Imbalances of Chromosome Arm 1p in Pediatric andAdult Germ Cell Tumors Are Caused by True AllelicLoss: A Combined Comparative GenomicHybridization and Microsatellite Analysis

Susanne Zahn,1 Sonja Sievers,1,2 Katayoun Alemazkour,1 Sandra Orb,1 Dieter Harms,3

Wolfgang A. Schulz,4 Gabriele Calaminus,1 Ulrich Gobel,1 and Dominik T. Schneider1*

1Clinic of Pediatric Oncology,Haematology and Immunology,Heinrich-Heine-University Dˇsseldorf,Germany2Max Planck Institute of Molecular Physiology,Dortmund,Germany3Kiel PediatricTumor Registry,Institute of Pediatric Pathology,Christian-Albrechts-University,Kiel,Germany4Clinic of Urology,Heinrich-Heine-University Dˇsseldorf,Germany

Previous studies on childhood germ cell tumors (GCTs) report highly variable frequencies of losses at chromosome arm 1p.

Since deletions at 1p portend a poor prognosis in other embryonal tumors, this study aims to clarify the question of the fre-

quency of true allelic loss at 1p and whether it constitutes a prognostic parameter. We analyzed 13 GCTs from different go-

nadal and extragonadal sites of children (4 teratomas, 9 malignant GCTs) and 18 GCTs of adolescents and adults (3 teratomas;

15 malignant GCTs) using automated microsatellite analysis with 23 polymorphic markers and chromosomal ‘‘high resolution’’

comparative genomic hybridization (HR-CGH). With this combined approach, we detected loss of heterozygosity (LOH) at

1p in 8/9 childhood malignant GCTs with concordant data from HR-CGH and microsatellite analyses. In contrast, LOH at 1p

was not detected in childhood teratomas (0/4) and constituted a rare event in GCTs of adolescence and adulthood (3/18).

The commonly deleted region was located at distal 1p36-pter, with a proximal boundary between the markers D1S450 and

D1S2870. These data unequivocally demonstrate that deletion at 1p is common in childhood GCTs and results in allelic loss.

This observation argues for the presence of a classical tumor suppressor at distal 1p. Considering the high frequency of LOH

at 1p and the overall favorable prognosis of childhood GCTs, a prognostic impact of LOH at 1p in childhood GCTs appears

unlikely. However, since two postpubertal tumors with LOH at 1p progressed, a prognostic relevance in this age group seems

possible, warranting a prospective evaluation. VVC 2006 Wiley-Liss, Inc.

INTRODUCTION

Germ cell tumors (GCTs) constitute a heteroge-

neous group of embryonal tumors, which originate

in early embryonic development. They may develop

at any age from the fetal period to adulthood and

can arise both in the gonads and at extragonadal

midline sites such as the coccyx, the mediastinum,

and the pineal region (Calaminus et al., 2004;

Schneider et al., 2004). Imprinting studies have

demonstrated that the cell of origin of both gonadal

and nongonadal GCTs is most probably a primitive

germ cell, the primordial germ cell. Its developmen-

tal potential differs according to its stage of matura-

tion and pattern of genomic imprinting (van Gurp

et al., 1994; Schneider et al., 2001(b); Oosterhuis et

al., 2005; Sievers et al., 2005). Thus, the origin of

nongonadal GCTs can be interpreted as resulting

from an abnormal migration of primordial germ cells

during embryogenesis.

GCTs show a distinctive age distribution that

separates a group of teratomas and yolk sac tumors

(YST) that arise during infancy and childhood from

the more diverse GCTs of adolescent and adult

patients, presenting as teratomas, germinomas

(seminomas, dysgerminomas), embryonal carcino-

mas, choriocarcinomas, or compositions of these

histological entities (Gobel et al., 2000; Schneider

et al., 2004; Oosterhuis et al., 2005). In addition,

cystic ovarian teratomas and spermatocytic semino-

mas of the elderly are considered distinct entities

(Oosterhuis et al., 2005). Characteristically, the ma-

jority of childhood GCTs are diagnosed at nongo-

nadal sites, while in adults gonadal tumors (der-

*Correspondence to: Dominik T. Schneider, M.D., Clinic ofPediatric Oncology, Hematology and Immunology, Heinrich-Heine-University, Medical Center, Moorenstr. 5, 40225 Dusseldorf,Germany. E-mail: dominik.schneider@uni-duesseldorf.de

Supported by: German Cancer Aid (Deutsche Krebshilfe).

Received 31 March 2006; Accepted 26 June 2006

DOI 10.1002/gcc.20363

Published online 8 August 2006 inWiley InterScience (www.interscience.wiley.com).

VVC 2006 Wiley-Liss, Inc.

GENES, CHROMOSOMES & CANCER 45:995–1006 (2006)

moid cysts of the ovary and malignant GCTs of the

testis) predominate (Schneider et al., 2004).

These distinct epidemiological, clinical, and path-

ologic patterns correlate with differences in chromo-

somal alterations. Postpubertal malignant GCTs are

characterized by an isochromosome 12p or 12p

amplification, gain of the chromosomes 7, 8, and

21q, loss of 11, 13, and 18, and other less frequent

imbalances (Oosterhuis et al., 2005). Prepubertal

malignant GCTs demonstrate imbalances at chromo-

some 1 (loss at 1p, gain at 1q), loss at 6q, and gain of

20q, whereas the genetic changes characteristic of

postpubertal tumors are lacking (Perlman et al.,

1994, 2000; Bussey et al., 1999; Mostert et al., 2000;

