Cancer Science Report Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays Junko Takita 1 MD, PhD, Yuyan Chen 1 MD, PhD, Motohiro Kato 1 MD PhD, Kentaro Ohki 1 MD PhD, Shigeru Ohta 3 , MD, PhD, Riki Nishimura 1 MD, PhD, Noriko Hoshino 1 MD, Masafumi Seki 1 MD, Masashi Sanada 4 MD, Akira Oka 1 , MD, PhD, Yasuhide Hayashi 6 MD, PhD, and Seishi Ogaw 4 MD, PhD 1 Department of Pediatrics 1 , Cell Therapy and Transplantation Medicine 2 , and 3 Department of Pediatrics, Shiga University of Medical school, 4 Cancer Genomics Project, Graduate school of Medicine, University of Tokyo, 5 Division of Hematology/Oncology, Saitama Children’s Medical Center, and 6 Gunma Children’s Medical Center Address reprint requests to Junko Takita MD, PhD Department 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: [email protected]1
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Cancer Science
Report
Genome-wide approach to identify second gene targets for
malignant rhabdoid tumors using high-density oligonucleotide
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: [email protected]
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.
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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
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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
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.
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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.
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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.
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