Supplementary data. Exome sequencing of hepatoblastoma reveals novel mutations and. cancer genes in the Wnt pathway and ubiquitin ligase complex

Transcript

1 Supplementary data Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex Deshui Jia 1,*, Rui Dong 2,*, Ying Jing 1, 3,*, Dan Xu 1,*, Qifeng Wang 4, Lei Chen 5, Qigen Li 6, Yuping Huang 1, Yuannv Zhang 1, Zhenfeng Zhang 1, Li Liu 1, Shan Zheng 2, Qiang Xia 6, Hongyang Wang 5,, Kuiran Dong 2,, Xianghuo He 1, To whom correspondence should be This WORD file includes: Supplementary Materials and Methods Supplementary Figures S1 through S5 Other Supplementary Information for this manuscript includes the following: Supplementary Tables S1 through S9 1

2 Materials and Methods Ethics Statement Hepatoblastoma primary tumors and matched peripheral blood lymphocytes and adjacent non-tumor tissues were obtained from affected individuals undergoing surgical resection at the Children s Hospital of Fudan University, the Second Military Medical University and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Participants that these samples were obtained from provided their written informed consent to participate in the study, and the Ethical Review Committee of the WHO Collaborating Center for Research in Human Production authorized by the Shanghai Municipal Government approved this study as well as the consent procedure. Sample selection and preparation for sequencing analyses DNA from 6 primary hepatoblastoma tumors and matched peripheral blood lymphocytes were obtained from affected individuals undergoing surgical resection at the Children s Hospital of Fudan University (Table 1 and Supplementary Table S1). None of the patients had received chemotherapy prior to resection. The tumor tissues were immediately frozen for short-term storage at -80 C. RNA was isolated using TRIzol reagent (Invitrogen) and DNA was isolated using the Qiagen reagents. Genomic DNA from tumor samples weighing up to 10 mg was extracted using the QIAamp DNA Micro Kit (Qiagen 56304) following the manufacturer s instructions. 2

3 Blood lymphocytes were derived from the same patient as the tumor DNA, separated and frozen for short-term storage at -80 C. Lymphocyte DNA was isolated using the QIAamp DNA Mini Kit (Qiagen 51304) and utilized as the normal germline control for genomic analyses. Illumina library construction and Illumina sequencing All methods for the library construction and whole-exome sequencing have been described previously 1. Genomic libraries were prepared using the Illumina TruSeq Sample DNA sample Preparation kit following the manufacturer s instructions. Enrichment was performed as previously described using the Illumina TruSeq Enrichment 62 Mb kit following the manufacturer s recommended protocol. Each exome was sequenced using the 100-bp paired-end protocol on an Illumina HiSeq 2000 DNA Analyzer to produce approximately Gb of sequence per exome. The average exome sequencing coverage was 87.91% at 20 or above. Sequencing reads were aligned to the human reference genome (NCBI Build 37) using the Burrows-Wheeler Aligner (BWA) algorithm with the default setting. Reads that were unmapped, PCR-derived duplicates and reads mapping outside the targeted region of the genome were excluded from the analysis. The genotype files from Affymetrix SNP 6.0 microarrays were used for coverage analysis and for QC purposes. Identification and validation of somatic mutations A mismatched base was identified as a somatic mutation only when it was not present 3

4 in the matched normal sample. All putative somatic mutations were validated visually using the Integrated Genomics Viewer (IGV) and predicted to affect protein function by Polyphen ( All non-synonymous mutations that were identified in the tumor samples were confirmed by independent PCR and sequencing in each tumor and its normal counterpart 2. Validated non-synonymous sequence variations were displayed using the Circos program 3. The 24 identified mutations within 21 somatically altered genes were further sequenced in a validation set composed of an independent series of 24 additional HB tumors and matched controls obtained from three hospitals, including the Children s Hospital of Fudan University (12 cases), the Second Military Medical University (8 cases) and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (4 cases) (Supplementary Table S1). PCR amplification and Sanger sequencing analyses were performed following previously described protocols 2. SPOPmutations have previously been detected in various malignancies, and SPOP has clear mutational hotspots at specific codons in the region encoding the MATH domain 4-6. Therefore, we sequenced all SPOP exons in the validation set. However, resequencing all SPOP coding exons failed to identify any additional tumors with a somatic mutation in this gene. This result suggests that a somatic mutation in the SPOP gene is rare in HB tumors, consistent with previously reported results for breast, lung and liver cancer 7. In addition, we examined mutation status of the 24 identified mutations in two HB cell lines (HepG2 and HUH-6) and did not find any of those novel mutations in the two cell lines, except for the known β-catenin gene mutations 4

