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
1

Department of Pediatrics1, Cell Therapy and Transplantation Medicine 2, and Department of Pediatrics, Shiga University of Medical school, 4Cancer

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

5

Division of Hematology/Oncology, Saitama Childrens Medical Center, and Gunma Childrens 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: jtakita-tky@umin.ac.jp

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.

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 neuroblastoma cell
4

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 manufacturers 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 manufacturers 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..

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

5

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 MRT. In our analysis, 3 cases did not show bi-allelic inactivation of SMARCB1. Among them, two 2 cases
6

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 genes alterations in large number ofmany MRT samples would be necessary to
7

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). Methylation analysis was also performed in 20 neuroblastoma cell lines, but we did not detected any
8

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-azadeoxycytidine. 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.

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 Childrens 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

References

[1]J.B. 1 Beckwith JB, N.F. Palmer NF., Histopathology and prognosis of Wilms tumors: results from the First National Wilms' Tumor Study. Cancer 1978; 41: (1978) 1937-1948. [2]M.R. 2 Wick MR, J.H. Ritter JH, L.P. Dehner LP., Malignant rhabdoid tumors: a clinicopathologic review and conceptual discussion. Seminars in diagnostic pathologySemin Diagn Pathol 1995; 12: (1995) 233-248. [3]M. 3 Bhattacharjee M, J. Hicks J, L. Langford L, et al.R. Dauser, D. Strother, M. Chintagumpala, M. Horowitz, L. Cooley, H. Vogel, Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood. Ultrastructural pathology Pathol 1997; 21: (1997) 369-378. [4]I. 4 Versteege I, N. Sevenet N, J. Lange J, et al. M.F. Rousseau-Merck, P. Ambros, R. Handgretinger, A. Aurias, O. Delattre, Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 1998; 394: (1998) 203-206. [5]J.A. 5 Biegel JA, B. Fogelgren B, L.M. Wainwright LM, J.Y. Zhou JY, H. Bevan H, L.B. Rorke LB., Germline INI1 mutation in a patient with a central nervous system atypical teratoid tumor and renal rhabdoid tumor. Genes, Chromosomes & cCancer 2000; 28: (2000) 31-37. [6]J.M. 6 Carter JM, C. O'Hara C, G. Dundas G, et al. D. Gilchrist, M.S. Collins, K. Eaton, A.R. Judkins, J.A. Biegel, A.L. Folpe, Epithelioid malignant peripheral nerve sheath tumor arising in a schwannoma, in a patient with "neuroblastoma-like" schwannomatosis and a novel germline SMARCB1 mutation. Am J Surg Pathol The American journal of surgical pathology 2012; 36: (2012) 154-160. [7]T.J.7 Hulsebos TJ, A.S. Plomp AS, R.A. Wolterman RA, E.C. Robanus-Maandag EC, F. Baas F, P. Wesseling P., Germline mutation of INI1/SMARCB1 in familial schwannomatosis. American journal of human geneticsAm J Hum Genet 2007; 80: (2007) 805-810. [8]J. 8 Calderaro J, J. Moroch J, G. Pierron G, et al F. Pedeutour, C. Grison, P. Maille, P. Soyeux, A. de la Taille, J. Couturier, A. Vieillefond, M.C. Rousselet, O. Delattre, Y. Allory, SMARCB1/INI1 inactivation in renal medullary carcinoma. Histopathology 2012; 61: (2012) 428-435. [9]Y. 9 Chen Y, J. Takita J, Y.L. Choi YL, et al.M. Kato, M. Ohira, M. Sanada, L. Wang, M. Soda, A. Kikuchi, T. Igarashi, A. Nakagawara, Y. Hayashi, H. Mano, S. Ogawa, Oncogenic mutations of ALK kinase in neuroblastoma. Nature 2008; 455: (2008) 971-974. [10]M. 10 Kato M, M. Sanada M, I. Kato I, et al.Y. Sato, J. Takita, K. Takeuchi, A. Niwa, Y . Chen, K. Nakazaki, J. Nomoto, Y . Asakura, S. Muto, A. Tamura, M. Iio, Y . Akatsuka, Y. Hayashi, H. Mori, T. Igarashi, M. Kurokawa, S. Chiba, S. Mori, Y . Ishikawa, K. Okamoto, K. Tobinai, H. Nakagama, T. Nakahata, T. Yoshino, Y. Kobayashi, S. Ogawa, Frequent inactivation of A20 in B-cell lymphomas. Nature 2009; 459: (2009) 712-716. [11]E.M. 11 Jackson EM, A.J. Sievert AJ, X. Gai X, et al.H. Hakonarson, A.R. Judkins, L. Tooke, J.C. Perin, H. Xie, T.H. Shaikh, J.A. Biegel, Genomic analysis using highdensity single nucleotide polymorphism-based oligonucleotide arrays and multiplex ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors. Clin Cancer ResClinical cancer research : an official journal of the American Association for Cancer Research 2009; 15: (2009) 1923-1930. [12]M.C. 12 Fruhwald MC, M. Hasselblatt M, S. Wirth S, et al.G. Kohler, R. Schneppenheim, J.I. Subero, R. Siebert, U. Kordes, H. Jurgens, J. Vormoor, Non-linkage of familial rhabdoid tumors to SMARCB1 implies a second locus for the rhabdoid tumor predisposition syndrome. Pediatric blood &

