Cancer Science

Report

Genome-wide approach to identify second gene targets for

malignant rhabdoid tumors using high-density oligonucleotide

microarrays

Junko Takita1 MD, PhD, Yuyan Chen1 MD, PhD, Motohiro Kato1 MD PhD,

Kentaro Ohki1 MD PhD, Shigeru Ohta3, MD, PhD, Riki Nishimura1 MD, PhD,

Noriko Hoshino1 MD, Masafumi Seki1 MD, Masashi Sanada4 MD, Akira

Oka1, MD, PhD, Yasuhide Hayashi6 MD, PhD, and Seishi Ogaw4 MD, PhD

1Department of Pediatrics1, Cell Therapy and Transplantation Medicine2,

and 3Department of Pediatrics, Shiga University of Medical school, 4Cancer

Genomics Project, Graduate school of Medicine, University of Tokyo,

5Division of Hematology/Oncology, Saitama Children’s Medical Center, and

6Gunma Children’s Medical Center

Address reprint requests to

Junko Takita MD, PhDDepartment of Pediatrics,Graduate School of Medicine, 　University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan　TEL: +813-3815-5411 Ex33462　FAX: +813-3816-4108 Email: jtakita-tky@umin.ac.jp

1

Summary

Malignant rhabdoid tumor [Remark 1](MRT) is a rare and highly

lethal cancer that mainly affects infants and young children.　 The vast

majority of malignant rhabdoid MRT tumors are characterized by loss of

function of the SMARCB1 gene on chromosome 22q11.2. However, little is

known about other genetic changes than SMARCB1 alterations that are

responsible for the development and/or progression of malignant rhabdoid

tumorsMRT. To explore additional gene targets in malignant rhabdoid

tumorsMRT, we analyzed 21 malignant rhabdoid tumor MRT specimens

using high-density single nucleotide polymorphism (SNP)–genotyping

microarrays (Affymetrx® GeneChip®). Although malignant rhabdoid

tumor MRT genomes are characterized by common 22q11.2 deletions

affecting the SMARCB1 locus with a frequency of 95.2% (20/21

specimens), other genetic changes were have been less frequent. Of the

20 specimens with deletions of 22q11.2, 8 specimens showed uniparental

disomy of the SMARCB1 locus with homozygous deletions or gene

mutations. High-resolution analysis also disclosed the recurrent

hemizygous/homozygous deletions of 7q35-q36.1 involving CNTNAP2

locus in 3 specimens. Mutations analysis of CNTNAP2 exhibited a novel

R157C missense mutation in a primary case, and methylation analysis

showed recurrent hypermethylation of CNTNAP2 in 3 of /9 cell lines.

These results demonstrated that CNTNAP2 is one of the additional gene

targets other than SMARCB1 in malignant rhabdoid tumorsMRT.

2

Introduction

Malignant rhabdoid tumor (MRT) is an extremely rare and highly

aggressive neoplasm that typically develops in infancy or early childhood..

MRT was initially described as ‘rhabdomyosarcomatoid’, [Remark 2] an

aggressive type of [Remark 3] tumor of the kidney, , and subsequent

studies have revealed that this tumorMRT occurs in various sites including

the central nervous system (CNS), lung, liver, skin, and soft tissues.. The

most frequent common location of the tumors is the kidneys, followed by

the CNS, and the tumors originatinge from the latter site being are

referred to as atypical teratoid/rhabdoid tumor (AT/RT).. Cytogenetic and

molecular analyses for of MRT have shown recurrent deletions at 22q11.2,

which resulted in identification of SMARCB1 [(OMIN #601607)] as a

characteristic gene abnormality of this tumor.. Germ-line and somatic

mutations/deletions of SMARCB1 have been recently [Remark 4] reported

in AT/RT, as well as in epithelioid sarcoma, familial schwannomatosis, and

renal medullary carcinoma.. The SMARCB1 gene is a member of the ATP-

dependent SWF/SNF chromatin-remodeling complex, and is recruited to

promoters of genes that regulate cell cycle, growth, and differentiation.. In

MRT, SMARCB1 appears to function as a classic tumor suppressor gene,

such that germ-line mutations and deletions predispose to the development

of these malignancies;, and somatic loss or mutation of the other allele

constitutes the second hit.

