1 SUPPLEMENTARY INFORMATION FOR THE GENOMIC LANDSCAPE OF HYPODIPLOID ACUTE LYMPHOBLASTIC LEUKEMIA Linda Holmfeldt 1,30 , Lei Wei 1,30 , Ernesto Diaz-Flores 2 , Michael Walsh 3 , Jinghui Zhang 4 , Li Ding 5,6 , Debbie Payne-Turner 1 , Michelle Churchman 1 , Anna Andersson 1,7 , Shann-Ching Chen 1 , Kelly McCastlain 1 , Jared Becksfort 4 , Jing Ma 1 , Gang Wu 4 , Samir N. Patel 1,29 , Susan L. Heatley 1,29 , Letha A. Phillips 1 , Guangchun Song 1 , John Easton 8 , Matthew Parker 4 , Xiang Chen 4 , Michael Rusch 4 , Kristy Boggs 8 , Bhavin Vadodaria 8 , Erin Hedlund 4 , Christina Drenberg 9 , Sharyn Baker 9 , Deqing Pei 10 , Cheng Cheng 10 , Robert Huether 4 , Charles Lu 5 , Robert S. Fulton 5,6 , Lucinda L. Fulton 5,6 , Yashodhan Tabib 5 , David J. Dooling 5,6 , Kerri Ochoa 5 , Mark Minden 11 , Ian D. Lewis 12 , L. Bik To 12 , Paula Marlton 13 , Andrew W. Roberts 14 , Gordana Raca 15 , Wendy Stock 15 , Geoffrey Neale 16 , Hans G. Drexler 17 , Ross A. Dickins 18 , David W. Ellison 1 , Sheila A. Shurtleff 1 , Ching-Hon Pui 3 , Raul C. Ribeiro 3 , Meenakshi Devidas 19 , Andrew J. Carroll 20 , Nyla A. Heerema 21 , Brent Wood 22 , Michael J. Borowitz 23 , Julie M. Gastier-Foster 24,25,26 , Susana C. Raimondi 1 , Elaine R. Mardis 4,5,27 , Richard K. Wilson 4,5,27 , James R. Downing 1 , Stephen P. Hunger 28 , Mignon L. Loh 2 , and Charles G. Mullighan 1 1 Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 2 Department of Pediatrics, University of California School of Medicine, San Francisco, California, USA 3 Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 4 Department of Computational Biology and Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 5 The Genome Institute at Washington University, St Louis, Missouri, USA 6 Department of Genetics, Washington University School of Medicine, St Louis, Missouri, USA 7 Department of Clinical Genetics, Lund University Hospital, Lund, Sweden 8 Pediatric Cancer Genome Project, St. Jude Children’s Research Hospital, Memphis, Tennessee USA 9 Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 10 Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 11 Princess Margaret Hospital/University Health Network, University of Toronto, Ontario, Canada 12 Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia 13 Oncology/Haematology Unit, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia 14 Department of Clinical Haematology and Bone Marrow Transplant, Royal Melbourne Hospital, Melbourne, Victoria, Australia 15 Hematology/Oncology, University of Chicago Medicine, Chicago, Illinois, USA 16 The Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 17 Department of Human and Animal Cell Cultures, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany 18 Molecular Medicine Division, Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 19 Department of Biostatistics, College of Medicine, University of Florida, Gainesville, Florida, USA 20 Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA Nature Genetics: doi: 10.1038/ng.2532
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1
SUPPLEMENTARY INFORMATION FOR
THE GENOMIC LANDSCAPE OF HYPODIPLOID ACUTE LYMPHOBLASTIC LEUKEMIA
Linda Holmfeldt1,30, Lei Wei1,30, Ernesto Diaz-Flores2, Michael Walsh3, Jinghui Zhang4, Li Ding5,6, Debbie Payne-Turner1, Michelle Churchman1, Anna Andersson1,7, Shann-Ching Chen1, Kelly McCastlain1, Jared Becksfort4, Jing Ma1, Gang Wu4, Samir N. Patel1,29, Susan L. Heatley1,29, Letha A. Phillips1, Guangchun Song1, John Easton8, Matthew Parker4, Xiang Chen4, Michael Rusch4, Kristy Boggs8, Bhavin Vadodaria8, Erin Hedlund4, Christina Drenberg9, Sharyn Baker9,
Deqing Pei10, Cheng Cheng10, Robert Huether4, Charles Lu5, Robert S. Fulton5,6, Lucinda L.
Fulton5,6, Yashodhan Tabib5, David J. Dooling5,6, Kerri Ochoa5, Mark Minden11, Ian D. Lewis12, L. Bik To12, Paula Marlton13, Andrew W. Roberts14, Gordana Raca15, Wendy Stock15, Geoffrey Neale16, Hans G. Drexler17, Ross A. Dickins18, David W. Ellison1, Sheila A. Shurtleff1, Ching-Hon Pui3, Raul C. Ribeiro3, Meenakshi Devidas19, Andrew J. Carroll20, Nyla A. Heerema21, Brent Wood22, Michael J. Borowitz23, Julie M. Gastier-Foster24,25,26, Susana C. Raimondi1, Elaine R. Mardis4,5,27, Richard K. Wilson4,5,27, James R. Downing1, Stephen P. Hunger28, Mignon L. Loh2, and Charles G. Mullighan1
1Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 2Department of Pediatrics, University of California School of Medicine, San Francisco, California, USA 3Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 4Department of Computational Biology and Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
5The Genome Institute at Washington University, St Louis, Missouri, USA
6Department of Genetics, Washington University School of Medicine, St Louis, Missouri, USA
7Department of Clinical Genetics, Lund University Hospital, Lund, Sweden
8Pediatric Cancer Genome Project, St. Jude Children’s Research Hospital, Memphis, Tennessee USA 9Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
10Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA 11Princess Margaret Hospital/University Health Network, University of Toronto, Ontario, Canada 12Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia 13Oncology/Haematology Unit, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia 14Department of Clinical Haematology and Bone Marrow Transplant, Royal Melbourne Hospital, Melbourne, Victoria, Australia
15Hematology/Oncology, University of Chicago Medicine, Chicago, Illinois, USA 16The Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
17Department of Human and Animal Cell Cultures, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany 18Molecular Medicine Division, Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 19Department of Biostatistics, College of Medicine, University of Florida, Gainesville, Florida, USA 20Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
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21Department of Pathology, College of Medicine, Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA 22Department of Laboratory Medicine, Seattle Children’s Hospital, Seattle, Washington, USA 23Division of Hematologic Pathology, Johns Hopkins Hospital, Baltimore, Maryland, USA 24Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, Ohio, USA 25Department of Pathology, Ohio State University, Columbus, Ohio, USA 26Department of Pediatrics, Ohio State University, Columbus, Ohio, USA 27Siteman Cancer Center, Washington University, St Louis, Missouri, USA 28Section of Pediatric Hematology/Oncology/Bone Marrow Transplantation and Center for Cancer and Blood Disorders, University of Colorado Denver School of Medicine, Children’s Hospital Colorado, Aurora, Colorado, USA 29Present addresses: Weill Cornell Medical College, Cornell University, New York, New York, USA (S.N.P.) and Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia (S.L.H.)