11

cancerPediatr Blood Cancer 2006; 47: (2006) 273-278. [13]J. 13 Takita J, Y . Chen Y, J. Okubo J, M. Sanada M, et al. M. Adachi, K. Ohki, R. Nishimura, R. Hanada, T. Igarashi, Y. Hayashi, S. Ogawa, Aberrations of NEGR1 on 1p31 and MYEOV on 11q13 in neuroblastoma. Cancer Sciscience 2011; 102: (2011) 1645-1650. [14]K. 14 Uno K, J. Takita J, K. Yokomori K, et al.Y . Tanaka, S. Ohta, H. Shimada, F.H. Gilles, K. Sugita, S. Abe, M. Sako, K. Hashizume, Y. Hayashi, Aberrations of the hSNF5/INI1 gene are restricted to malignant rhabdoid tumors or atypical teratoid/rhabdoid tumors in pediatric solid tumors. Genes, c Chromosomes & cCancer 2002; 34: (2002) 33-41. [15]C. 15 Zweier C, E.K. de Jong EK, M. Zweier M, et al. A. Orrico, L.B. Ousager, A.L. Collins, E.K. Bijlsma, M.A. Oortveld, A.B. Ekici, A. Reis, A. Schenck, A. Rauch, CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila. American journal of human genetics Am J Human Genet 2009; 85: (2009) 655-666. [16]J. 16 Takita J, M. Ishii M, S. Tsutsumi S, et al. Y. Tanaka, K. Kato, Y. Toyoda, R. Hanada, K. Yamamoto, Y. Hayashi, H. Aburatani, Gene expression profiling and identification of novel prognostic marker genes in neuroblastoma. Genes, cChromosomes & cCancer 2004; 40: (2004) 120-132. [17]J. 17 Takita J, H.W. Yang HW, Y .Y. Chen YY, et al. R. Hanada, K. Yamamoto, T. Teitz, V. Kidd, Y . Hayashi, Allelic imbalance on chromosome 2q and alterations of the caspase 8 gene in neuroblastoma. Oncogene 2001; 20: (2001) 44244432. [18]J.18 Takita J, Y . Hayashi Y, T. Nakajima T, et al. J. Adachi, T. Tanaka, N. Yamaguchi, Y . Ogawa, R. Hanada, K. Yamamoto, J. Yokota, The p16 (CDKN2A) gene is involved in the growth of neuroblastoma cells and its expression is associated with prognosis of neuroblastoma patients. Oncogene 1998; 17: (1998) 3137-3143. [19]D. 19 Muller D, R. Millon R, S. Theobald S, et al. T. Hussenet, B. Wasylyk, S. du Manoir, J. Abecassis, Cyclin L1 (CCNL1) gene alterations in human head and neck squamous cell carcinoma. British journal of cancer Br J Cancer 2006; 94: (2006) 1041-1044. [20]A.J. 20 Ramsay AJ, V. Quesada V, M. Foronda M, et al. L. Conde, A. MartinezTrillos, N. Villamor, D. Rodriguez, A. Kwarciak, C. Garabaya, M. Gallardo, M. Lopez-Guerra, A. Lopez-Guillermo, X.S. Puente, M.A. Blasco, E. Campo, C. Lopez-Otin, POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia. Nature gGenetics 2013; 45: (2013) 526-530. [21]N. 21 Omura N, C.P. Li CP, A. Li A, et al. S.M. Hong, K. Walter, A. Jimeno, M. Hidalgo, M. Goggins, Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma. Cancer Biol Therbiology & therapy 2008; 7: (2008) 1146-1156. [22]M. 22 Tsikitis M, Z. Zhang Z, W. Edelman W, D. Zagzag D, G.V. Kalpana GV., Genetic ablation of Ccyclin D1 abrogates genesis of rhabdoid tumors resulting from Ini1 loss. Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Soc U S A 2005; 102: (2005) 12129-12134. [23]P. 23 Nair P, K. De Preter K, J. Vandesompele J, F. Speleman F, R.L. Stallings RL., Aberrant splicing of the PTPRD gene mimics microdeletions identified at this locus in neuroblastomas. Genes, c Chromosomes & cCancer 2008; 47: (2008) 197-202. [24]D.A. 24 Solomon DA, J.S. Kim JS, J.C. Cronin JC, et al. Z. Sibenaller, T. Ryken, S.A. Rosenberg, H. Ressom, W. Jean, D. Bigner, H. Yan, Y. Samuels, T. Waldman, Mutational inactivation of PTPRD in glioblastoma multiforme and malignant melanoma. Cancer Resresearch 2008; 68: (2008) 10300-10306. [25]S. 25 Poliak S, L. Gollan L, R. Martinez R, et al. A. Custer, S. Einheber, J.L. Salzer, J.S. Trimmer, P. Shrager, E. Peles, Caspr2, a new member of the neurexin