In recent years, genome-wide copy number analysis using single

nucleotide polymorphism (SNP) arrays (SNP-chip) has shown to have an

outstanding power to reveal detailed profiles of genomic abnormalities and

to identify new genetic targets in various cancers.. A previous report

3

showed that high-resolution SNP-chip analysis could detect bi-allelic

alterations in SMARCB1 in almost of all MRT cases, which suggests that

SMARCB1 is the primary mutational gene target responsible for the

development of MRT and provided further evidence for the clinical utility of

molecular diagnostic testing.. However, some MRT cases retain expression

of the protein, even with in a fraction of familial MRTs have been reported

that are not associated with a SMARCB1 inactivation..[Remark 5] These

findings suggest the possibility of additional relevant genetic loci distinct

from SMARCB1. However, the detailed genetic abnormalities in MRT other

than chromosome 22 have not been fully understood. Therefore, to clarify

the additional genetic lesions involved in the pathogenesis of MRTs, we

performed SNP-chip analysis for of 21 MRT samples.

Materials and Methods

Specimens

This study was approved by the ethicsal board of the University of Tokyo

(Approval Number 1598). Primary tumor specimens were obtained at the

time of the initial surgery or biopsy from patients who were diagnosed as

having MRT or AT/RT at collaborating hospitals. In total, 14 primary MRT

specimens (4 samples of AT/RT) and 9 cell lines derived from patients with

MRT patinets (KYM-1, TM87-16, TTC-1240, TTC-549, TTC-642, TTN-45,

YAMRT, RTK(J)-4N, and STM-91-01) were analyzed in this study. The TCC

TTC series and STM-91-01 were generous gifts from Dr. Ohta. KYM-1,

YAMRT, TTNN-45, and RTK(J)-4N were generous gifts from Dr. Inoue, Dr.

Sugita, Dr. Kanegane and Dr. Yokomori. [Remark 6] [Remark 7]. A

4

neuroblastoma cell line, SJNB-1, was used as a control in the methylation

analysis. All cell lines were cultured in RPMI-1640 supplemented with 9%

fetal bovine serum..

Microarray analysis

High molecular weight DNA was isolated from tumor specimens and

subjected to SNP array analysis using Affymetrix® GeneChip® Mapping

50K and/or 250K arrays (Affymetrix, Inc.)[Remark 8] according to the

manufacturer’s protocol (STable S1). After appropriate normalization of

mean array intensities, signal ratios between tumor and normal cells were

calculated, and allele-specific copy numbers were inferred from the

observed signal ratios based on the hidden Markov model using

CNAG/AsCNAR software (http://www.genome.umin.jp)..

Mutation and expression analyses of SMARCB1 and CNTNAP2

Direct sequencing analyses of all coding exons of SMARCB1 and CNTNAP2

was were carried out in all samples as previously described. . The primer

sequences and conditions of PCRs reactions for mutation analyses of these

genes are have been described in the previous papers. . Total RNA was

extracted from the 9 cell lines using Isogen reagent (Nippon Gene, Osaka,

Japan) according to the manufacturer’s instructions and subjected to

reverse-transcriptionRT reactions to synthesize cDNA using the

SuperScript Preamplification System for first-strand cDNA synthesis (Life

Technologies, Inc., Rockville, MD, USA). Semi-quantitative RT-PCR analysis

for CNTNAP2 expression was performed as described previously..

5

Methylation-specific PCR and 5-aza-2-deoxycytidine treatment.

Bisulfate modification of genomic DNA was performed as previously

described. . For methylation-specific PCR (MSP), approximately 10 ng of

bisulfite-treated DNA was amplified with primers for both the methylated

and unmethylated sequences. Reaction products were separated by

electorophoresis on a 2.0% agarose gel. 5-Aza-2-deoxycytidine (Sigma

Chemical[Remark 9) was dissolved in cold RPMI 1640 immediately before

use. Cells were exposed to 0.5 and 1.0 mM of 5-aza-2-deoxycytidine for 3

days, with the medium and drug being replaced in every 24 h. . Then

cCells then were harvested and used for RT-PCR analysis.