5 identified previously in HepG2 and HUH-6 8. SNP array analysis and data process The experiment was performed as previously described 1, 9. Primary HB tumors and paired lymphocyte samples were obtained from 6 HB patients (Supplementary Table S1). DNA was extracted, amplified and hybridized onto an Affymetrix Genome-Wide Human SNP array 6.0 according to the manufacturer s instructions (Affymetrix Inc.). The overall hybridization quality was estimated by the call rate index obtained from Genotyping Console Software (GTC 3.0, birdseed algorithm using default parameter settings). Partek Genomics Suite version 6.4 (Partek Inc., St. Louis, MO) was used for copy number analysis. Regions of CNAs were detected using a Hidden Markov Model (HMM) algorithm in the standard Partek workflow for paired samples. The following criteria were used to define significant CNAs were as follows: a gain or loss was inferred for a copy number of greater than 2.5 or less than 1.5, respectively; and the minimum physical length of the putative CNA was more than 100 kb. Gene annotation and overlap were conducted using the National Center for Biotechnology Information build 36.1 and the University of California, Santa Cruz (UCSC) hg18. Significant focal regions of amplification and deletion were identified by applying GISTIC algorithm with a threshold of 3.6/1.2 (log2 ratio of 0.848/20.737) as described previously 10. Focal regions of copy number alterations were displayed using the Circos program 3. 5

6 Gene expression analysis Total RNA was prepared from the HepG2 cells overexpressing vector control, SPOP wide-type and S119N mutant using TRIzol reagent (Invitrogen). The RNA was then labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer s instructions. Differential expression profiling was performed using an RVM (Random Variance Model) algorithm comparing the SPOP wide-type or mutant with vector control to identify differentially expressed genes (DEGs) (fold change 2). Gene Ontology and pathway enrichment analysis Pathway enrichment analyses of genes harboring somatic SNVs or CNA-related genes were performed using molecule annotation system (MAS), a web-based software toolkit ( Gene Ontology and pathway enrichment analyses were employed to determine the significant functions and pathways of the genes. Fisher s exact test was utilized to select the significant pathways and the threshold of significance was defined as a p-value<0.05 and q-value< Cell lines and cell culture Two HB cell lines (HepG2 and HUH-6) and HEK293T cells were cultured at 37 C in a 5% CO 2 atmosphere in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were regularly certified as being free of mycoplasma contamination. 6

7 Lentiviral vector preparation and infection The lentiviral shrna vectors and the scrambled control shrna were purchased from Open Biosystems (Huntsville, AL, USA). Details of the shrna information are provided in the Supplementary Table 8. The lentiviral constructs for the SPOP, CTNNB1 and CAPRIN2 genes were constructed as described previously 9. The entire coding sequence of the target cdnas was amplified and cloned into the pwpxl vector, which was obtained from Addgene. The SPOP (S119N), CTNNB1 (S33F/G34V and G512V) and CAPRIN2 (R968H/S969C) mutants were generated using a Site-Directed Mutagenesis Kit (Stratagene). Details of the primer sequences and restriction sites are provided in the Supplementary Table 9. Lentivirus production was performed by cotransfecting the viral plasmids with 2 packaging plasmids with Lipofectamine 2000 into HEK293T cells. Supernatants containing the lentiviral particles were harvested at 48 h post-transfection and filtered. Infections were performed with viral supernatant in the presence of 8 μg/ml polybrene (Sigma-Aldrich). Quantitative PCR mrna expression levels were quantified using a 7500 Real-Time PCR System with SDS 2.3 software (Applied Biosystems) according to the manufacturer's instructions. Total RNA was extracted from 33 paired HB primary tumor and adjacent non-tumor tissues, which were reversely transcribed to complementary DNA (cdna) and then 7