Schneider et al., 2001(a), 2002; van Echten et al.,

2002). Notably, in these reports no other recurrent

aberration has been reported at such considerably

variable frequency as loss at 1p, with reported fre-

quencies ranging from less than 10% to more than

80%. It is unclear, whether this variability is attribut-

able to technical or biological differences. Thus, it

should be noted that although the loss of a chromo-

some leads to LOH in diploid cells, a chromosome

copy number loss in triploid or high-ploidy tumors

such as GCTs may not lead to LOH. Another poten-

tial source of the discrepant reports could be the use

of different techniques such as microsatellite analy-

sis, fluorescence in situ hybridization (FISH), com-

parative genomic hybridization (CGH), and cytoge-

netic analysis. Of note, the latter techniques detect

chromosomal loss, which is not necessarily related to

true loss of heterozygosity in poly- or aneuploid

tumors such as GCTs. Therefore, the terminology

‘‘chromosome loss’’ and ‘‘loss of heterozygosity

(LOH)’’ must be used in a sensible fashion. How-

ever, because LOH at 1p is of prognostic signifi-

cance in other embryonal tumors (e.g., neuroblas-

toma, nephroblastoma), this issue is of considerable

interest in pediatric GCTs. To clarify the question of

the frequency of true allelic loss in GCTs and to

evaluate a potential prognostic relevance in this tu-

mor entity, we combined CGH with an extensive

microsatellite analysis. These two independent ex-

perimental approaches provide complementary in-

formation. To take the biological heterogeneity into

account we included GCTs from different clinical,

histological, and genetic subgroups into the study.

MATERIALS ANDMETHODS

Patients

Paired tumor and constitutional samples (periph-

eral blood lymphocytes or adjacent non-cancerous

tissue) of 31 patients (20 males, 11 females) were an-

alyzed. Formalin-fixed and paraffin-embedded

archival tumor tissue was obtained from the Kiel Pe-

diatric Tumor Registry in Kiel. In addition, institu-

tions participating in the German prospective proto-

col for nontesticular GCTs in children and adoles-

cents (MAKEI 96) (Gobel et al., 2000) provided

fresh-frozen samples that were collected in the

MAKEI GCT tissue bank with support of the Bio-

Case project of the Competence Network of Pediat-

ric Oncology. For nine adult GCTsamples, extracted

DNA from tumor and matched non-cancerous testic-

ular tissue was available from a former study per-

formed at the Clinic of Urology, Heinrich-Heine

University, Dusseldorf, Germany (Schmidt et al.,

2001). Since these patients were not MAKEI proto-

col patients, no clinical follow-up data were avail-

able. All tumors were histologically classified accord-

ing to the WHO classification of GCTs (Mostofi

et al., 1993). In teratoma, the grade of immaturity

was evaluated as described by Gonzalez-Crussi et al.

(1978). The clinical and pathologic characteristics of

the tumors are summarized in Table 1. The labora-

tory investigators were blinded to the clinical follow-

up data of the patients.

For statistical analysis and presentation, the sam-

ples were divided into teratomas and YSTs of infants

and children as well as GCTs of adolescents and

adults (seminomas and nonseminomas). According

to their potentially malignant clinical presentation

and their association with testicular intratubular neo-

plasia (syn. carcinoma in situ), testicular teratomas

were included in the group of non-seminomatous

GCTs of adolescents and adults.

The group of childhood GCTs consisted of 13

samples, including one immature and three mature

teratomas (one testicular, one ovarian, and two coc-

cygeal) and nine yolk sac tumors (three testicular

and six coccygeal), which showed additional tera-

tomatous elements in two samples. The group of

GCTs of adolescents and adults included 18 tumors.

Among these, there were three testicular seminomas

and one ovarian dysgerminoma. In addition, the

group of non-seminomatous GCTs included three

testicular mature teratomas, four testicular embry-

onal carcinomas, one mediastinal choriocarcinoma,

and six mixed malignant GCTs (two testicular, four

ovarian). Among the latter group, a pericardial re-

lapse of an ovarian mixed malignant GCTand a lung

metastasis of a testicular mixed malignant GCTwere

analyzed. Both samples were collected after chemo-

therapy, while all other samples were collected prior

to cytostatic therapy.

H&E stained sections were evaluated prior to

DNA extraction to estimate the proportion of tu-

Genes, Chromosomes & Cancer DOI 10.1002/gcc

996 ZAHN ETAL.

mor in paraffin or frozen tissue blocks, respectively.

If tumor content was >80%, the tissue was extracted

completely. Otherwise, areas containing pure tu-

mor and pure non-cancerous tissue were manually

dissected from unstained sections and collected

separately.

The present study was reviewed and approved

by the Local Ethics Committee of the Heinrich-

Heine University, Dusseldorf, Germany.

DNA Extraction from Fresh-Frozen Tissue

and Peripheral Blood Lymphocytes

Genomic DNA was extracted using a DNeasy

Tissue Kit (Qiagen, Hilden, Germany) for tissue

samples and a QIAamp Blood Kit (Qiagen) for

blood samples according to the manufacturer’s

instructions.

DNA Extraction from Archival Tissue

Five 5 lm sections were deparaffinized with xy-

lene and rehydrated through an alcohol series. For

dissection of tumor and surrounding non-cancerous

tissue, five to ten 10 lm sections on glass slides

were manually dissected after deparaffinization

with a razor blade using a stained section as a

guide. The tissue was incubated at 558C for 72 h

with SDS/proteinase K (1.5 mg/ml, new enzyme

added after 24 and 48 hr) (Isola et al., 1994), fol-

lowed by DNA extraction using the DNeasy Tis-

sue Kit (Qiagen).

Chromosomal Comparative Genomic

Hybridization Analysis

Labeling of Tumor and Reference DNA

Male tonsil DNA was used as reference for CGH.

Prior to labeling, the presence of high-molecular

weight DNA and absence of overwhelming DNA

degradation (in archival tissues) were confirmed by

1% agarose gel electrophoresis, and the amount

of DNA was determined with UV spectroscopy.

Tumor and reference DNA (2 lg) were directly la-

beled with Spectrum-Green- and Spectrum-Red-

dUTP (Vysis, Downers Grove, Illinois) using nick

translation, as previously described (Riopel et al.,

1998; Perlman et al., 2000; Schneider et al., 2002).