D, diagnosis; R, relapse; PH, BCR-ABL1 positive; H50, >50 chromosomes; H47, >47 chromosomes; IMVS, Institute of Medical and Veterinary Science, Adelaide, Australia; PAH, Princess Alexandra Hospital, Woolloongabba, Australia; RMH, Royal Melbourne Hospital, Parkville, Australia; UHN, University Health Network, Toronto, Canada; CALGB, The Cancer and Leukemia Group B.
SJHYPO001 D 36,203,643,314 98.2% 0.18 98.0 96.3 94.6
SJHYPO001 G 23,676,803,494 89.8% 0.096 93.4 89.2 85.4
SJHYPO005 D 18,219,143,560 98.6% 0.15 97.0 94.3 91.9
SJHYPO005 G 19,460,765,244 98.4% 0.13 96.9 94.2 91.8
SJHYPO009 D 17,764,641,338 98.5% 0.25 96.7 93.7 90.7
SJHYPO009 G 14,040,901,830 98.0% 0.12 96.4 93.1 90.1
SJHYPO009 R 8,842,150,848 99.1% 0.23 93.3 87.1 78.0
SJHYPO012 D 14,348,121,812 96.9% 0.49 89.9 82.4 74.5
SJHYPO012 G 15,199,382,132 95.2% 0.36 92.1 87.1 81.9
SJHYPO014 D 23,025,245,932 98.2% 0.13 97.6 95.5 93.6
SJHYPO014 G 20,245,193,662 98.7% 0.15 97.6 95.4 93.1
SJHYPO016 D 14,199,789,458 96.5% 0.23 91.9 86.7 81.5
SJHYPO016 G 10,731,147,788 95.1% 0.19 94.6 90.2 85.8
SJHYPO019 D 7,772,516,004 98.6% 0.27 91.7 84.0 73.4
SJHYPO019 G 9,554,828,260 98.8% 0.2 94.2 90.2 84.7
SJHYPO024 D 14,791,275,674 99.1% 0.18 95.3 93.1 90.6
SJHYPO024 G 20,608,688,016 99.2% 0.24 95.7 94.1 92.4
SJHYPO032 D 16,899,506,042 98.0% 0.094 93.7 89.6 86.0
SJHYPO032 G 14,253,139,392 98.4% 0.15 93.7 89.3 85.1
SJHYPO036 D 25,996,903,080 99.1% 0.21 96.2 95.1 94.0
SJHYPO036 G 16,179,316,452 99.2% 0.18 95.7 94.1 92.1
SJHYPO037 D 11,345,339,292 98.1% 0.56 91.5 83.0 71.0
SJHYPO037 R 11,900,972,410 99.0% 0.5 93.2 87.5 79.1
SJHYPO039 D 11,917,553,580 90.1% 0.11 89.1 81.8 74.9
SJHYPO039 G 12,215,196,742 98.2% 0.12 92.8 87.8 82.6
SJHYPO041 D 5,202,479,296 98.7% 0.33 87.0 70.6 50.2
SJHYPO041 G 8,122,168,106 99.1% 0.23 93.9 88.8 81.1
SJHYPO045 D 10,228,524,600 86.0% 0.048 82.8 78.6 74.2
SJHYPO045 G 12,959,649,308 77.4% 0.054 82.7 78.6 74.6
SJHYPO047 D 11,252,109,626 99.0% 0.21 94.8 91.7 87.5
SJHYPO047 G 14,875,868,628 99.2% 0.27 95.2 92.9 89.9
SJHYPO052 R 9,029,261,630 98.7% 0.26 93.2 87.1 78.4
SJHYPO116 D 5,890,611,060 95.7% 0.041 90.9 80.3 65.9
SJHYPO116 G 4,953,854,700 96.9% 0.042 90.7 81.0 67.7
SJHYPO117 D 16,385,912,760 99.2% 0.48 94.6 91.2 86.3
SJHYPO117 R 9,911,419,870 98.5% 0.53 89.6 78.6 64.3
SJHYPO124 D 10,739,280,914 99.2% 0.48 92.8 85.2 73.6
SJHYPO124 G 4,325,139,968 98.9% 0.31 84.3 63.8 40.8
SJHYPO125 D 15,139,696,586 96.4% 0.05 91.9 86.5 81.4
SJHYPO125 G 14,135,196,844 95.9% 0.088 91.5 86.3 81.3
SJHYPO126 D 14,091,114,990 99.2% 0.33 95.1 92.2 87.6
SJHYPO126 G 8,951,109,244 98.4% 0.42 91.4 82.2 69.5
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Supplementary Table 5: Validation frequency of next-generation sequencing data.
Total number of mutations/alterations per next generation sequencing (NGS) technique is indicated as well as validation percentages. WES, whole exome sequencing; WGS, whole genome sequencing; SNV, single nucleotide variation; Indel, insertion/deletion mutation.
NGS Technique
Mutation type
Somatic Somatic
% Non-
tumor Non-
tumor % Wild-type
Wild-type %
WES SNV/Indel 229 72.9 33 10.5 52 16.6
WES SV 1 50 0 0 1 50
WGS SNV/Indel 417 89.7 33 7.1 15 3.2
WGS SV 95 86.4 10 9.1 5 4.5
WES & WGS All 742 83.3 76 8.5 73 8.2
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Supplementary Table 6: Number of sequence mutations, copy number alterations and structural variants identified by next-generation sequencing.
Tier1: Coding synonymous, nonsynonymous, splice site, and non-coding RNA variants; Tier2: Conserved variants; Tier3: Variants in non-repeat masked regions; Tier4: Remaining SNVs. SNV, single nucleotide variation; Indel, insertion/deletion mutation; CDS, Coding DNA sequence; HQ, high quality; SV, structural variation; CNA, copy number alteration; N, number; Mb, megabases; AA, amino acid; UTR, untranslated leader region; Amp, amplification; Del, deletion. * Including numbers based on whole chromosome gain and loss.
Supplementary Table 7: Mutations identified by next-generation sequencing.
See Excel Table: “Table_S7_NGS_SNVs_Indels.xlsx”
Somatic and putative germline deleterious single nucleotide variations (SNVs) and insertion/deletion mutations (Indels) identified by whole genome- and whole exome sequencing of a subset of the hypodiploid ALL cohort. Putative germline variants are highlighted in yellow. D, diagnosis; R, relapse. Column definition is listed below: A. GeneName: HUGO gene symbol B. VarType: SNV, single nucleotide variation; Indel, insertion/deletion mutation C. Sample: Hypodiploid ALL sample ID D. Chr: chromosome E. Position: chromosome position in hg19 coordinates F. Class: classification based on amino acid change pattern. ‘exon’, mutation in non-coding RNA genes; ‘splice_region’ mutation not directly affecting the canonical splice sites but located within 10bp of the canonical splice sites. G. AAChange: predicted amino acid change for the mutation H. ProteinGI: NCBI protein GI number I. mRNA_acc: RefSeq accession number J. Mut Reads Diagnosis: number of NGS reads containing mutant allele (diagnosis) K. Total Reads Diagnosis: number of NGS reads covering the site (diagnosis) L. Mut Reads Relapse: number of NGS reads containing mutant allele (relapse) M. Total Reads Relapse: number of NGS reads covering the site (relapse) N. Mut Reads Normal: number of NGS reads containing mutant allele (normal) O.Total Reads Normal: number of NGS reads covering the site (normal) P. Reference Allele: the allele represented in the reference human genome. Reference allele is marked as ‘–‘ for an insertion. Q. Non-reference Allele: the mutated allele R. Flanking: 20bp [reference allele/mutant allele] 20bp S. Status: somatic or germline mutation (germline referring to non-tumor cells) T. SIFTResult: ‘deleterious status’ assigned by SIFT U. pph2result: ‘deleterious status’ assigned by polyPHEN2
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Supplementary Table 8: Structural variations identified by whole genome sequencing.