Results and Discussion[Remark 10]

Our SNP-chip analysis revealed a high frequency of deletion at 22q11.2 in

the MRT genome, as previously described.. In total, 20 out of 21

specimens (95.2%) had loss of heterozygousity (LOH) at 22q11.2 involving

the SMARCB1 locus (Fig.ure 1A, B). In 8 samples, uniparental disomy

(UPD) of 22q segments caused homozygous mutations/deletions of

SMARCB1 (Fig.ure 1B). Ten samples had homozygous focal deletions

commonly involving a 175 kb region (ch22:22,353,181-22,528,353), which

exclusively included SMARCB1. Subsequent mutation analysis revealed

that 4 samples with heterozygous deletion or UPD at the 22q11.2 locus had

mutations in the SMARCB1 gene. An MRT-derived cell line, with 22qUPD

(YAMRT) harbored a small deletion involving exons 1-3, which was not

detectable by SNP array analysis. In total, 18 out of the 21 MRT samples

had bi-allelic aberrations of SMARCB1, indicating genetic homogeneity of

6

MRT. In our analysis, 3 cases did not show bi-allelic inactivation of

SMARCB1. Among them, two 2 cases with MRT of the kidney showed

heterozygous deletions of the 22q11.2 locus, but the remaining case with

AT/RT did not have any genetic alterations in the SMARCB1 locus.

Immunohistochemical analyses of these 3 cases showed positive results for

vimentin, but negative findings for muscle lineage markers and SMARCB1,

supporting the diagnosis of MRT or AT/RT. Thus, genetic and/or epigenetic

alterations of promoter regions of SMARCB1 should be considered for the

possible alterations for inactivation of SMARCB1 in these cases.

Although recurrent copy number changes other than 22q11.2 deletions

were less frequent in MRT (Fig.ure 1A), 7 loci of gains and 8 loci of losses

were commonly detected in multiple samples (Table 2). Importantly, some

of the regions contained potential gene targets which that were known to

be associated with tumorigenesis of other cancers, such as CCNL1, POT1,

CNTNAP2, and PRPTD (Table 2).. In the previous reports [Remark 11],

heterozygous knockout mice developed tumors consistent with MRT,

beginning as early as 5 weeks of age (ref)[Remark 12], but crossed mice

with SMARCB1 +/- mice and CCND1 -/- mice [Remark 12]did not develop

any spontaneous tumor. . Thus, these findings suggested that CCND1,

located at 11q13.3, may be a key mediator involved in the genesis of MRT.

Interestingly, our analysis identified recurrent gains at CCNL1, which

functions in association with cyclin-dependent kinases, including CCND1,

suggestinged that CCNL1 acts as one of the mediators involved in the

development of MRT. The PTPRD and POT1 were mutated or down

regulated in several human cancers, including neueroblastoma, lung

cancer, and chronic myeloid leukemia ;, and thus, further analysis of these

7

genes alterations in large number ofmany MRT samples would be

necessary to assess their involvements in the pathogenesis of MRT.

Homozygous deletion is very informative for identifying gene targets.

Indeed, SMARCB1 was identified from homozygously deleted regions at

22q11.2.. In this the present study, we found recurrent homozygous

deletions of CNTNAP2 locus at 7q35-q36 in the 2 specimens, and another

one case exhibited hemizygous deletion in this region (Fig.ure 1Aa).

CNTNAP2 encodes a single-pass transmembrane protein mediating cell-cell

interactions in the nervous system [Remark 13]. . This gene is located in a

common fragile site which that is inactivated in different types of cancers

including brain tumor, ovarian cancer, and breast cancer. . Furthermore,

methylation of the promoter region of CNTNAP2 has been reported in

pancreatic adenocarcinoma. . Although these results suggest that

CNTNAP2 is implicated in tumor suppressor genes for several cancers, the

oncogenic role of this gene in the MRT has been poorly studied. Thus, to

assess the involvements of CNTNAP2 this gene in the MRT pathogenesis,

we further performed mutation, expression, and methylation analyses in

our series. Through mutation analysis of coding region of CNTNAP2, we

found a novel R157C missense mutation in a fresh tumor of MRT, which

was not found in 60 healthy volunteers, not registered in dbSNP 137

(http://www.ncbi.nlm.nih.gov/projects/SNP/) and in 1000 genomes

(http://www.1000genomes.org/) (Fig.ure 2Bb). This single nucleotide

change was scored as "probably damaging" or "damaging" by two 2

computational prediction software packagess (i.e., SIFT and PolyPhen-2).

Furthermore, 3 of 9 cell lines lacking CNTNAP2 expression displayed

methylation of the CpG island of the CNTNAP2 (Fig.ure 2Bb, Cc).