The amounts of DNase and DNA-Polymerase

(Invitrogen, Karlsruhe, Germany) and the reaction

time were carefully adjusted to obtain labeled

DNA fragments of 500 to 1,500 bp length on a 1%

agarose gel.

Precipitation, Hybridization, and

Post-Hybridization Washes

Eight hundred nanograms of labeled tumor and

reference DNA were ethanol coprecipitated with

20 lg of Cot-1 DNA (Invitrogen). The precipitate

was resuspended in hybridization buffer containing

50% formamide, 2xSSC, and 10% dextran sulfate.

The probe was denatured for 10 min at 758C and

partially reannealed for 30 min at 378C. The probe

was hybridized for three days at 378C to normal

male metaphase chromosomes (Vysis) that previ-

ously had been thawed out from –208C, washed for

5 min in 2xSSC, and denatured for 2 min at 708Cin 70% formamide, 2xSSC, pH 7.0. Post-hybridiza-

tion washes were performed in 2xSSC at 708C,378C, and at room temperature, for 5 min each.

The chromosomes were counterstained with 0.1

lg/ml 406/diamidino-2-phenylindole-2HCl (DAPI)

(Sigma, Munchen, Germany) and covered with

Glycerol/PBS solution AF1 (Science Services,

Munchen, Germany). The experimental protocol

TABLE 1. Clinical and Pathologic Characteristics of the 31 Patients Included in this Study

Age groupa Site Histology N S

Infants/children Teratoma Testis Mature teratoma 1Ovary Immature teratoma 1Coccyx Mature teratoma 2 4

Malignant GCT Testis Yolk sac tumor 3Coccyx Yolk sac tumor 4

Yolk sac tumor + Teratoma 2 9Adolescents/adults Seminoma Testis Seminoma 3

Ovary Dysgerminoma 1Non-Seminoma Testis Embryonal carcinoma 4

Mixed malignant GCT 2b

Mature teratoma 3Ovary Mixed malignant GCT 4c

Mediastinum Choriocarcinoma 1 18

aInfants/children, 0–7 years; adolescents/adults, �8 years.bIncluding a lung metastasis of a testicular mixed malignant germ cell tumor (embryonal carcinoma, choriocarcinoma).cIncluding a progressive ovarian mixed malignant germ cell tumor (yolk sac tumor, growing immature teratoma).

Genes, Chromosomes & Cancer DOI 10.1002/gcc

997LOH 1P IN CHILDHOOD GERM CELL TUMORS

was validated using control samples that had been

characterized by conventional cytogenetic analysis.

Microscopic Analysis

Gray-level images were acquired for each fluores-

cent dye with a charge-coupled device camera on a

Zeiss Axioscope epifluorescence microscope. These

images were analyzed with the Applied Imaging

Corporations’s dedicated Cytovision soft- and hard-

ware for ‘‘high resolution’’ CGH (HR-CGH, ac-

cording to the protected designation by Cytovision

Inc., Santa Clara, California) (Ness et al., 2002). In

each case, 15 metaphases were captured and after

exclusion of overlapping chromosomes, at least

12 non-overlapping autosomal chromosomes (and

10 sex chromosomes) were included for profiling.

After subtraction of background fluorescence, the

green/red ratio of each entire metaphase was nor-

malized to 1.0. According to the designated high-

resolution modus for CGH analysis, average fluo-

rescence ratios and 95% confidence intervals were

calculated and compared with dynamic standard

reference intervals, thus increasing the sensitiv-

ity and spatial resolution of chromosomal CGH to

*5 MB (Kirchhoff et al., 2001). According to this

method, an experimental bias in the analysis of

individual samples is reduced by scaling poten-

tially systematic deviations by a constant factor

that fits the standard reference intervals to the

ratios of the test slide at locations representing nor-

mal DNA. Chromosomal regions were considered

imbalanced if the 95% confidence intervals of

hybridization of tumor DNA did not overlap with

the dynamic standard reference intervals, corre-

sponding to a statistical significance of P < 0.05.

Microsatellite Analysis

To estimate whether the imbalances at chromo-

some arm 1p detected with HR-CGH are caused

by true allelic losses, we performed automated

microsatellite analysis.

Degenerate Oligonucleotide-Primed (DOP)-PCR

For the further analysis of dissected tissue samples,

DOP-PCR was applied prior to microsatellite anal-

ysis to universally amplify the tumor and normal

DNA obtained by SDS/proteinase K treatment.

The protocol was modified from a published

method (Telenius et al., 1992) and was initially

developed for genome amplification prior to CGH

analysis. In this context, the method has been vali-

dated by comparing CGH profiles of samples with

defined cytogenetically characterized aberrations

that have been used with and without amplifica-

tion of the starting material. Since there was no dif-

ference in the CGH profiles, an amplification bias

caused by DOP-PCR could be excluded. Based on

this experience, the same DOP-PCR protocol was

used prior to microsatellite analysis. DOP-PCR

was carried out in a final volume of 20 ll containing1 ng of template DNA (tumor or non-cancerous tis-

sue), 1.65 mM MgCl2, 0.2 mM of each dNTP, 1 lMprimer 6 MW (50-CCG ACT CGA GNN NNN

NAT GTG G-30), and 2.5 U Expand Long Tem-

plate enzyme mixture (Roche, Mannheim, Germany).

The cycling conditions were 5 min at 958C, fol-lowed by 5 cycles of 30 sec at 948C, 30 sec at 308C,and 1.5 min at 728C, followed by 35 cycles of

30 sec at 948C, 30 sec at 438C, and 1.5 min at 728C.The reaction was completed by a final extension

step of 7 min at 728C. The PCR product was

checked on a 1% agarose gel.

PCR Amplification of Polymorphic Markers

Mapped to 1p

A total of 23 chromosomal loci were evaluated for

allelic imbalances (AI) by PCR amplification of

locus specific (CA)n-repetitive microsatellite DNA.