See Excel Table: “Table_S8_WGS_SVs.xlsx”
Somatic structural variations identified by whole genome sequencing of a subset of the hypodiploid ALL cohort. CDS, coding DNA sequence.
Column definition is listed below: A. Sample: Hypodiploid ALL sample ID B. ChrA: Chromosome for breakpoint A C. PosA: Position of breakpoint A D. OrientationA: + Region to the left of PosA is included in mutant genotype - Region to the right of PosA is included in mutant genotype E. ChrB: Chromosome for breakpoint B F. PosB: Position of breakpoint B G. OrientationB: + Region to the right of PosB is included in mutant genotype - Region to the left of PosB is included in mutant genotype H. Type: INS, insertion; DEL, deletion; INV, inversion; ITX, intrachromosomal
translocation; CTX, interchromosomal translocation I. Usage: GENIC: Both endpoints were in genes: checked for fusion
HALF_INTERGENIC: One endpoint was in a gene: checked for truncation INTERGENIC / INTRONIC: Neither endpoint was in a gene or both were in the same intron of a gene; no gene fusion or truncation INVERTED_REPEAT: Both endpoints were in the same gene, but in opposite orientations: checked for truncation
J. Gene: Fusion or truncated gene that would result from structural variation K. Chromosomes: Chromosomes involved in the rearrangement L. Tx: Number of predicted fusion transcripts M. Valid CDS: Number of predicted fusion transcripts with an annotated CDS start and stop N. In-Frame CDS: Number of “Valid CDS” transcripts with a CDS length divisible by three. O. Mod. In-Frame CDS: Number of “In-Frame CDS” transcripts that are not identical to an
existing annotated transcript. P. mutA: Number of reads supporting the structural variation at breakpoint A Q. mutB: Number of reads supporting the structural variation at breakpoint B R. Validation Status:
Valid: The SV has been experimentally validated Putative: The SV has yet to be validated
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Supplementary Table 9: Genes resequenced.
CDS, coding DNA sequence.
Gene Genbank Accession Number Exons sequenced
CBL NM_005188.2 8-9
CRLF2 NM_022148.2 6
ETV6 NM_001987.4 CDS
FLT3 NM_004119.2 14, 20 and coding region of 24
IKZF1 NM_006060.3 CDS
IKZF2 NM_016260.2 CDS
IKZF3 NM_012481.3 CDS
JAK1 NM_002227.2 13-18
JAK2 NM_004972.3 13-24 and coding region of 25
KIF2B NM_032559.4 CDS
KRAS NM_033360.2 2-3
MAPK1 NM_138957.2 CDS
NF1 NM_000267.2 CDS
NRAS NM_002524.3 2-3
PAG1 NM_018440.3 CDS
PAX5 NM_016734.1 CDS
PTPN11 NM_002834.3 3, 4 and 13
RB1 NM_000321.2 CDS
TP53 NM_000546.4 CDS
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Supplementary Table 10: Regions of copy number alterations and copy-neutral loss-of-heterozygosity in hypodiploid ALL. See Excel Table: “Table_S10_SNP_data.xlsx” The table lists all regions of copy number alterations (CNAs) identified by manual curation of circular binary segmentation data for the hypodiploid ALL cohort, and copy-neutral loss-of-heterozygosity (CN LOH) identified by dChip using the Hidden Markov Model algorithm. CNAs smaller than 8 SNP and/or copy number probes have been filtered. Final lesion listings exclude DNA gains and losses arising from antigen receptor gene rearrangements at 2p11.2 (IGK@), 7p14.1 (TRG@), 7q34 (TRB@), 14q11.2 (TRA@), 14q32.33 (IGH@) and 22q11.22 (IGL@). The workbook contains 5 sheets, with the near haploid, masked near haploid, low hypodiploid, masked low hypodiploid and near diploid cases in different sheets. Del, deletion; Homo, homozygous; Hemi, hemizygous; Subpop, subpopulation; R, relapse. “Duplicated genome” indicates lesions and whole chromosomal events affected by reduplication of the hypodiploid genomic complement in the masked near haploid and low hypodiploid ALL cases. Masked hypodiploid cases do here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. The analyses were initially performed in human assembly hg18, on which the Affymetrix SNP 6.0 microarray is based, and then mapped to hg19 using a method described previously90. Both hg18 and hg19 coordinates are included in the table.
A. ID: Sample B. Comment: lesion type C. Chrom: Chromosome D. Cytoband: Sublocation on chromosome E. loc.start_hg18: Chromosomal start position of lesion based on Human genome build 18 F. loc.end_hg18: Chromosomal end position of lesion based on Human genome build 18 G. loc.start_hg19: Chromosomal start position of lesion based on Human genome build 19 H. loc.end_hg19: Chromosomal end position of lesion based on Human genome build 19 I. LiftOverStatus: One of the following: “complete” (all bases in the original hg18 segment are
successfully remapped to hg19); “partial” (not all, but >50% of the bases are remapped, with the ratio of remapping); “suspicious” (>50% of the bases are remapped, but did not pass subsequent QA); “failed” (>50% of the bases cannot be remapped).
J. num.mark: Number of probes included in the segment K. seg.mean: log2 ([normalized tumor signal]/[normalized normal signal]) L. seg.observedCN: Absolute copy number M. seg.size (kb): Size of segment in kilobases N. total # of gene in the segment: Number of genes included in segment O. first 10 genes in segment: Lists the names of the first 10 genes in the segment P. total # of miRNA in the segment: Number of miRNAs included in segment Q. first 10 miRNAs in segment: Lists the names of the first 10 miRNAs in the segment
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Supplementary Table 11: Mutations identified by Sanger sequencing in the hypodiploid ALL cohort. See Excel Table: “Table_S11_Sanger_mutations.xlsx” Somatic and putative germline deleterious single nucleotide variations (SNVs) and insertion/deletion mutations (Indels) identified by Sanger sequencing of the entire hypodiploid ALL cohort. Column definition is listed below: A. Gene Name: HUGO gene symbol B. VarType: SNV, single nucleotide variation; Indel, insertion/deletion mutation C. Sample: Hypodiploid ALL sample ID D. Chr: chromosome E. Position: chromosome position in hg19 coordinates F. Class: classification based on amino acid change pattern. ‘exon’, mutation in non-coding
RNA genes; ‘splice_region’ mutation not directly affecting the canonical splice sites but located within 10bp of the canonical splice sites.