8

Methylation analysis was also performed in 20 neuroblastoma cell lines,

but we did not detected any methylation of CpG islands of the CNTNAP2

(data not shown). To elucidate whether hypermethylation of CNTNAP2

blocked gene expression in MRT cells, we subjected all 3 cell lines to 3

days’ exposure of to 5-aza-deoxycytidine. As shown in Fig.ure 2Dd,

CNTNAP2 expression was induced by 5-aza-deoxycytidine treatment in the

3 cell lines, indicating that methylation of the CpG island is a direct

mechanism for CNTNAP2 silencing in MRT.

Consistent with other reports, our results illustrated the fact that

SMARCB1 is the primary gene implicated in the pathogenesis of MRT.

Although frequencies of recurrent genetic changes other than SMARCB1

locus were low, our findings suggest that CNTNAP2 is one of the potential

second gene targets for MRT. Further studies will beare necessary to

unravel the oncogenic effects of CNTNAP2 in MRT.

9

Acknowledgements

We are grateful to Ms. Matsumura, Ms. Hoshino, Ms. Yin, Ms. Saito, Ms.

Mori, and Ms. Ogino for their excellent technical assistance. We also wish

to express our appreciation to Dr. Sugita, Yamanashi Medical University;,

Dr. Yokomori, Graduate School of Medicine, University of Tokyo;, Dr.

Kanegane, Toyama University;, and Dr. A. Inoue, St. Jude Children’s

Research Hospital, for their generous gifts of MRT cell lines. This work

was supported by Research on Measures for Intractable Diseases, Health,

and Labor Sciences Research Grants, Ministry of Health, Labor and

Welfare; by Research on Health Sciences focusing on Drug Innovation; by

the Japan Health Sciences Foundation; by Core Research for Evolutional

Science and Technology, Japan Science and Technology Agency; and by

Project for Development of Innovative Research on Cancer Therapeutics.

Disclosure statement

The authors have no conflicts of interest to declare.

10

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Figure Legends

Figure 1. Copy number changes detected in malignant rhabdoid

tumors. A: Characteristics of copy number alterations in malignant

rhabdoid tumors. Regions showing statistically significant increase or

decrease in genomic copy number were detected using the GISTIC

algorithm based on SNP array analysis. B: Overall representation of

aberrations of chromosome 22q11.2 in malignant rhabdoid tumors.

Specimens indicated by red are cell lines. Pink bar indicates uniparental

disomy, and yellow and green bars indicate heterozygous deletion and

homozygous deletion, respectively. The minimum overlapping deleted

region was expanded in 175kb in chromosome 22q11.2 including

SMARCB1 and other 6 genes. SMARCB1 status is also indicated at the

right. MRT-12 and MRT-14 shows heterozygous deletion of SMARCB1

locus, and wild-type allele of SMARCB1 was retaineds in each case. UPD:

uniparental disomy, HD: homozygous deletion, mt: mutation, and del:

deletion.

Figure 2. Recurrent deletions of chromosome 7q35-q36 and

CNTNAP2 alterations in malignant rhabdoid tumor. A: Deletions of

chromosome 7q35-36 in 3 specimens detected by SNP array. For each

panel, total copy numbers (tCNs) (red dots), moving averages of tCNs for

five 5 consecutive SNPs (blue line), an ideogram of the relevant

chromosome, location of heterozygous SNP calls (green bars), and allele-

specific copy numbers (AsCNs) averaged for five 5 consecutive SNPs (red

and green lines for larger and smaller alleles, respectively) are plotted. B:

Expression and mutation analyses of rhabdomyosarcoma. Upper panel

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shows RT-PCR analysis of CNTNAP2 in 9 cell lies. Sequence chromatogram

of R157C missense mutation detected in a fresh tumor, MRT-8, is shown in

the lower lower panel. C: Bisulfate modification- and methylation- specific

PCR for CNTNAP2 in cell lines. Hypermethylation of CgG island in KYM-1

cell line is shown in the upper panel. The Llower panel shows control.

CpG islands are marked by asterisks. D: Representative results of re-

expression of transcriptionally silenced CNTNAP2 after treatment of 5-aza-

deoxycytidine in malignant rhabdoid tumor cell lines. RT-PCR analysis of

RTK(J)-4N cell line harvested following 72 h incubation with control media

(-) and 5 μM 5-aza-2-deoxycytidine (+). SJNB-1 neuroblastoma cell line,

which expressed abundant CNTNAP2, was used as a positive control.

