, consistent with entry into meiosis.
One may speculate whether the extragonadal environment may have
contributed to the establishment of an aberrant female gamete-specific
imprinting pattern in a male. This hypothesis is strongly supported by
studies (60) of the development of extragonadal germ cells in mice,
which in both sexes develop into oocytes.
In conclusion, gonadal and nongonadal germ cell tumors are derived from PGCs, and therefore, the terminology “germ cell tumors”
is also appropriate for nongonadal tumors. The IGF-2 and H19 imprinting pattern is to some extent different from testicular GCTs in
adult patients, suggesting that pediatric GCTs arise from a different
stage of germ cell development. Ovarian and rare nongonadal terato-
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IMPRINTING ANALYSIS OF PEDIATRIC GERM CELL TUMORS
mas develop from germ cells that are more advanced in their development. Future studies of imprinting control mechanisms in GCTs, as
well as the timely order and biochemical mechanisms of imprint
erasure and reestablishment during germ cell development, will provide important information that will eventually elucidate the early
steps of GCT development.
ACKNOWLEDGMENTS
We thank Prof. Dr. D. Harms, Institute for Pediatric Pathology University
of Kiel, Germany, for reviewing and contributing specimens from the German
Children’s Tumor Registry to this analysis. We also thank all of the clinical
centers contributing clinical data to the Maligne Keimzelltumaren and
Pediatric Oncology Group trials for GCT in children.
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Multipoint Imprinting Analysis Indicates a Common Precursor
Cell for Gonadal and Nongonadal Pediatric Germ Cell Tumors
Dominik T. Schneider, Amy E. Schuster, Michael K. Fritsch, et al.
Cancer Res 2001;61:7268-7276.
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