G. AAChange: predicted amino acid change for the mutation H. ProteinGI: NCBI protein GI number I. mRNA_acc: RefSeq accession number J. Mutant peak intensity (%): Mutant peak size of total peaks K. Reference Allele: the allele represented in the reference human genome. Reference allele
is marked as ‘–‘ for an insertion. L. Non-reference Allele: the mutated allele M. Flanking: 10bp [reference allele/mutant allele] 10bp N. Status: somatic or germline mutation (germline referring to non-tumor cells) O. SIFTResult: ‘deleterious status’ assigned by SIFT P. SIFTScore Q. pph2result: ‘deleterious status’ assigned by polyPHEN2 R. pph2score
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Supplementary Table 12: Copy number alterations and mutations. See Excel Table: “Table_S12_Specific_lesion_information.xlsx” Unless otherwise stated, genes were not sequenced. Paired or unpaired indicate whether a matched normal DNA sample was available. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. NH, near haploid (24-31 chromosomes); mNH, masked near haploid (2x24-31 chromosomes); LH, low hypodiploid (32-39 chromosomes); mLH, masked low hypodiploid (2x32-39 chromosomes); ND, near diploid (44-45 chromosomes); CNA, copy number alteration; Seq mut, sequence mutation; Del, deletion; Amp, amplification; Homo, homozygous; Het, heterozygous; e, exon; i, intron; LOH, loss-of-heterozygosity; US, upstream; DS, downstream; † Concomitant deletion of the other corresponding chromosomal copy, giving rise to a bi-allelic mutational event; § Mutation present in matched remission sample; §§ Directly upstream of gene in question; ¥ Deleted as part of rearrangement of the immunoglobulin lambda light chain locus at 22q11.22.
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Supplementary Table 13: Association between aneuploidy and lesions.
P values were calculated by an Exact Chi-Square test (2x5) and values <0.05 are highlighted, as well as the subgroup(s) that are associated with a high frequency of the lesion in question. Signaling indicates genes involved in RTK- and/or Ras signaling. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. CNA, copy number alteration; Seq. mut., sequence mutation; pwy, pathway; NH, near haploid; mNH, masked near haploid; LH, low hypodiploid; mLH, masked low hypodiploid; ND, near diploid.
Differential expression analysis performed by limma with estimation of false discovery rate (FDR) at 0.05 between near haploid (NH) and masked near haploid (mNH) cases. No statistically significant differences were identified between those two hypodiploid subgroups. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%.
Differential expression analysis performed by limma with estimation of false discovery rate (FDR) at 0.05 between low hypodiploid (LH) and masked low hypodiploid (mLH) cases. No statistically significant differences were identified between those two hypodiploid subgroups. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%.
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Supplementary Table 16: TP53 mutations in adult ALL.
LH, low hypodiploid; H47, >47 chromosomes; NH, near haploid; D, diagnosis; R, relapse; Homo, homozygous; Het, heterozygous; LFS, Li-Fraumeni Syndrome.
Seq ID ALL subgroup
Status TP53 mutation (NM_000546.4)
Homo or het
Status LFS associated or sporadic
Predicted effect Domain Dominant negative
ADT003 LH D R248Q Homo Somatic LFS and sporadic DNA contact DNA binding Yes
ADT017 LH D V173M Homo Somatic LFS and sporadic Deleterious DNA binding Yes
ADT018 LH D V173M Homo No germline LFS and sporadic Deleterious DNA binding Yes
ADT027 LH D Y220H Homo No germline Sporadic Deleterious DNA binding No
ADT028 LH D R249T Homo Somatic Sporadic Deleterious DNA binding N/A
ADT040 T-ALL D and R R175H Homo No germline LFS and sporadic Conformational DNA binding Yes
ADT044 N/A R L265P Homo No germline LFS and sporadic Deleterious DNA binding Yes
ADT044 N/A R R267Q Homo No germline LFS and sporadic Deleterious DNA binding N/A
ADT074 H47 R R213* Homo No germline LFS and sporadic Deleterious DNA binding No
ADT076 Other R R282fs Het No germline Sporadic Truncating
ADT084 Other R L145P Het Somatic Sporadic Deleterious DNA binding N/A
ADT084 Other R G187_E6splice_region Homo Somatic
ADT085 LH D Y220C Homo Somatic LFS and sporadic Deleterious DNA binding Yes
ADT121 Other D GinsR282 Het No germline
ADT122 Other D P219L Het No germline Sporadic Deleterious DNA binding No
ADT122 Other D R273C Het No germline LFS and sporadic Deleterious DNA binding Yes
Adult_Hypo2 LH D R273H Homo No germline LFS and sporadic DNA contact DNA binding Yes
Adult_Hypo3 LH D R249S Het No germline Sporadic Conformational DNA binding Yes
Adult_Hypo4 LH D R273fs Homo No germline Sporadic Truncating
Adult_Hypo5 NH D R290fs Het No germline Sporadic Truncating
Adult_Hypo6 LH D G187_E6splice_region Homo No germline Sporadic
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Supplementary Table 17: Copy number alterations and mutations in hypodiploid ALL vs non-hypodiploid ALL.
Abnormalities are deletions unless otherwise indicated. The St. Jude (SJ) cohort was studied in Ref29 and consisted of 258 childhood ALL cases divided into high hyperdiploid (H50; >50 chromosomes; n=44), TCF3-PBX1 (n=17), ETV6-RUNX1 (n=50), MLL-rearranged (n=24), BCR-ABL1 (PH; n=21), hypodiploid (Hypo; mainly near diploid cases with a dicentric chromosome; n=10), and other (n=92). The hypodiploid ALL cohort is divided into the near haploid (NH), low hypodiploid (LH) and near diploid (ND) subgroups. § Genes that were sequenced in the hypodiploid ALL cohort but not in the SJ cohort (genes that only are targeted by sequence mutations and not by copy number alteration and that were not sequenced in the SJ cohort are shaded in gray); † Copy number alteration (CNA); *Sequence mutation; **B cell pathway lesions include deletions or sequence mutations involving BLNK, EBF1, IKZF1, IKZF2, IKZF3, LEF1, PAX5, RAG1/2, and TCF3. VPREB1 may be considered part of the B cell pathway but is located in the immunoglobulin lambda light chain locus at 22q11.22, and is commonly deleted upon rearrangement of this locus. The biologic significance of VPREB1 deletions in B-ALL is thereby unclear, and the frequency of B cell pathway lesions is thus shown excluding and including VPREB1 alterations. iAmp21, internal amplification of chromosome 21; pwy, pathway.