8 subjected to q-pcr analysis of the mrna expression of candidate genes. First-strand cdna synthesis and amplification were performed using Reverse Transcription Reagents (Takara) according to the manufacturer's instructions. cdna templates were combined with SYBR Green premix containing Rox (Takara) to perform quantitative PCR reactions. β-actin was used as an endogenous control for mrna level optimization. Details of the primers sequences are provided in the Supplementary Table 9. The 33 paired HB primary tumor and adjacent non-tumor were obtained from three hospitals (Supplementary Table S1), including the Children s Hospital of Fudan University (16 cases), the Second Military Medical University (15 cases) and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (2 cases). Cell proliferation assays Cells were seeded at 2000 cells/well in 96-well plates and incubated. An aliquot of 10 μl of Cell-Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan) was added to triplicate wells and incubated for 2 h. Subsequently, the absorbance was measured at 450 nm to calculate the number of viable cells in each well. Each measurement was performed in triplicate and the experiments were repeated twice. Colony formation assays Cells were seeded in 6-well plates at a concentration of 500 cells per well and cultured at 37 C for 2 wk. At the end of the incubation, the cells were fixed with 8

9 100% methanol and stained with 0.1% crystal violet. Megascopic cell colonies were counted by Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD). Each measurement was performed in triplicate and each experiment was performed at least three times. Immunohistochemistry The experiments were performed as previously described 11. Immunohistochemistry (IHC) was performed on 5-mm sections of formalin-fixed paraffin-embedded (FFPE) tissue from 42 HB individuals (obtained from the surgical specimen archives of the Children s Hospital of Fudan University) by heat-induced epitope retrieval in EDTA (ph 9.0) using a pressure cooker treatment for 2 min. Samples were incubated at room temperature for 1 h with an antibodies against β-catenin (1:100; BD Biosciences), SPOP (1:600; Novus) and Caprin2 (1:00; Novus) followed by incubation with the secondary antibody (Dako); sections were counterstained with hematoxylin. Positive staining was defined according to the percentage of stained cells (0 = no cells, 1 = 1%-4%, 2 = 5%-19%, 3 = 20%-39%, 4 = 40%-59%, 5 = 60%-79%, 6 = 80%-100% of positive cells) and intensity (0-3). Percentage scores were multiplied by intensity scores to yield an overall score. A cytoplasmic and nuclear β-catenin score of >3 were considered as positive, whereas score of 0 to 3 was considered as negative. Cell cycle analysis Cells were fixed into 70% ethanol at 20 for 24 hours, stained with 50 μg/ml 9

10 propidium iodide (Kaiji, NanJing, China), and analyzed using Cytomics FC 500 MPL System (Beckman Coulter, CA). The results were analyzed using CXP software (Beckman Coulter, CA). Reporter Assay The experiments were performed as previously reported 12. Briefly, HUH-6 cells overexpressing GFP, CAPRIN2 WT and mutant constructs were seeded as triplicates in 24-well plates, respectively. Cells were transiently transfected with TOPFLASH or FOPFLASH plasmids (Millipore, Billerica, MA) with Renilla luciferase control vector (Promega, Madison, MI) using Lipofectamine 3000 (Invitrogen). Luciferase activity was measured with a dual-luciferase kit (Promega). Co-immunoprecipitation (Co-IP) Co-IP experiments were performed with the Pierce Co-Immunoprecipitation (Co-IP) Kit (Thermo Scientific) according to the manufacturer's instructions. Whole cell lysates were collected from the specified cells and subjected to co-ip analysis. The antibody against Androgen receptor (Santa Cruz Biotechnology) was used for co-ip. Nuclear and cytoplasmic protein extraction Nuclear and cytoplasmic protein extractions were performed by following the manufacturer s instructions of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). HepG2 and HUH6 cells overexpressing GFP, CAPRIN2 WT and 10

11 mutant constructs were used to extract nuclear and cytoplasmic proteins. Specific antibodies against α-tubulin and Lamin A/C were used as cytoplasmic and nuclear protein control. Immunoblotting analysis For immunoblotting analysis, cells were lysed and protein concentration was determined by the Bradford assay. The cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with specific primary antibodies against SPOP (Thermo Scientific), β-catenin (BD), α-tubulin (Cell Signaling Technology), Lamin A/C (Cell Signaling Technology), Androgen receptor (Cell Signaling Technology), Caprin2 (Proteintech), CDKN2B (Abcam), Ubiquitin (Abcam) or β-actin (Sigma). Statistical analysis Statistical analysis and graphical presentation were performed with GraphPad Prism 5.0. The results are presented as the mean ± s.e.m and were evaluated using an unpaired Student s t-test (two-tailed; p<0.05 was considered to be significant), unless specified otherwise (paired t-test and non-parametric test). Statistical analysis of differences between study groups was performed using an unpaired Student s t-test and p<0.05 was considered statistically significant. A paired Student s t-test was used to analyze differences in the expression of mrna levels among tumors and paired non-tumor tissues in q-pcr analysis. 11