Eleven of these markers mapped to 1p36.1-p36.3

(D1S243, D1S468, D1S2660, D1S2795, D1S2870,D1S450, D1S244, D1S2667, D1S507, D1S2644,D1S2843) and eight mapped to 1p13.2-1p35.2

(D1S247, D1S427, D1S2788, D1S2829, D1S2876,D1S206, D1S2651, D1S2881), resulting in an aver-

age resolution of 1.9 Mb in the distal and 10.5 Mb

in the more proximal parts of chromosome arm 1p.

Tumor samples harboring a segmental allelic

imbalance were evaluated with four additional

markers in 1p34.1-p34.3 (D1S2656, D1S2729,D1S2706, D1S2713) for a precise determination

of the proximal extent of the unbalanced region.

The sequences and mapping information for the

primers D1S243, D1S468, D1S2660, D1S2795,D1S2870, D1S508, D1S450, and D1S244 have been

described by Dib et al., (Dib et al., 1996). The in-

formation for the remaining primers were obtained

from the Genome Database (http://gdb.org) and

UCSC Genome Browser (http://genome.ucsc.edu,

May 2004 Freeze). The primers were purchased

from MWG Biotech (London, United Kingdom),

with each forward primer fluorescence-labeled

with 6-FAM.

PCR was performed with 10 ng of template

DNA, 1.5 mM MgCl2, 0.2 mM of each dNTP,

0.5 lM of each primer, and 0.5 U Taq polymerase

(Qiagen) in a final volume of 20 ll. All primers

were applied under the same conditions using a

Genes, Chromosomes & Cancer DOI 10.1002/gcc

998 ZAHN ETAL.

touch-down PCR approach with an annealing tem-

perature ranging from 63 to 548C with a gradual

reduction of 18C per cycle. The cycling conditions

were 5 min at 958C, followed by 10 cycles of 30 sec

at 958C, 30 sec at the designed annealing tempera-

ture, and 30 sec at 728C, followed by 26 cycles of

30 sec at 958C, 30 sec at 548C, and 30 sec at 728C.The reaction was completed by a final extension

step of 2 min at 728C.PCR products were first run on a 2% agarose gel

to check the efficacy of PCR amplification.

Depending on band intensities, PCR products

were diluted 1:1 to 1:25 in H2O, 1 ll of which was

mixed with 0.5 ll of an internal standard-size

marker (GS-400-HD ROX size standard, Applied

Biosystems, Foster City, California) and 12 llHiDi-formamide (Applied Biosystems), denatured

for 2 min at 958C, chilled on ice, and fractionated

by capillary electrophoresis using an ABI 3130 sys-

tem (Applied Biosystems). Data were collected

automatically and analyzed using GeneMapper 3.7

software (Applied Biosystems), which provides

quantification based on peak height and area. Data

were transferred into Excel data sheets, and statis-

tical analysis was performed with the SPSS 12.0

software package (SPSS Inc., Chicago, Illinois). A

paired sample was considered to be evaluable if at

least five markers were polymorphic in the consti-

tutional DNA. To exclude a DOP-PCR amplifica-

tion bias, the experimental protocol has been vali-

dated for each individual marker using original

DNA and DOP-PCR amplified DNA as control-

templates for microsatellite analysis.

Quantification and Thresholds

Allelic imbalance was quantitatively assessed by

calculating the AI index, which was defined as the

allele ratio in the tumor sample divided by the al-

lele ratio in the corresponding normal sample. The

allele ratio was calculated as the peak height of

the smaller allele divided by the peak height of

the larger allele. AI indices >1 were converted to

be in the range of 0–1. The cutoff value for consid-

ering an allelic imbalance was set at �0.5. To

delineate the deleted chromosomal region in spe-

cific tumors, we included the microsatellite loci

that showed AI as well as their interstitial (and

telomeric) non-informative loci located within the

region harboring the allelic imbalance (Fig. 1).

The location of the AI border on the cytogenetic

map was then obtained from the UCSC database

and the region of the aberrant chromosome was

reconstructed.

RESULTS

Among the 31 tumors included in this study, 20

were derived from male and 11 from female

patients. Age at diagnosis ranged from neonates to

39 years. For comparison of the different age

groups, a age cutoff of eight years was chosen. In

accordance with the general epidemiological pat-

terns of GCTs, most tumors of infancy and child-

hood were nongonadal primaries, whereas all but

one tumor of adolescent and adult patients were

gonadal. Furthermore, the histological subtypes

characteristic of the different age groups were

adequately represented. Thus, teratomas and yolk

sac tumors were analyzed in the childhood group

of tumors, while seminomas and nonseminomatous

tumors of the different histological entities were

included in the group of older patients. Table 2

summarizes the clinical and pathologic data in rela-

tion to the chromosomal imbalances detected with

chromosomal CGH and microsatellite analyses,

sorted by tumor histology and patient’s age.

CGH Analysis

GCTs of nine pediatric and 11 adolescent/adult

patients were examined for chromosomal imbalan-

ces using CGH. Chromosomal imbalances were

revealed in four of five analyzed malignant pediat-

ric GCTs with a loss at chromosome arm 1p seen in

all altered tumors. The common region of overlap

mapped to 1p35-pter. In one tumor (Case 8), an in-

terstitial gain at 1p12-p31 was detected in addition

to the terminal deletion at this chromosome arm.

Other recurrent aberrations in this age group

included concomitant gain of 1q as well as gain of

chromosome 20 or 20q (three tumors each) and a

loss at 6q (two tumors). In contrast, all pure terato-

mas as well as one malignant tumor of this age

group, which consisted of 95% teratoma and 5%

YST histology (Case 5), showed normal CGH pro-

files.