SJHYPO055-D LH T125R Homo Somatic LFS and sporadic Deleterious DNA-binding
SJHYPO061-D LH R174_C176>R Homo Somatic DNA-binding
SJHYPO062-D NH A88fs Homo Somatic In COSMIC at aa Truncating Proline-rich
SJHYPO063-D LH R280K Homo Non-tumor LFS and sporadic DNA binding DNA-binding Yes
SJHYPO064-D LH R282fs Homo Somatic In COSMIC at aa Truncating DNA-binding
SJHYPO068-D LH R248W Het Somatic LFS and sporadic DNA contact DNA-binding Yes
SJHYPO074-D LH R273C Homo Somatic LFS and sporadic DNA contact DNA-binding
SJHYPO077-D LH Y163N Homo Somatic Sporadic Deleterious DNA-binding Yes
SJHYPO078-D LH R306* Homo Somatic LFS and sporadic Truncating None
SJHYPO079-D LH L130H Homo Non-tumor Sporadic Deleterious DNA-binding
SJHYPO080-D LH R273H Homo Somatic LFS and sporadic DNA contact DNA-binding Yes
SJHYPO083-D LH GinsR282 Homo Somatic DNA-binding
SJHYPO084-D LH Y220C Homo Somatic LFS and sporadic Deleterious DNA-binding Yes
SJHYPO093-D LH R248W Het Somatic LFS and sporadic DNA contact DNA-binding Yes
SJHYPO096-D ND R282>RLA Homo Somatic DNA-binding
SJHYPO119-D LH R273H Homo Non-tumor LFS and sporadic DNA contact DNA-binding Yes
SJHYPO120-D LH R280S Homo Non-tumor Sporadic DNA binding DNA-binding Yes
SJHYPO126-D LH I162fs Homo Non-tumor Sporadic Truncating DNA-binding
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Supplementary Table 19: IKZF1, IKZF2 and RB1 deletions in adult ALL.
Number (N) and percentage of cases with deletion in the respective gene are indicated. PH, BCR-ABL1 positive; H47, >47 chromosomes; H50, >50 chromosomes; MLL, mixed lineage leukemia; LH, low hypodiploid.
ALL subtype IKZF1
deletion (N)
IKZF1 deletion
(%)
IKZF2 deletion
(N)
IKZF2 deletion
(%)
RB1 deletion
(N)
RB1 deletion
(%)
Low hypodiploid (N=11) 0 0 3 27.3 2 18.2
Near haploid (N=1) 0 0 1 100 1 100
H50 (N=4) 0 0 0 0 0 0
MLL (N=3) 0 0 0 0 0 0
PH (N=31) 17 54.8 0 0 4 12.9
ERG (N=4) 1 25 0 0 2 50
H47 (N=2) 0 0 0 0 0 0
Other (N=40) 7 17.5 0 0 3 7.5
Bi-phenotypic (N=1) 0 0 0 0 0 0
N/A (N=4) 0 0 0 0 0 0
T-ALL (N=16) 0 0 0 0 0 0
Total non-LH (N=106) 25 23.6 1 0.9 10 9.4
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Supplementary Table 20: Alterations targeting histone modifiers in next-generation sequenced hypodiploid ALL.
SNV, single nucleotide variant; Indel, insertion/deletion mutation; CNV, copy number variation; DEL, deletion; D, diagnosis; R, relapse; G, remission; NH, near haploid; LH, low hypodiploid.
Sample Gene
Mutation type Mutation class Status Histone modifier type
Hypodiploid ALL subgroup
SJHYPO002-D CREBBP91
Indel K389_M395>K Somatic Histone writer NH
SJHYPO006-D CREBBP Focal CNV DEL Somatic Histone writer NH
Mutation type Mutation class Status Histone modifier type
Hypodiploid ALL subgroup
SJHYPO039-D CDC6, Ref83
SNV E402Q Somatic Histone DNA modifier NH
SJHYPO046-D TET1, Ref83
SNV E12_UTR_3 Somatic Histone DNA modifier NH
SJHYPO120-D MBD5, Ref85
SNV S1097I Somatic Histone DNA modifier LH
SJHYPO125-D TET3, Ref83
SNV G795D Somatic Histone DNA modifier LH
SJHYPO046-D ARID1A87
SNV P1384S Somatic Histone reorder chromatin NH
SJHYPO006-D HIST1H2BK SNV G14S Somatic Histone NH
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Supplementary Table 21: Differential expression analysis – NH versus LH.
See Excel Table: “Table_S21_NH_vs_LH_Limma.xlsx”
Differential expression analysis performed by limma with estimation of false discovery rate (FDR) at 0.05 between near haploid (NH) and low hypodiploid (LH) cases. More than 15,000 probesets showed differential expression between these two hypodiploid subgroups.
Supplementary Table 22: Gene set enrichment analysis (GSEA) – NH versus LH.
See Excel Table: “Table_S22_NH_vs_LH_GSEA.xlsx”
GSEA analysis comparing near haploid (NH) and low hypodiploid (LH) ALL, leaving 671 gene sets significant with an FDR cutoff at 0.25.
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Supplementary Table 23: Ex vivo drug study of PI3K/mTOR and MEK inhibitors on hypodiploid ALL cells.
IC50 values for the respective samples and drugs are shown. The concentration range tested for the different drugs are indicated. “>” indicates if an IC50 was not reached with the highest concentration tested. NT, not tested.
Hypodiploid subgroup
Generic ID Bez235 (0.03125-1uM)
GDC-0941 (0.02-5uM)
Mek162 (0.03125-1uM)
PD0325901 (0.1024-10uM)
near haploid SJHYPO037-X1 0.095 0.067 0.209 NT
near haploid SJHYPO037-X2 0.096 0.041 0.244 NT
near haploid SJHYPO054-X2 NT NT NT >10
near haploid SJHYPO054-X3 0.033 0.027 0.514 >10
near haploid SJHYPO123-X1 NT 0.079 >1 >10
near haploid SJHYPO123-X2 0.075 0.093 >1 >10
near haploid SJHYPO123-X3 0.075 NT NT NT
low hypodiploid SJHYPO077-X1 NT 0.027 NT <0.1024
low hypodiploid SJHYPO120-X1 0.109 0.501 >1 0.637
low hypodiploid SJHYPO120-X3 0.072 0.499 >1 0.155
near haploid NALM-16 0.261 0.673 >1 >10
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Supplementary Table 24: Sequences of shRNAs.
shRNA ID Sequences of the guide strands of the shRNA
Supplementary Table 25: Single nucleotide variations identified by mRNA seq of NALM-16.