12 Supplementary Figures Supplementary Figure S1. The genomic landscape of somatic copy number alterations in 6 HB samples. Chromosomes are depicted one by one. Samples (HB2, HB3, HB5, HB6, HB8 and HB9) are represented in each chromosome. Amplifications are shown in blue, while deletions are shown in red. 12

13 Supplementary Figure S2. shrna-mediated loss-of-function screening of mutant genes on HB cell proliferation. A. CCK-8 assays depicting representative cell proliferation results in HepG2 and HUH-6 cells after lentivirus-mediated knockdown of OR5I1 and CDC20B genes. B. Colony formation assay showing the effects of knockdown of the SPOP, OR5I1 and CDC20B genes on the proliferation of HepG2 and HUH-6 cells. C. Colony formation assay showing the effects of knockdown of the CTNNB1 and CAPRIN2 genes on the proliferation of HepG2 and HUH-6 cells. D. 13

14 Representative results of CCK-8 assays for the effects of knockdown of KLHL22, GLTSCR1 and TMC5 genes on the in vitro proliferation of HepG2 cells by lentivirus-mediated knockdown. E. Representative results of CCK-8 assays for the effects of knockdown of VCAM1, TRPC4AP and FGGY genes on the in vitro proliferation of HUH-6 cells by lentivirus-mediated knockdown. The results are shown as mean ± s.e.m, *, p<0.05; **, p<0.01; ***, p<

15 Supplementary Figure S3. q-pcr analyses of mrna levels of mutant genes in 33 paired HB and adjacent non-tumor tissues. q-pcr analyses of the expression levels of 6 mutant genes (VCAM1, RNF169, HDCA9, FGGY, C2orf57, GLTSCR1, CAPRIN2, TRPC4AP and KLHL22). p< 0.05 was considered significant. 15

16 Supplementary Figure S4. SPOP S119N is a loss-of-function mutation in HB. A. Representative images of colony formation assays showing GFP, wild-type (WT) SPOP or SPOP S119N overexpression in HepG2 and HUH-6 cells. B. Representative results of cell cycle analyses by FACS. Knockdown of the SPOP promotes the cell cycle progression from G1 to S phase in HepG2 cells. C. q-pcr analyses of the knockdown of SPOP gene on the expression levels of CDKN2B in HepG2 and HUH-6 cells, respectively. 16

17 Supplementary Figure S5. SPOP promotes ubiquitination of Androgen receptor. Overexpression of SPOP increases ubiquitination of endogenous AR, whereas overexpression of SPOP S119N mutant inhibits the ubiquitination of AR. HepG2 cells empty vector (HepG2-GFP), SPOP WT (HepG2-SPOP WT) and SPOP S119N (HepG2-SPOP S119N) were treated with 10 mm MG132 for 8 hr. Immunoprecipitated AR proteins were analyzed by WB for ubiquitination. 17

18 Supplementary Tables Supplementary Table S1. Numbers of HB tumors analyzed by each platform Supplementary Table S2. Somatic sequence variations identified in 6 HB genomes by whole-exome sequencing. Supplementary Table S3. Valdiation of 21 identified mutations in an additional set of 24 HB tumors by sanger sequencing. Supplementary Table S4. Somatic copy number alterations identified in 6 HB genomes by SNP 6.0 arrays. Supplementary Table S5. Focal regions of amplifications and deletions identified in 6 HB genomes. Supplementary Table S6. Immunohistochemical analysis of SPOP and Caprin2 proteins in HB tumors. Supplementary Table S7. The identification of differentially expressed genes in HepG2 cells overexpressing empty vector, SPOP WT and S119N mutant by microarray. In order to identify regulators that mediate SPOP-induced cell cycle arrest, we employed gene expression microarray to explore candidate genes among HepG2 cells overexpressing empty vector (GFP), SPOP wide type (WT) and SPOP S119N mutant. The HepG2-GFP cells were used as reference. Upregulation, fold change 2 and downregulation, fold change 0.5. Supplementary Table S8. The sequences of sirnas and shrna plasmids used. Supplementary Table S9. Primers used for q-pcr and subclone. 18