In malignant GCTs of adolescent and adult

patients, chromosomal aberrations were found in

eight of 11 analyzed tumors with two showing loss

at 1p. The most frequent chromosomal imbalance

in GCTs of this age group was a gain of 12p, which

was found in six tumors. Notably, chromosomal

imbalances were also found in both analyzed tes-

ticular mature teratomas of adult patients, includ-

ing a gain of 12p, but no imbalance at 1p.

Microsatellite Analysis

To evaluate whether the chromosomal imbalan-

ces at chromosome arm 1p detected by CGH in

Genes, Chromosomes & Cancer DOI 10.1002/gcc

999LOH 1P IN CHILDHOOD GERM CELL TUMORS

this and previous studies are indeed related to true

allelic loss, we performed automated microsatellite

analysis using a total of 19 polymorphic markers

mapped to 1p13-pter. We additionally expanded the

series by including nine cases of adult testicular can-

cer. Thus, 31 GCTs and matched constitutional sam-

ples were investigated. The results are summarized

in Figure 1 and Table 2. At least eight markers were

informative for each constitutional sample. Using a

stringent cutoff value for detection of LOH, eight of

nine YSTs of infants and children displayed LOH.

In these tumors, the region of imbalance extended

to the most distal informative marker, indicating a

terminal deletion, while the proximal boundaries

varied throughout the chromosome arm. Using four

additional microsatellite markers, the minimal com-

monly deleted region was defined by tumor 14 with

the proximal boundary located between D1S450 in

1p36.22 to D1S2870 in 1p36.31, resulting in a dele-

tion size between 6.2 and 9.5 Mb. In accordance

with its balanced CGH profile, the only malignant

tumor without LOH was the tumor with predomi-

nant teratoma histology (Case 5). Otherwise, no

LOH at 1p was detected in pure teratomas of

infancy and childhood.

In contrast to the childhood GCTs, only three out

of 18 analyzed malignant GCTs of adolescent and

adult patients showed terminal LOH at 1p, with the

most distal boundary marked by D1S507 and a

resulting aberration size of at least 14.9 Mb. Notably,

two patients with LOH were ovarian GCTs and

showed tumor progression or relapse. One of three

testicular mature teratomas was also determined to

have LOH at chromosome arm 1p (case 23). This tu-

mor displayed LOH for five of 12 informative loci

with the AI indices exceeding the cutoff value of

0.5, while the remaining seven loci had borderline

indices, making this tumor a borderline case, when

the stringent cutoff criteria are applied.

Comparison of CGH Profiles and AI Patterns

of Chromosome Arm 1p

As shown in Table 2, 20 tumors were investigated

with both chromosomal CGH and microsatellite

analysis. With the exception of one tumor, concord-

ant results were obtained by both techniques. In

Figure 1. Results of the microsatellite analysis of 23 markers mapped to 1p13.2-1p36.3. Each columnrepresents the data of a single tumor sorted by age and histology with each line corresponding to onemarker. The cytogenetic position of the markers is provided on the left. Open boxes, both alleles retained;black boxes, allelic imbalance (AI); grey boxes, AI presumed because of imbalance of neighboring markers;n.i., not informative.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

1000 ZAHN ETAL.

detail, chromosomal imbalances at 1p were identi-

fied by both methods in six and no imbalances at 1p

in 13 tumors. In a testicular teratoma with borderline

AI indices (case 23), CGH showed a balanced state

of this chromosome region but detected other chro-

mosomal imbalances characteristic of adult GCTs

such as gain of 12p (Table 2). Both analyses were

performed on the same DNA sample.

All six GCTs harboring a deletion at 1p detected

with CGH also showed LOH at the same chromo-

TABLE 2. Summary of the Clinical, Pathologic, and Genetic Data of the 31 Patients Included in this Study,Sorted by Histology and Age

No. Tissue Age Sex Site Histologya Status AI 1p CGH Losses CGH Gains

1 FFT 0 mo M Coccyx MT NED – – –2 FFPET 1 mo M Testis MT NED – – –3 FFT 14 mo F Ovary IT NED – –4 FFT 14 mo F Coccyx MT n.d. – – –5b FFT 0 mo M Coccyx TER, YST NED – – –6 FFT 6 mo M Testis YST NED 1p13.2-pter 1p, 16p12-13.2,

18q, 20p12–

7 FFPET 9 mo M Testis YST NED 1p13.3-pter n.a. n.a.8 FFT 18 mo F Coccyx YST NED 1p13.2-pter 1p35-pter,

2q34-qter,4q, 6q24-qter,16, 19, 20p

1p12-p31,1q21-qter, 2p,2q14-q24,3p21-pter, 3q, 12,14q23-qter,20q13-qter,

9 FFPET 20 mo M Testis YST NED 1p21.2-pter n.a. n.a.10 FFPET 22 mo F Coccyx YST, TER NED-2 1p36.1-pter n.a. n.a.11 FFPET 22 mo M Coccyx YST NED 1p36.3-pter n.a. n.a.12 FFT 27 mo F Coccyx YST NED 1p34.2-pter 1p34-pter,

6q24-qter,18q21-qter

1q32-qter,20q11-13

13 FFT 24 mo M Coccyx YST NED 1p34.3-pter 1p35-pter,5q, 10p,13q32-qter, 16

1q, 2p, 3q, 4p,13q12-q31,14, 18q, 20, 21

14 FFPET 9 y M Mediastinum CHC NED – n.a. n.a.15c FFT 9 y F Ovary-Relapse

(Pericardium)IT, YST AWD 1p36.1-pter 1p36-pter –

16 FFT 10 y F Ovary YST, DYS, IT NED – – –17 FFT 10 y F Ovary DYS, CHC NED – – –18 FFT 12 y F Ovary YST, IT NED – – 319 FFT 12 y F Ovary DYS NED-2 1p36.2-pter 1p35-pter,

4q, 13, 2112p

20 FFT 15 y M Testis EC, YST NED – – 12p21d FFT 15 y M Testis + lung

metastasisEC, CHC NED – – –

22 FFT 16 y M Testis EC NED – – 12p23 FFT 22 y M Testis MT n.d. 1p13.2-pter 18q21-qter 7q11.2-21,

7q32-36, 12p24 FFT 24 y M Testis MT n.d. – n.a. n.a.25 FFT 26 y M Testis MT n.d. – 10q 12p26 FFT 39 y M Testis SE n.d. – n.a. n.a.27 FFT 27 y M Testis SE n.d. – n.a. n.a.28 FFT 27 y M Testis SE n.d. – n.a. n.a.29 FFT 22 y M Testis EC n.d. – n.a. n.a.30 FFT 27 y M Testis EC n.d. – – 12p31 FFT 27 y M Testis EC n.d – n.a. n.a.