See Excel Table: “Table_S25_NALM-16_mRNA-seq_variations.xlsx”
RNA-seq (transcriptome sequencing) was carried out for the near haploid ALL cell line NALM-16. Single nucleotide variations are listed. Assessment of functional impact of missense variations calculated by POLYPHEN and SIFT is included, as well as comparisons with a local database of sequence variations obtained from whole genome sequencing of tumor and normal DNA from 254 children (The SJCRH – Washington University Pediatric Cancer Genome Project (PCGP)93. Column definition is listed below: A: GeneName: HUGO gene symbol B: Chr: Chromosome C: HG19_Pos: Chromosome position in hg19 coordinates D: Class: Classification based on amino acid change pattern. Exon refers to variations in non-coding RNA genes. E: AAChange: Predicted amino acid change for the variation F: ProteinGI: NCBI protein GI number G: mRNA_acc: Refseq accession number H: ReferenceAllele: The allele represented in the reference human genome. Reference allele is marked as – for an insertion. I: MutantAllele: Mutant allele J: Flanking: 20bp[reference allele/mutant allele]20bp K: Freq: Frequency of reads with the variation L: SIFTResult: Deleterious status assigned by SIFT M: SIFTScore: SIFT score N: pph2result: Deleterious status assigned by polyPHEN2 O: pph2score: PolyPHEN2 score P: PCGP+-2: Variation identified not at the specific site, but within 2bp, in at least two whole genome sequenced sample from the PCGP. Q: COSMIC_OMIM_VALID_CLINIC+-2: Variation identified not at the specific site, but within 2bp, in COSMIC or OMIM. R: COSMIC_OMIM_VALID_CLINIC_pmid: Pubmed ID
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Supplementary Table 26: Primer sequences used for targeted gene resequencing and NF1 deletion mapping.
See Excel Table: “Table_S26_Primers.xlsx”
Supplementary Table 27: Murine lymphoid precursor cells used for gene expression profiling.
Bone marrow harvested from wild-type C57BL/6 mice was flow sorted based on the surface marker scheme below.
Differentiation stage
Explanation Surface markers used for flow cytometric cell sorting
CLP Common lymphoid precursor Sca1low
, Lin-, IL7Rα
+, cKIT
low
Hardy Fraction A pre-pro-B B220+, CD43
+, CD24
-, BP-1
-
Hardy Fraction B Pro-B B220+, CD43
+, CD24
+, BP-1
-
Hardy Fraction C Pre-B early B220+, CD43
+, CD24
+, BP-1
+
Hardy Fraction D Pre-B late B220+, CD43
-, IgM
-, IgD
-
Hardy Fraction E Immature B cells B220+, CD43
-, IgM
+, IgD
-
Hardy Fraction F Mature B cells B220-bright, CD43-, IgM
+, IgD
+
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Supplementary Table 28: Antibodies used for biochemical studies.
CD16/CD32 553142 BD Biosciences Used to block Fc receptors
sc-68, Neurofibromin (N) sc-68 Santa Cruz Biotechnology, Inc. Immunoblotting; N-terminal epitope of NF1
sc-67, Neurofibromin (D) sc-67 Santa Cruz Biotechnology, Inc. Immunoblotting; C-terminal epitope of NF1
Aiolos sc-101982 Santa Cruz Biotechnology, Inc. Immunoblotting
Helios sc-9866 Santa Cruz Biotechnology, Inc. Immunoblotting
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Supplementary Table 29: Association between aneuploidy and event free survival (EFS).
Estimated EFS in percentage with standard error within parenthesis. Information is missing for 17 hypodiploid ALL cases. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%.
P values calculated by a Log-rank Test. NH, near haploid; LH, low hypodiploid. Masked and non-masked cases separated
Hypodiploid subgroup n= Year 1 Year 2 Year 5 P value
Near haploid 43 79.6 (6.5) 57.1 (8.2) 54.2 (13.0) 0.11
Masked near haploid 17 87.1 (8.7) 77.4 (13.0) 77.4 (26.0)
Supplementary Table 30: Association between aneuploidy and cumulative incidence of any relapse. Cumulative incidence (CIN) of any relapse in percentage with standard error within parenthesis. Information is missing for 17 hypodiploid ALL cases. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. P values calculated by Gray's Test. NH, near haploid; LH, low hypodiploid.
Masked and non-masked cases separated
Hypodiploid subgroup n= Year 1 Year 2 Year 5 P value
Near haploid 43 20.4 (6.5) 37.3 (8.1) 40.2 (8.3) 0.10
Masked near haploid 17 12.9 (8.9) 20.2 (10.9) 20.2 (10.9)
Supplementary Table 31: Association between aneuploidy and minimal residual disease.
Number of cases with negative (Neg) and positive (Pos) minimal residual disease (MRD), respectively. Information is missing for 33 hypodiploid ALL cases. Masked hypodiploid cases here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. NH, near haploid; LH, low hypodiploid. P values were calculated by an Exact Chi-Square test.
Comparison between near haploid and low hypodiploid ALL, excluding near diploid ALL (P = 0.82) Hypodiploid subgroup n= MRD Neg (< 0.01%) MRD Pos (≥0.01%)
NH and masked NH 55 60% 40%
LH and masked LH 21 57.1% 42.9%
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Supplementary Table 32: Association between copy number alterations/mutations and event free survival (EFS).
Estimated EFS in % with standard error (SE) within parenthesis. P values calculated by a Log-rank Test. Signaling indicates that gene is involved in RTK- or Ras signaling.
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Factors n= Year 1 Year 2 Year 5 P value
CDKN2A/B
Normal 70 81.6 (4.8) 64.0 (6.5) 61.8 (10.6) 0.81
Altered 37 77.9 (7.0) 71.6 (8.0) 59.9 (10.1)
Histone cluster (6p22)
Normal 93 78.3 (4.5) 64.8 (5.7) 57.6 (8.0) 0.35
Altered 14 92.9 (6.6) 78.6 (10.5) 78.6 (16.3)
FLT3 (Signaling)
Normal 102 79.4 (4.2) 65.2 (5.3) 58.9 (7.6) 0.16
Altered 5 100 (0.0) 100 (0.0) 100 (0.0)
KRAS (Signaling)
Normal 105 80.8 (4.0) 67.0 (5.1) 60.7 (7.6) 0.54
Altered 2 50.0 (25.0) 50.0 (25.0) 50.0 (25.0)
NF1 (Signaling)
Normal 76 81.1 (4.6) 67.5 (5.9) 59.3 (8.7) 0.92
Altered 31 77.6 (8.2) 64.3 (9.9) 64.3 (13.6)
NRAS (Signaling)
Normal 94 78.8 (4.4) 66.4 (5.4) 59.6 (7.9) 0.45
Altered 13 90.9 (8.3) 68.2 (14.5) 68.2 (19.2)
PTPN11 (Signaling)
Normal 104 80.6 (4.0) 67.7 (5.1) 61.4 (7.5) 0.13
Altered 3 66.7 (22.2) 33.3 (19.2) 33.3 (27.2)
Signaling combined
Normal 57 80.5 (5.2) 68.9 (6.6) 58.8 (9.4) 0.98
Altered 50 79.9 (6.1) 63.8 (7.8) 63.8 (11.6)
IKZF1 (B pathway)
Normal 104 79.6 (4.1) 66.7 (5.2) 60.3 (7.8) 0.77
Altered 3 100 (0.0) 66.7 (22.2) 66.7 (22.2)
IKZF2 (B pathway)
Normal 94 84.1 (4.0) 68.4 (5.4) 63.9 (7.5) 0.043
Altered 13 53.8 (12.9) 53.8 (13.8) 26.9 (23.0)
IKZF3 (B pathway)
Normal 97 78.1 (4.4) 63.4 (5.4) 58.3 (7.4) 0.12
Altered 10 100 (0.0) 100 (0.0) 83.3 (34.0)
PAX5 (B pathway)
Normal 90 77.4 (4.6) 62.0 (5.9) 53.9 (9.2) 0.014
Altered 17 94.1 (5.5) 88.2 (7.6) 88.2 (9.1)
VPREB1 (B pathway)
Normal 97 78.2 (4.4) 65.6 (5.4) 58.7 (8.0) 0.30
Altered 10 100 (0.0) 77.8 (13.9) 77.8 (16.4)
B pathway combined
Normal 61 76.4 (5.8) 57.7 (7.1) 52.7 (9.7) 0.076
Altered 46 84.7 (5.3) 77.4 (6.7) 69.9 (10.6)
RB1
Normal 89 81.1 (4.3) 68.0 (5.4) 61.1 (7.6) 0.62
Altered 18 77.4 (10.6) 60.3 (14.4) 60.3 (26.9)
TP53
Normal 83 83.1 (4.3) 68.4 (5.7) 63.3 (7.8) 0.31
Altered 24 70.8 (9.0) 61.1 (11.0) 45.8 (19.5)
PAG1
Normal 100 81.0 (4.1) 69.8 (5.2) 63.3 (7.5) 0.034
Altered 7 71.4 (15.6) 28.6 (13.9) No Data
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Supplementary Table 33: Association between copy number alterations/mutations and cumulative incidence (CIN) of any relapse.