19 References 1. Barbieri CE, Baca SC, Lawrence MS, Demichelis F, Blattner M, Theurillat JP, White TA, et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat Genet 2012;44: Huang J, Deng Q, Wang Q, Li KY, Dai JH, Li N, Zhu ZD, et al. Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma. Nat Genet 2012;44: Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, et al. Circos: an information aesthetic for comparative genomics. Genome Res 2009;19: Geng C, He B, Xu L, Barbieri CE, Eedunuri VK, Chew SA, Zimmermann M, et al. Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover. Proc Natl Acad Sci U S A 2013;110: Kim MS, Je EM, Oh JE, Yoo NJ, Lee SH. Mutational and expressional analyses of SPOP, a candidate tumor suppressor gene, in prostate, gastric and colorectal cancers. APMIS 2013;121: Le Gallo M, O'Hara AJ, Rudd ML, Urick ME, Hansen NF, O'Neil NJ, Price JC, et al. Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Nat Genet 2012;44: Kim MS, Yoo NJ, Lee SH. Somatic mutation of SPOP tumor suppressor gene is rare in breast, lung, liver cancers, and acute leukemias. APMIS Cairo S, Armengol C, De Reynies A, Wei Y, Thomas E, Renard CA, Goga A, et al. Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer. Cancer Cell 2008;14: Jia D, Wei L, Guo W, Zha R, Bao M, Chen Z, Zhao Y, et al. Genome-wide copy number analyses identified novel cancer genes in hepatocellular carcinoma. Hepatology 2011;54: Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, Lin WM, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 2007;450: Lachenmayer A, Alsinet C, Savic R, Cabellos L, Toffanin S, Hoshida Y, Villanueva A, et al. Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib. Clin Cancer Res 2012;18: Li J, Zhou BP. Activation of beta-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer 2011;11:49. 19

20 Supplementary Table S1 Numbers of HB tumors analyzed by each platform Data type Primary tumors Lymphocytes adjacent Exome sequencing SNP 6.0 array Expression array a Exome sequencing, SNP 6.0 array b Exome sequencing, SNP 6.0 and Expression array c PCR + Sanger Sequencing d quantitative-pcr (q-pcr) e Immunohistochemistry (IHC) f a, Expression arrays were used to analyze the 5 paired HB primary tumors and adjacent non-tumor liver tissues. b, Exome livers sequencing and SNP 6.0 array were used to analyze the same 6 paired HB primary tumors and lymphocytes. c, 3 HB primary tumors were analyzed by Exome sequencing, SNP 6.0 array and Expression arrays. d, PCR amplification and Sanger sequencing were examined in an additional set of 24 paired HB primary tumors and lymphocytes or adjacent non-tumor tissues. e, q-pcr was employed to examine the relative expression levels of 21 mutant genes in 33 paired HB primary tumors and adjacent non-tumor tissues. f, IHC was employed to examine the protein expression levels of β-catenin in an overlapping or additional set of 42 HB primary tumors and 10 non-tumor tissues.

21 Supplementary Table S2. Somatic sequence variations identified in 6 HB genomes by whole-exome sequencing. Chromosome Start End REF ALT QUAL Samples Gene_location Exon_syno Gene chr G C HB2 exonic nonsynonymous SNV C1orf94 chr C T HB2 intronic NA LTBP1 chr C T 788 HB2 ncrna_intronic NA LOC chr G A HB2 ncrna_intronic NA LOC chr T C HB2 exonic synonymous SNV ERBB4 chr C T HB2 intronic NA ATIC chr C T HB2 intronic NA ATIC chr C G HB2 intronic NA XRCC5 chr C T HB2 exonic synonymous SNV TNS1 chr G A HB2 UTR3 NA PNKD chr C A HB2 intronic NA C2orf62 chr T C HB2 intronic NA DNPEP chr A T HB2 exonic nonsynonymous SNV NYAP2 chr C A HB2 UTR3 NA NYAP2 chr C T HB2 intronic NA COL4A4 chr C T HB2 UTR3 NA COL4A3 chr A G HB2 exonic synonymous SNV MFF chr C A HB2 UTR3 NA TM4SF20 chr G A HB2 intergenic NA CCL20(dist=52676),WDR69(dist=1371) chr G T HB2 intronic NA WDR69 chr A G HB2 intronic NA SPHKAP chr C T HB2 intronic NA SP140L chr C T HB2 exonic nonsynonymous SNV C2orf57 chr C G HB2 intergenic NA ALPPL2(dist=7423),ALPI(dist=37986)