FFT, fresh frozen tissue; FFPET, formalin fixed and paraffin embedded tissue; mo, month; y, years; M, male; F, Female; n.a., not analyzed; AI, allelic imbal-

ance; MT, mature teratoma; IT, immature teratoma; DYS, dysgerminoma; SE, seminoma; EC, embryonal carcinoma; YST, yolk sac tumor; CHC, chorio-

carcinoma; MGCT, mixed malignant germ cell tumor; NED, no evidence of disease; NED-2, no evidence of disease after recurrence; AWD, alive with

disease; n.a., not analyzed; n.d., no data.aIn patients with mixed malignant GCTs, the predominant histology of the analyzed tissue block is provided in bold letters.bPredominant teratoma histology (95%) with YST microfoci (5%).cPrimary ovarian MGCT (YST, IT), tissue extracted after initiation of chemotherapy.dA lung metastasis of a testicular MGCT (EC, CHC) was analyzed, tissue was extracted after initiation of chemotherapy.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

1001LOH 1P IN CHILDHOOD GERM CELL TUMORS

some region, suggesting that the aberrant CGH

profiles at 1p seen in our patients represent true

allelic loss. The close correlation between the

CGH profiles—evaluated according to the high-

resolution technology (Applied Imaging)

(Kirchhoff et al., 2001)—and the AI patterns

obtained using microsatellite analysis is shown in

Figure 2a (Case 12). In this representative experi-

Figure 2. Comparison of HR-CGH pro-files and allelic imbalance patterns detectedwith microsatellite analysis for cases 12 (a)and 8 (b). The different microsatellitemarkers and their corresponding cytogeneticpositions on chromosome arm 1p, the calcu-lated AI indices, and the resulting allele statusare listed on the left and compared to theHR-CGH profile on the right. The HR-CGHprofile includes the mean and 95% confi-dence intervals ratio-profiles of each experi-ment calculated over at least 15 metaphases.The 95% standard reference intervals arepositioned underneath the confidence inter-vals. The vertical lines represent green/redfluorescence ratios of 0.5, 0.75, 1.0, 1.25, and1.75. Deviations to the left and to the right ofthe ideogram indicate chromosomal gainsand losses, respectively. (a) Detection ofLOH at 1p34-pter in case 12. With a stand-ard chromosomal CGH algorithm, this termi-nal deletion of 1p would have escaped detec-tion, because the profile does not cross thetraditional cutoff of 0.75. However, throughthe introduction of dynamic standard refer-ence intervals in the HR-CGH algorithm, thisimbalance becomes apparent. The HR-CGHfinding is supported by LOH analysis. (b)Detection of LOH at 1p35-pter and gain at1p31-qter in case 8. At both regions, thechromosomal imbalances can be visualized byHR-CGH and result in allelic imbalance,which could be misinterpreted as LOH with-out the knowledge of the HR-CGH profile.However, only at 1p35-pter, the allelic imbal-ance corresponds to LOH.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

1002 ZAHN ETAL.

ment, CGH demonstrated a terminal deletion at

1p34-pter, which is associated with a true allelic

loss across exactly the same region.

In one tumor, the CGH profile displayed a loss

at the distal and a gain at the proximal region of 1p

(Fig. 2b, Case 8). To a certain extent, this remarka-

ble CGH profile is also reflected in the allelic ratio

retrieved with microsatellite analysis. This analysis

demonstrated a pronounced loss of one allele at

distal 1p (mean, 0.11, range 0.06–0.22). In contrast,

PCR products from loci mapped to proximal 1p

exhibited both alleles with a less pronounced

change in the allelic ratio (mean 0.43, range 0.26–

0.65). Notably, the difference of the AI levels at

both chromosomal regions was statistically signifi-

cant (student’s t test, P < 0.001). However, even

with the use of stringent cutoffs that by far exceed

those used in the majority of LOH studies, the

imbalances at proximal 1p would have been con-

sidered LOH without the knowledge of the CGH

profile.

DISCUSSION

Deletions at chromosome arm 1p have been

implicated in the pathogenesis of a broad spectrum

of human malignant diseases, including embryonal

cancers such as neuroblastoma and nephroblastoma

(Schwab et al., 1996). The most widely studied

embryonal tumor that shows loss at 1p is neuro-

blastoma, in which it is highly correlated with

MYCN amplification and an unfavorable prognosis,

suggesting the presence of one or more tumor sup-

pressor genes within this region that modulate

response to therapy (Brodeur and Nakagawra,

1992; Maris and Matthay, 1999; Attiyeh et al.,

2005).