CIN of any relapse in % with SE within parenthesis. P values calculated by Gray's Test. Signaling indicates that gene is involved in RTK- or Ras signaling.
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Factors n= Year 1 Year 2 Year 5 P value
CDKN2A/B
Normal 70 18.4 (4.8) 34.0 (6.2) 36.2 (6.4) 0.26
Altered 36 17.1 (6.5) 20.5 (7.1) 27.7 (8.2)
Histone cluster (6p22)
Normal 92 19.7 (4.3) 30.6 (5.2) 35.5 (5.6) 0.55
Altered 14 7.1 (7.1) 21.4 (11.4) 21.4 (11.4)
FLT3 (Signaling)
Normal 101 18.7 (4.0) 30.7 (4.9) 34.9 (5.2) 0.16
Altered 5 0 0 0
KRAS (Signaling)
Normal 105 18.2 (3.9) 29.7 (4.8) 33.8 (5.1) 0.47
Altered 1 0 0 0
NF1 (Signaling)
Normal 75 16.4 (4.4) 28.5 (5.5) 34.1 (5.9) 0.81
Altered 31 22.4 (8.3) 31.6 (9.6) 31.6 (9.6)
NRAS (Signaling)
Normal 93 19.2 (4.2) 29.2 (5.0) 33.7 (5.3) 0.64
Altered 13 9.1 (9.1) 31.8 (16.4) 31.8 (16.4)
PTPN11 (Signaling)
Normal 104 18.4 (3.9) 28.9 (4.8) 33.1 (5.1) 0.67
Altered 2 0 50.0 (50.0) 50.0 (50.0)
Signaling combined
Normal 57 17.7 (5.1) 27.2 (6.1) 34.1 (6.7) 0.93
Altered 49 18.4 (6.0) 32.3 (7.6) 32.3 (7.6)
IKZF1 (B pathway)
Normal 103 18.5 (4.0) 29.2 (4.8) 33.5 (5.1) 0.90
Altered 3 0 33.3 (33.3) 33.3 (33.3)
IKZF2 (B pathway)
Normal 93 15.0 (3.9) 28.2 (5.1) 32.7 (5.4) 0.30
Altered 13 38.5 (14.2) 38.5 (14.2) 38.5 (14.2)
IKZF3 (B pathway)
Normal 96 20.0 (4.2) 32.3 (5.1) 35.3 (5.3) 0.15
Altered 10 0 0 16.7 (16.7)
PAX5 (B pathway)
Normal 90 21.4 (4.5) 33.9 (5.4) 39.2 (5.8) 0.0086
Altered 16 0 6.3 (6.3) 6.3 (6.3)
VPREB1 (B pathway)
Normal 96 19.8 (4.2) 30.0 (5.0) 34.6 (5.4) 0.42
Altered 10 0 22.2 (14.8) 22.2 (14.8)
B pathway combined
Normal 61 23.6 (5.8) 38.1 (6.9) 43.1 (7.2) 0.033
Altered 45 11.2 (4.8) 18.6 (6.0) 21.5 (6.5)
RB1
Normal 88 18.0 (4.2) 28.5 (5.1) 33.3 (5.5) 0.81
Altered 18 16.7 (9.1) 33.8 (13.8) 33.8 (13.8)
TP53
Normal 82 15.9 (4.2) 27.8 (5.4) 32.9 (5.8) 0.59
Altered 24 25.0 (9.1) 34.7 (10.4) 34.7 (10.4)
PAG1
Normal 99 17.1 (3.9) 25.9 (4.7) 30.3 (5.1) 0.023
Altered 7 28.6 (18.6) 71.4 (20.0) No Data
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Supplementary Table 34: Multivariable analysis of copy number alterations/mutations, clinical features and association with cumulative incidence of any relapse.
Fine & Gray’s modeling of CIN of any relapse identified PAG1 alteration as the only gene alteration independently associated with poor outcome. WBC, white blood cell count; MRD, minimal residual disease. HR, hazard ratio; CI, confidence interval.
Supplementary Figure 1: Coverage plots for next-generation sequenced hypodiploid ALL cases.
a-c, Different colors represent different fold coverage, as indicated. SJHYPO052-D and –G underwent whole genome sequencing, while exome sequencing was performed for SJHYPO052-R, explaining the gap seen around SJHYPO052 in panel c.
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Supplementary Figure 2: Circos plots of whole genome sequenced hypodiploid ALL.
Circos94 plots depicting structural genetic variants, including DNA copy number alterations, intra- and inter-chromosomal translocations, and sequence alterations. Loss-of-heterozygosity, orange; amplification, red; deletion, blue; Sequence mutations in Refseq genes: non-silent single nucleotide variants, brown; insertion/deletions, red; genes at structural variant breakpoints: genes involved in in-frame fusions, pink; others, blue.
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Supplementary Figure 3: Mutation spectrum of next-generation sequenced hypodiploid ALL.
Next-generation sequenced cases are depicted from left to right. Colored bars represent number of specific lesions identified in each case as indicated. WGS, whole genome sequencing; WES, whole exome sequencing; SNV, single nucleotide variation; aa, amino acid; CDS, coding DNA sequence; UTR, untranslated leader region; HQ, high quality; CNV, copy number variation; Amp, amplification; Del, deletion; Mb, megabases.
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Supplementary Figure 4: Protein domain and alteration plots for targets of sequence mutations in hypodiploid ALL.