22 chr C G HB2 upstream NA TRPM8 chr G A HB2 intronic NA TRPM8 chr C T HB2 intronic NA SNED1 chr C A HB2 UTR3 NA SNED1 chr A G HB2 intronic NA PASK chr A G HB2 intronic NA PASK chr G A HB2 intronic NA PASK chr C T HB2 intronic NA PASK chr G C HB2 intronic NA PPP1R7 chr G A HB2 intronic NA XYLB chr G T HB2 exonic nonsynonymous SNV CTNNB1 chr T C HB2 intronic NA CCDC171 chr G A HB2 intergenic NA LOC642929(dist=6349),FAM75A6(dist=472669) chr G T HB2 intronic NA JAKMIP3 chr G T HB2 exonic nonsynonymous SNV RNF169 chr T C HB2 exonic synonymous SNV CACNA1G chr A C HB2 ncrna_exonic NA LOC chr T A HB2 UTR3 NA AP1M1 chr G C HB2 UTR3 NA AP1M1 chr T A HB2 UTR3 NA AP1M1 chr G A HB2 exonic nonsynonymous SNV GLTSCR1 chr T A HB2 exonic nonsynonymous SNV KLHL22 chr C T HB2 UTR5 NA KLHL22 chrx C T HB2 intronic NA WDR45 chrx C T HB2 intronic NA XAGE3 chrx A G HB2 intronic NA XAGE3

23 chr G A 2303 HB3 ncrna_exonic NA ESPNP chr T C HB3 exonic nonsynonymous SNV VCAM1 chr C T HB3 intergenic NA NONE(dist=NONE),LOC375010(dist=26304) chr G A HB3 intergenic NA NONE(dist=NONE),LOC375010(dist=26271) chr A C HB3 intergenic NA NONE(dist=NONE),LOC654342(dist=29328) chr C T HB3 exonic synonymous SNV RNF123 chr G A HB3 exonic nonsynonymous SNV CDC20B chr C T HB3 UTR3 NA ZNF346 chr T G HB3 ncrna_exonic NA MUTED-TXNDC5 chr A G HB3 intronic NA SYNE1 chr C A HB3 intronic NA COL28A1 chr G A HB3 intronic NA GLCCI1 chr C A 93.3 HB3 intronic NA ABCB5 chr T C HB3 UTR3 NA SLC26A4 chr C A HB3 intergenic NA ODZ4(dist=547089),MIR4300(dist= ) chr C T HB3 exonic nonsynonymous SNV SPOP chr A G HB3 UTR3 NA WDR45L chr A T HB3 exonic nonsynonymous SNV SLC12A5 chry A G HB3 intergenic NA NONE(dist=NONE),GYG2P1(dist= ) chrun_gl G A HB3 intergenic NA NONE(dist=NONE),NONE(dist=NONE) chr T A HB5 exonic nonsynonymous SNV PANK3 chr C T HB5 exonic nonsynonymous SNV TMEM14B chr C T HB5 intergenic NA ZCCHC6(dist=21005),GAS1(dist=568870) chr C T HB5 intergenic NA ZCCHC6(dist=21011),GAS1(dist=568864) chr T A HB5 UTR5 NA SEMA4G chr A G HB5 intronic NA EML1

24 chr C T HB5 UTR3 NA C17orf63 chrun_gl C A HB5 intergenic NA NONE(dist=NONE),NONE(dist=NONE) chrun_gl G A 76.1 HB5 intergenic NA LOC (dist=7443),RN5-8S1(dist=21858) chrun_gl A G HB5 intergenic NA LOC (dist=7447),RN5-8S1(dist=21854) chr A T HB6 intronic NA ATG4C chr C T HB6 exonic synonymous SNV SPRR3 chr C A HB6 intronic NA HNRNPU chr C T HB6 exonic nonsynonymous SNV CTNNB1 chr G T HB6 exonic nonsynonymous SNV CTNNB1 chr T G HB6 intronic NA ADAMTS6 chr C A HB6 intronic NA ENPP1 chr A T HB6 intronic NA VPS13B chr G A HB6 intronic NA VPS13B chr T A HB6 intronic NA MSANTD3-TMEFF1,TMEFF1 chr C T HB6 upstream NA CNNM1 chr G T HB6 intronic NA AMPD3 chr G A HB6 exonic nonsynonymous SNV OR5I1 chr C T HB6 intronic NA C15orf41 chr T C HB6 exonic nonsynonymous SNV TMC5 chrx T C HB6 intronic NA ATRX chrx G A 525 HB6 intronic NA BRCC3 chr C T HB8 exonic nonsynonymous SNV SLC1A7 chr C T HB8 intergenic NA LOC (dist= ),PRMT6(dist= ) chr T C HB8 intronic NA AXDND1 chr A T HB8 exonic synonymous SNV TPR chr C T HB8 UTR3 NA KIF14