Chromosomal loss at 1p has also been noted in

several cases of adolescent testicular and ovarian

GCTs and in pediatric GCTs (Perlman et al., 1994;

Bussey et al., 1999; Perlman et al., 2000; Mostert et

al., 2000; Schneider et al., 2001(a); Bussey et al.,

2001; Schneider et al., 2002; van Echten et al.,

2002). Of note, these studies were based on FISH,

CGH, or cytogenetic analyses, which detect chro-

mosomal copy number loss but do not determine

true allelic loss. However, the incidence of loss at

1p in pediatric GCTs varies significantly between

the different studies. In the largest cytogenetic

analysis of pediatric GCTs including 81 patients,

deletion at 1p was observed in only one of 20 ana-

lyzed malignant childhood GCTs (Bussey et al.,

1999). In contrast, Perlman et al. detected loss at

1p in five of seven cases analyzed with conven-

tional cytogenetic techniques, indicating signifi-

cant observer-dependent differences in the assess-

ment of 1p alterations (Perlman et al., 1994). Com-

plementary FISH analyses using a 1p36 cosmid

probe coupled with a centromeric or 1q probe

reported a relative reduction in the number of

1p36 signals compared to the centromeric or 1q sig-

nals in up to 80% of childhood YSTs (Jenderny et

al., 1996; Perlman et al., 1996; Stock et al., 1996;

Bussey et al., 2001). However, since conventional

chromosomal CGH revealed infrequent loss at dis-

tal 1p but frequent gain of 1q, including the cen-

tromeric region, there has been some debate

whether the results of the FISH studies were

related to gain of 1q rather than loss of 1p (Perlman

et al., 2000; Schneider et al., 2001(a), 2002). On the

other hand, the terminal region of chromosome

arm 1p is apparently difficult to evaluate with both

conventional G-Banding or conventional chromo-

somal CGH for technical reasons (Kirchhoff et al.,

1998). In consequence, smaller deletions at 1p may

escape detection with conventional chromosomal

CGH.

These technical limitations could be overcome

with complementary techniques such as the

recently developed genome-wide array CGH tech-

nology (Solinas-Toldo et al., 1997; Pinkel et al.,

1998). A recent array CGH study surveyed chromo-

somal imbalances in childhood YSTs and observed

a 50% (4/8) prevalence of loss at distal 1p in malig-

nant pediatric GCTs, with a minimal region of

overlap mapped to 1p35-pter (Veltman et al.,

2005).

To investigate the frequency of loss at 1p in our

cohort, we performed a multimodal experimental

approach that combined microsatellite analysis and

the improved chromosomal high resolution CGH

technique (Kirchhoff et al., 1998). The latter tech-

nique expands the concept of conventional chro-

mosomal CGH by the use of an optimized algo-

rithm based on dynamic standard reference inter-

vals (Applied Imaging). This technique results in a

threefold increase of sensitivity and spatial resolu-

tion compared to conventional CGH (Kirchhoff

et al., 1998; Kristensen et al., 2003).

With this approach, we confirm the previously

reported distinct cytogenetic patterns of pediatric

and adult GCTs. These chromosomal patterns cor-

relate with age rather than tumor site or histologi-

cal composition. More specifically, we found the

characteristic losses at 1p and 6q and gains of 1q

and 20q in childhood YSTs, irrespective of testicu-

lar or coccygeal primary site. In adolescent and

adult GCTs, we detected the characteristic gain of

12p, which constitutes the biological hallmark of

Genes, Chromosomes & Cancer DOI 10.1002/gcc

1003LOH 1P IN CHILDHOOD GERM CELL TUMORS

GCTs of this age group and also appears irrespec-

tive of gonadal or nongonadal site (Schneider et al.,

2006). Focusing on 1p, we detected chromosomal

losses in four of five analyzed childhood YSTs, with

a commonly deleted region mapped to 1p35-pter,

identical to that observed with array CGH

(Veltman et al., 2005). Thus, our data support the

aforementioned FISH studies that demonstrated a

high frequency of deletion at distal 1p in malignant

pediatric GCTs. Furthermore, our findings support

the assumption that conventional chromosome-

based CGH may indeed underestimate the occur-

rence of deletions at distal 1p. As illustrated in Fig-

ure 2a, the deletion at 1p would escape detection if

analyzed with the standard CGH algorithm using

fixed thresholds, e.g. 0.75 and 1.25 (Kallioniemi

et al., 1992).

Nevertheless, it has to be pointed out, that none

of the aforementioned techniques is able to detect

true allelic loss. However, if a potential tumor sup-

pressor gene is located at distal 1p, true allelic loss

should occur and result in concordant information

from CGH and microsatellite analysis. Indeed, two

previous LOH studies showed LOH at 1p in 5/11

and 6/8 informative tumors, respectively (Hu et al.,

2000; Kato et al., 2003). However, since both stud-

ies included only two polymorphic markers at dis-

tal 1p, the precise extent of the allelic loss was not

determined.

The present more detailed microsatellite analy-

sis of chromosome arm 1p covers the distal region

of 1p (1p36.1-36.3) at a mean interval of 1.9 Mb

and the more proximal region (1p13.2-35.2) at a

mean interval of (6.9Mb). We identified a highly

prevalent allelic loss at 1p in childhood YSTs (8/9

tumors), irrespective of their anatomical localiza-

tion in the testes (3 YSTs) or at the coccyx (5

YSTs). Of note, the only malignant childhood tu-

mor without LOH consisted of predominant tera-

toma histology with only microscopic foci of YST,

and pure teratomas in children are considered to

be chromosomally normal in almost all cases.

Moreover, the technical aspects of microsatellite

analysis have also to be considered critically. In

general, the ratio between two alleles in tumor tis-

sue is calculated and compared to the allelic ratio

in reference tissue. On one hand, in tumors with

inhomogeneous cell populations, LOH may not

result in a complete disappearance of the affected

allele, and LOH may remain undetected if bulk

DNA is analyzed. On the other hand, particularly

in poly- or aneuploid tumors such as GCTs, differ-

ential gain of a certain chromosomal region may

mimic allelic loss of the homologous chromosomal

region. Accordingly, we observed a considerable

variability of the allelic ratios in adult testicular

GCTs (mean 1.05, standard variation 0.22, range

0.65–1.45), even in the absence of LOH or chro-

mosomal loss demonstrated by HR-CGH. This

problem can at least in part be overcome either by

a microsatellite analysis that uses a retained allele

as an internal control in a multiplex PCR approach

(Hogarty et al., 2000; Bauer et al., 2001) or by strin-

gent cutoff levels—a precaution not frequently

taken in microsatellite studies. However, the com-

bination of microsatellite analysis with alternative

experimental techniques such as CGH certainly

represents the best possible control. This issue is

highlighted by the profile illustrated in Figure 2b.