12

superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels. Neuron 1999; 24: (1999) 1037-1047. [26]S. McAvoy S, S.C. Ganapathiraju SC, A.L. Ducharme-Smith AL, et al. J.R. Pritchett, F. Kosari, D.S. Perez, Y. Zhu, C.D. James, D.I. Smith, Non-random inactivation of large common fragile site genes in different cancers. Cytogenet ic and gGenome Res research 2007; 118: (2007) 260-269.

13

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 allelespecific 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 shows RT-PCR analysis of CNTNAP2 in 9 cell lies. Sequence chromatogram
14

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 M 5-aza-2deoxycytidine (+). SJNB-1 neuroblastoma cell line, which expressed abundant CNTNAP2, was used as a positive control.

15

Table 1. Mutations of SMARCB1 (SNF5/INI1) in malignant rhabdoid tumor. Sample name KYM-1 TM87-16 TTC-1240 TTC-549 TTC-642 TTN-45 YAMRT STM-91-01 RTK(J) -4N MRT-2 MRT-3 MRT-4 MRT-5 MRT-6 MRT-7 MRT-8 MRT-9 MRT-10 MRT-12 MRT-13 MRT-14 Attribute Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary 22q homoD homoD UPD homoD UPD UPDhomoD UPD homoD homoD homoD homoD homoD UPD+homoD UPD+homoD homoD UPD+homoD UPD normal heteroD heteroD heteroD Mutation of SMARCB1 homoD homoD G646T (E216 stop) homoD C118T (R40 stop) homoD del exons 1-3 homoD homoD homoD homoD homoD homoD homoD homoD homoD 27bp to ex6 553 del ag to aa intron 5, D224 stop

16

Table 2. Recurrent gains and losses in malignant rhabdoid tumor. chromosome Gain 4 (CL: TM87-16, 1 2 3 120,804,640 41,745,732 158,297,982 245,519,990 42,067,857 158,507,647 124,715 322 209 TTC-549, YAMRT, P:MRT-8) 1 (CL:TTC1240) 2 (CL:TM87-16, P:MRT-8) 4 (CL:TM87-16, 7 123,981,822 124,568,564 587 TTC-1240, TTC-549, TTN-45) 7 7 16 Loss 2 3 3 4 7 7 9 16 9,582,260 71,464,505 10,072,420 71,708,905 490 244 140,875,055 60,099,620 78,740,688 182,649,464 69,446,093 141,241,576 60,538,672 78,995,893 182,977,255 69,593,327 366 439 255 328 147 1 (CL:STM-91-01) 3 (CL:TM87-16, TTC-642, YAMRT) 2 (CL:TM87-16, P:MRT-8) 3 (CL:TTC-642, KYM-1, RTK(J)-4N) 2 (CL:TM87-16, TTC-642) 3 (CL:STM-91-01, TTC-549, P:MRT-7) 3 (CL:TM87-16, TTC-1240, KYM-1) 2 (CL:TTC-642, P:MRT-8) LRP1B FHIT ROBO1 none genes AUTS2 CNTCAP2 PTPRD ATBF1 125,675,684 130,358,924 76,268,665 125,906,795 130,939,457 77,096,360 231 580 828 2 (CL:TM87-16, KYM-1) 3 (CL:TM87-16, TTC-1240, TTN-45) 2 (CL:TM87-16, KYM-1) GRM8 none gene CLEC3A, WWOX POT1 none gene CCNL1, VEPH1 many genes Position* start end Length (kb) Number of affected samples# Gene(s)

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

17

18

19

STable S1. Analyzed samples and SNP array platform Sample name Cell line / Primary Array KYM-1 Cell line XbaI 50K array TM87-16 Cell line NspI 250K array TTC-1240 Cell line XbaI 50K array TTC-549 Cell line XbaI 50K array TTC-642 Cell line NspI 250K array TTN-45 Cell line XbaI 50K array YAMRT Cell line NspI 250K array NspI 250K array STM-91-01 Cell line RTK(J) -4N MRT-2 MRT-3 MRT-4 MRT-5 MRT-6 MRT-7 MRT-8 MRT-9 MRT-10 MRT-12 MRT-13 MRT-14 Cell line Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary XbaI 50K array NspI 250K array XbaI 50K array XbaI 50K array XbaI 50K array NspI 250K array NspI 250K array NspI 250K array NspI 250K array NspI 250K array NspI 250K array NspI 250K array NspI 250K array

20