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Table 1. Mutations of SMARCB1 (SNF5/INI1) in malignant rhabdoid tumor.Sample name Attribute 22q Mutation of SMARCB1

KYM-1 Cell line homoD homoDTM87-16 Cell line homoD homoDTTC-1240 Cell line UPD G646T (E216 stop)TTC-549 Cell line homoD homoDTTC-642 Cell line UPD C118T (R40 stop)TTN-45 Cell line UPD＋homoD homoDYAMRT Cell line UPD del exons 1-3STM-91-01 Cell line homoD homoDRTK(J) -4N Cell line homoD homoDMRT-2 Primary homoD homoDMRT-3 Primary homoD homoDMRT-4 Primary homoD homoDMRT-5 Primary UPD+homoD homoDMRT-6 Primary UPD+homoD homoDMRT-7 Primary homoD homoDMRT-8 Primary UPD+homoD homoDMRT-9 Primary UPD 27bp to ex6 553 delMRT-10 Primary normalMRT-12 Primary heteroDMRT-13 Primary heteroD ag to aa intron 5, D224 stopMRT-14 Primary heteroD 　

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Table 2. Recurrent gains and losses in malignant rhabdoid tumor.

chromosomePosition†* Length

(kb)

Number of

affected samples‡ #Gene(s)

start end

Gain

1 120,804,640 245,519,990 124,715

4 (CL: TM87-16,

TTC-549, YAMRT,

P:MRT-8)

many genes

2 41,745,732 42,067,857 322 1 (CL:TTC1240) none gene

3 158,297,982 158,507,647 2092 (CL:TM87-16,

P:MRT-8)CCNL1, VEPH1

7 123,981,822 124,568,564 587

4 (CL:TM87-16,

TTC-1240, TTC-

549, TTN-45)

POT1

7 125,675,684 125,906,795 2312 (CL:TM87-16,

KYM-1)GRM8

7 130,358,924 130,939,457 580

3 (CL:TM87-16,

TTC-1240, TTN-

45)

none gene

16 76,268,665 77,096,360 8282 (CL:TM87-16,

KYM-1)CLEC3A, WWOX

Loss

2 140,875,055 141,241,576 366 1 (CL:STM-91-01) LRP1B

3 60,099,620 60,538,672 4393 (CL:TM87-16,

TTC-642, YAMRT)FHIT

3 78,740,688 78,995,893 2552 (CL:TM87-16,

P:MRT-8)ROBO1

4 182,649,464 182,977,255 3283 (CL:TTC-642,

KYM-1, RTK(J)-4N)none genes

7 69,446,093 69,593,327 1472 (CL:TM87-16,

TTC-642)AUTS2

73 (CL:STM-91-01,

TTC-549, P:MRT-7)CNTCAP2

9 9,582,260 10,072,420 4903 (CL:TM87-16,

TTC-1240, KYM-1)PTPRD

16 71,464,505 71,708,905 2442 (CL:TTC-642,

P:MRT-8)ATBF1

List of lesions detected in more than two 2 cases, and known genes in the regions.

*†NCBI build 35#‡ CL: cell line, P: primary sample

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STable S1. Analyzed samples and SNP array platformSample name Cell line / Primary ArrayKYM-1 Cell line XbaI 50K arrayTM87-16 Cell line NspI 250K arrayTTC-1240 Cell line XbaI 50K arrayTTC-549 Cell line XbaI 50K arrayTTC-642 Cell line NspI 250K arrayTTN-45 Cell line XbaI 50K arrayYAMRT Cell line NspI 250K array

STM-91-01 Cell line NspI 250K array

RTK(J) -4N Cell line XbaI 50K arrayMRT-2 Primary NspI 250K arrayMRT-3 Primary XbaI 50K arrayMRT-4 Primary XbaI 50K arrayMRT-5 Primary XbaI 50K arrayMRT-6 Primary NspI 250K arrayMRT-7 Primary NspI 250K arrayMRT-8 Primary NspI 250K arrayMRT-9 Primary NspI 250K array

MRT-10 Primary NspI 250K array

MRT-12 Primary NspI 250K arrayMRT-13 Primary NspI 250K arrayMRT-14 Primary NspI 250K array

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