Here, a clear-cut LOH of distal 1p is detected with

both HR-CGH and microsatellite analysis. How-

ever, HR-CGH revealed that proximal 1p is part of

a chromosomal gain that involves the centromeric

region and the long arm of chromosome 1. Notably,

microsatellite analysis of this region consistently

demonstrated allelic imbalances at levels that

could be misinterpreted as LOH if the CGH pro-

file is not determined.

Our combined experimental approach, which

allows a distinct differentiation between chromo-

some copy number loss and true allelic loss, pro-

vides clear evidence that deletions at chromosome

arm 1p previously reported at variable frequencies

are very frequent events in malignant pediatric

GCTs and indeed represent true allelic loss. This

suggests a fundamental role of LOH at 1p in the

pathogenesis of malignant GCTs of infants and

children and strongly indicates the presence of one

or more tumor suppressor genes in this chromo-

somal region.

In our investigation, the deleted region

extended more than one third of the short chromo-

some arm, with the proximal boundaries localized

close to the centromere (three tumors) or in 1p34-

p35 (four tumors). One tumor displayed a smaller

terminal deletion with the proximal breakpoint

located within the region between D1S450 in

1p36.22 and D1S2870 in 1p36.31, thus narrowing

the region of interest involved in the pathogenesis

of pediatric GCTs to distal 1p36. However, analysis

of a greater number of samples might allow for fur-

ther delineating the commonly deleted region, as

it has been accomplished in neuroblastomas (Hen-

rich et al., 2006). The commonly deleted region in

neuroblastomas is encompassed in the region

deleted in childhood GCTs. It currently includes

only two annotated genes, calmodulin binding

transcription activator 1 (CAMTA1) and FLJ10737

Genes, Chromosomes & Cancer DOI 10.1002/gcc

1004 ZAHN ETAL.

(Henrich et al., 2006). Current data suggest that

reduced expression of CAMTA1 at 1p36.31-1p36.23

portends a poor prognosis in neuroblastomas, irre-

spective of LOH 1p status (Hogarty et al., 2001).

This indicates that other mechanisms of down-reg-

ulation of gene expression may also be relevant.

Current research focuses on a potential coinci-

dence of genetic and epigenetic changes at distal

1p. But thus far, no mutations were found and an

imprinting mechanism was excluded (Caren et al.,

2005). Accordingly, there is no strong indication

that in neuroblastomas there is a cooperation

between allelic loss and epigenetic inactivation of

genes at 1p36 (Araki et al., 2002; Carling et al.,

2003; Lomas et al., 2004).

However, in other tumor entities such as squa-

mous cell carcinoma, meningeoma, and endocrine

cancer, genes at distal 1p may become subject to

epigenetic inactivation (Kato et al., 2003). One of

the potential candidate genes, the transcription

factor RUNX3 located at 1p36 outside the com-

monly deleted region of neuroblastomas, has previ-

ously been analyzed in a small study of infantile

yolk sac tumors. The authors observed a coinci-

dence of LOH at neighboring 1p36 sequences and

RUNX3 promoter hypermethylation in six tumors

(Kato et al., 2003; Bergthorsson et al., 2006).

Therefore, a synergy of genetic and epigenetic

events may occur, thus indicating for a different bi-

ological impact of changes at 1p in childhood yolk

sac tumors compared to other embryonal tumors

such as neuroblastomas.

The high frequency of LOH at 1p and the over-

all favorable prognosis of childhood GCTs indicate

that LOH at 1p is obviously not associated with

adverse outcome in childhood malignant GCTs.

This assumption is supported by the clinical data

summarized in Table 2. In fact, the high frequency

of LOH at 1p might additionally distinguish child-

hood YSTs from other embryonal tumors such as

neuroblastoma with frequencies between 25 and

35% and an association with unfavorable outcome.

Conversely, according to our and previous data,

deletion or LOH of 1p36 constitutes a significantly

less frequent event in malignant GCTs of adoles-

cence and adulthood (Oosterhuis et al., 2005).

Again, this finding indicates a different pathogenic

pathway of childhood GCTs compared to GCTs of

postpubertal patients. Notably, two adolescent girls

with LOH at 1p suffered a relapse or showed re-

fractory disease (MGCT with a growing teratoma

component). Therefore, a prognostic relevance of

LOH at 1p in this age group cannot be excluded,

warranting a prospective evaluation in a suitably

large group of patients enrolled onto prospective

therapeutic protocols. It is also important that, in

contrast to childhood teratomas, which were genet-

ically balanced, both analyzed testicular mature

teratomas of adult patients showed chromosomal

aberrations, which resemble those of malignant

tumors. This finding correlates with the histopa-

thologic observation that teratomas of the adult

testis may be associated with testicular intratubular

neoplasia, indicating a common pathogenesis with

other malignant nonseminomas (Ulbright, 2004).

Therefore, adult testicular teratomas should be

grouped together with malignant non-seminoma-

tous GCTs.

In summary, we demonstrate that LOH at 1p

constitutes a genetic parameter that distinguishes

different biological and clinical subgroups of GCTs

and opens perspectives for future biological and

clinical prognostic studies.

ACKNOWLEDGMENTS

Tissue samples have in part be collected through

the BioCase project of the Competence Network

Pediatric Oncology, funded by the German Minis-

try of Research and Technology. The HR-CGH fa-

cility has been donated by the Elterninitiative

Kinderkrebsklinik e.V. Dusseldorf. We thank Sus-

anne Koch for data management and Luiz-Fer-

nando Lopes for critical reading of the manuscript.

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Genes, Chromosomes & Cancer DOI 10.1002/gcc

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