25 chr C T HB8 upstream NA LOC chr T C HB8 UTR3 NA SEC62 chr T C HB8 UTR3 NA YEATS2 chr G A HB8 exonic nonsynonymous SNV NIPBL chr T A HB8 exonic nonsynonymous SNV HDAC9 chr C T HB8 exonic nonsynonymous SNV C8orf34 chr C G HB8 intergenic NA ZNF33A(dist=20129), ZNF37A(dist=14140) chr G A HB8 intronic NA PBLD chr C T HB8 UTR3 NA CHID1 chr C T HB8 exonic nonsynonymous SNV CD163L1 chr T A HB8 exonic nonsynonymous SNV CAPRIN2 chr C T HB8 exonic nonsynonymous SNV CAPRIN2 chr G A HB8 exonic nonsynonymous SNV RPGRIP1 chr G C HB8 UTR3 NA SCAMP5 chr T A HB8 UTR5 NA ST3GAL2 chr C T HB8 UTR3 NA GAS7 chr A T HB8 UTR3 NA H3F3B chr C T HB8 ncrna_exonic NA LINC00526 chr A G HB8 intronic NA ANKRD30B chr C T HB8 UTR3 NA AQP4 chr G A HB8 UTR3 NA BRWD1 chr C G HB8 ncrna_intronic NA CES5AP1 chrx A C HB8 ncrna_intronic NA SSX8 chrx C A HB8 intronic NA HUWE1 chrx T A HB8 downstream NA MIR384 chrx G C HB8 intronic NA ATP1B4

26 chrx C T HB8 intronic NA MTMR1 chrx C A HB8 intronic NA SLC6A8 chrx C A HB8 intronic NA SLC6A8 chr G T HB9 exonic nonsynonymous SNV FGGY chr C T HB9 intronic NA SEC22B chr T C HB9 exonic nonsynonymous SNV LGR6 chr T A HB9 ncrna_intronic NA RNU5F-1 chr C G HB9 UTR3 NA MAEA chr A G HB9 ncrna_exonic NA LINC00200 chr A T HB9 intronic NA TRPM7 chr T A HB9 intronic NA TRPM7 chr T C HB9 intronic NA RPRD1A chr C T HB9 intronic NA LIPG chr C T HB9 exonic nonsynonymous SNV TRPC4AP chrx A G HB9 UTR3 NA SAT1 chrx G A HB9 intronic NA MAGEB3

27 Supplementary Table S3 Valdiation of 21 identified mutations in an additional set of 24 HB tumors by sanger sequencing. Gene symbol Mutation prevalence Mutated residues CTNNB1 7/24 In-frame deletion (exon 3) OR5I1 2/24 In-frame deletion (exon 1) C2ORF57 1/24 R386C

28 Supplementary Table S4. Somatic copy number alterations identified in 6 HB genomes by SNP 6.0 arrays. Chromosome Cytoband Start End Length(bps) Copy number state Mean Sample ID chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q Amplification HB2 chr2 2q32.1-2q Amplification HB2 chr2 2q36.3-2q Amplification HB2 chr2 2q32.2-2q Amplification HB2 chr1 1q21.1-1q Amplification HB2 chr2 2q33.3-2q Amplification HB2 chr2 2q37.1-2q Amplification HB2 chr2 2q32.3-2q Amplification HB2 chr2 2q34-2q Amplification HB2 chr2 2q24.2-2q Amplification HB2 chr1 1q41-1q Amplification HB2 chr1 1q21.3-1q Amplification HB2 chr14 14q Amplification HB3 chr1 1q Amplification HB3 chr14 14q Amplification HB5 chr20 20q Amplification HB6 chr14 14q Amplification HB6 chr7 7q Amplification HB6 chr9 9p11.2-9q Amplification HB6 chry Yp Amplification HB8 chr17 17p Amplification HB8 chr12 12q Amplification HB8 chr17 17q Amplification HB8 chr1 1q Amplification HB8 chr10 10q Amplification HB8 chr6 6p Amplification HB8 chr12 12q Amplification HB8 chr13 13q q Amplification HB8 chr2 2q21.2-2q Amplification HB8 chr1 1q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr17 17p Amplification HB8 chr8 8q Amplification HB8 chr8 8q Amplification HB8 chr12 12q Amplification HB8

29 chr10 10p Amplification HB8 chr17 17q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr17 17p Amplification HB8 chr12 12p Amplification HB8 chr6 6p Amplification HB8 chr12 12q Amplification HB8 chr20 20q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr19 19q Amplification HB8 chr16 16q q Amplification HB8 chr8 8q Amplification HB8 chr17 17q Amplification HB8 chr6 6q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr10 10p Amplification HB8 chr2 2p Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr10 10p Amplification HB8 chr10 10q Amplification HB8 chry Yp Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr8 8q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1p Amplification HB8 chr12 12p Amplification HB8

30 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr20 20p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr13 13q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr12 12q q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr2 2q Amplification HB8 chr8 8q Amplification HB8 chr13 13q Amplification HB8 chr8 8q Amplification HB8 chr1 1q Amplification HB8 chr6 6p Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr17 17p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr2 2p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr2 2p Amplification HB8 chr12 12p Amplification HB8 chr6 6q Amplification HB8

31 chr16 16q Amplification HB8 chr1 1q Amplification HB8 chr10 10q Amplification HB8 chr17 17q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr6 6p24.3-6p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr6 6p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr17 17p Amplification HB8 chr12 12p Amplification HB8 chr12 12p Amplification HB8 chr21 21q Amplification HB8 chr17 17p Amplification HB8 chr8 8q Amplification HB8 chr12 12q Amplification HB8 chr8 8q Amplification HB8 chr1 1q Amplification HB8 chr2 2q Amplification HB8 chr2 2p Amplification HB8 chr5 5q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr5 5q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr2 2p Amplification HB8 chr12 12p Amplification HB8 chr19 19p Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr20 20q Amplification HB8

32 chr1 1p Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr16 16p Amplification HB8 chr6 6p Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr2 2q12.3-2q Amplification HB8 chr20 20q Amplification HB8 chr12 12q q Amplification HB8 chr16 16p Amplification HB8 chr12 12q Amplification HB8 chr1 1q31.3-1q Amplification HB8 chr12 12q Amplification HB8 chr17 17p Amplification HB8 chr1 1q Amplification HB8 chr6 6q Amplification HB8 chr12 12q Amplification HB8 chr1 1q42.2-1q Amplification HB8 chr16 16q13-16q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr21 21q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr8 8q Amplification HB8 chr17 17q Amplification HB8 chr12 12q Amplification HB8 chr17 17q q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr2 2p24.1-2p Amplification HB8 chr2 2p Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr2 2p Amplification HB8

33 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr17 17p Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr2 2p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr19 19p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr6 6p Amplification HB8 chr16 16p Amplification HB8 chr2 2p Amplification HB8 chr12 12q Amplification HB8 chr2 2p Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr2 2q Amplification HB8 chr12 12p Amplification HB8 chr1 1q Amplification HB8 chr17 17q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr17 17q Amplification HB8 chr12 12p Amplification HB8 chr19 19q Amplification HB8 chr1 1q32.3-1q Amplification HB8 chr12 12q Amplification HB8 chr2 2p23.3-2p Amplification HB8

34 chr6 6p Amplification HB8 chr1 1q Amplification HB8 chr1 1q Amplification HB8 chr16 16q Amplification HB8 chr12 12p p Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr12 12p Amplification HB8 chr12 12q q Amplification HB8 chr12 12p Amplification HB8 chr20 20p Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr12 12q Amplification HB8 chr1 1q21.1-1q Amplification HB8 chr12 12p Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr2 2p Amplification HB8 chr2 2p Amplification HB8 chr2 2q12.1-2q Amplification HB8 chr1 1q Amplification HB8 chr12 12p Amplification HB8 chr12 12p Amplification HB8 chr16 16q Amplification HB8 chr2 2q Amplification HB8 chr1 1q Amplification HB8 chr10 10q Amplification HB8 chr2 2q Amplification HB8 chr12 12q Amplification HB8 chr20 20q Amplification HB8 chr12 12q Amplification HB8 chr12 12q Amplification HB8 chr5 5q Amplification HB8 chr12 12q q Amplification HB8 chr20 20q Amplification HB8 chr12 12q Amplification HB8 chr1 1q Amplification HB8 chr16 16p p Amplification HB8 chr1 1q Amplification HB8 chr17 17q q Amplification HB8 chr1 1q Amplification HB8