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Supplementary Figure 5: Mapping of NF1 deletions. a, Top: Schematic of the NF1 gene with direction indicated by arrows and exons by vertical lines. The intragenic deletions and amplification of NF1 are depicted for each hypodiploid ALL case harboring a copy number alteration in this gene. Heterozygous deletions are shown as solid lines and homozygous deletions as dotted lines. The majority of focal deletions are accompanied by loss of the entire other chromosomal copy, leading to a bi-allelic loss. The intragenic NF1 amplification gives rise to copy-neutral loss-of-heterozygosity in SJHYPO120. b, Electropherogram showing the fusion point at the genomic level for one case. A 3 base pair (bp) insertion of non-consensus bases is present between the intron 14 to intron 35 fusion. Together with the presence of partially conserved heptamer recombination signal sequences (RSS) immediately internal to the genomic breakpoints, this is suggestive of a RAG mediated recombination event. c, Transcriptome sequencing (mRNA seq) data from the hypodiploid ALL cell line NALM-16. Read depth (red) and GC content (blue) for all NF1 exons are shown, with no coverage of exons 15-35. d, Schematic of the full length NF1 gene (top), NF1 gene with homozygous deletion from intron 14 to intron 35 (middle), and two putative open reading frames (ORF1 and ORF2) present in the NALM-16 NF1 transcript. Putative ORF1 is translated from the canonical NF1 start site, and has a premature stop codon in exon 36 downstream of the deletion. Putative ORF2 has an alternative start site in exon 37, downstream of the deleted region. The deleted region is depicted by a gray box, and ORFs as white boxes. e, NALM-16 NF1 RNA seq raw data presented in the Bambino viewer95. The corresponding paired reads spanning over the breakpoint between exon 14 (upper) and exon 36 (lower) are shown in the top and bottom panels, respectively. Read 1-5 indicate reads in the top and bottom panels that are the same read, spanning the splice site.
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Supplementary Figure 6: Immunoblot analysis of NF1. a-b, Immunoblot analysis on the cell lines NALM-16 and THP-1 using the antibody sc-68 (a), raised against the N-terminus of NF1, and sc-67 (b) raised against the C-terminus of NF1. The size of full length NF1 is 250kDa, and the predicted sizes of putative NF1 mutant ORFs in NALM-16 are 62kDa (mutant ORF1, N-terminal part of wild-type NF1) and 115kDa (mutant ORF2, C-terminal part of wild-type NF1), respectively. Full length, wild-type NF1 is not present in NALM-16, but detected in the control THP-1. Only a nonspecific band, present also in THP-1, is seen at the size of mutant putative ORF1 (a), and no band is detected for mutant putative ORF2 (b), indicating that the NF1 deletion leads to loss of NF1 protein production.
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Supplementary Figure 7: Validation of mutations in NRAS and PTPN11 in non-tumor samples in near haploid ALL.
a and b, Electropherograms of forward and reverse DNA sequences covering the NRAS p.Gly12Ser substitution in SJHYPO020 (a) and PTPN11 p.Gly503Arg in SJHYPO036 (b). The only available non-tumor DNA was obtained from hematopoietic cells from the respective patient, and it is thus not known if the mutations were inherited or acquired in the hematopoietic compartment prior to the development of leukemia. The respective mutated codons are shown in upper case letters. a, SJHYPO020-D (P1) is the sorted tumor population (CD45-dim, CD19 positive (+)); SJHYPO020-G is a remission bone marrow sample from this patient; P2 is the sorted CD45 positive, CD7 positive fraction from a bone marrow sample taken at diagnosis; P3 is the sorted CD45 positive, CD7 negative fraction from the same diagnosis sample. b, SJHYPO036-D and –G represent samples taken at diagnosis and remission, respectively. c, Fluorescence activated cell sorting (FACS) plots showing the gating for the cell sorting of an SJHYPO020 bone marrow sample taken at diagnosis.
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Supplementary Figure 8: PAG1 deletions correlate with PAG1 expression levels. a, Heatmap showing SNP microarray data for the area covering PAG1 on chromosome 8q21.12. A focal deletion in PAG1 was first detected in the relapse sample for SJHYPO056, while this deletion was not detected at diagnosis (D vs R*). b, Top: Schematic of the PAG1 gene with direction indicated by arrows and exons by vertical lines. WGL log2 ratio copy number data visualized in the UCSC web browser (http://genome.ucsc.edu/). The pink vertical lines indicate probe intensities, with lines below the respective black zero lines correspond to a loss of genetic material. Double-headed arrows indicate the extent of the deletions. Note the lack of focal deletion in SJHYPO056-D but deletion at relapse. c, Relative gene expression levels of the PAG1 transcript in hypodiploid ALL samples either wild-type (WT) for PAG1 or harboring a PAG1 deletion (Del) as indicated, as assessed by three PAG1 specific probe sets from Affymetrix GeneChip HT HG-U133+ PM microarrays.
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Supplementary Figure 9: Mutant p53 fails to stimulate p21 in hypodiploid ALL. a, Flow cytometry analysis of p53 (left panel) and p21 (right panel) levels in cells from hypodiploid ALL xenograft SJHYPO120-X, harboring a p.Arg280Ser p53 substitution, and the cell line Reh (TEL-AML1 ALL harboring wild-type p53). Cells were treated with increasing concentrations of etoposide to activate p53, as indicated. p53 levels were already high in the p53 mutant cells, and etoposide treatment did not lead to increased p21 levels in these cells, while stimulation was seen in Reh. b, Histological examination of sternum stained for p53 (left panel) and p21 (right panel) from mice xenografted with primary hypodiploid ALL tumor cells either mutant (upper) or wild-type (lower) for TP53 as indicated. Scale bar corresponds to 50 microns.
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Supplementary Figure 10: IKZF1 and IKZF2 deletions in adult ALL. a-b, SNP 6.0 microarray heatmaps showing focal deletions of IKZF1 (a) and IKZF2 (b) in the adult ALL cohort. Blue indicates DNA loss. PH+, Philadelphia chromosome (BCR-ABL1) positive ALL; LH, low hypodiploid; NH, near haploid.
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Supplementary Figure 11: Expression of Ikzf1, Ikzf2 and Ikzf3 during murine lymphoid development. a-c, Gene expression levels of the Ikaros family genes Ikzf1 (a), Ikzf2 (b) and Ikzf3 (c) in murine cells flow sorted into Hardy Fractions as assessed by 2-3 probesets per gene from Affymetrix GeneChip MG-430 2.0 microarrays. One-way analysis of variance was performed to test for significant differences between the groups. CLP, common lymphoid precursor; Hardy Fraction A, pre-pro-B; B, proB; C, preB early; D, preB late; E, immature B; F, mature B cells.
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Supplementary Figure 12: CD19 levels and degree of antigen receptor rearrangements in hypodiploid ALL.
a, Comparison of CD19 expression level on near haploid and low hypodiploid ALL tumor cells. All near haploid (N=47) and low hypodiploid (N=19) cases with available CD19 expression data from flow cytometry studies are compared. There is a significant association between the level of CD19 expression and hypodiploid ALL subgroup. b, Percentage of cases with a rearrangement in the antigen receptor loci at 2p11.2 (IGK@), 7p14.1 (TRG@), 7q34 (TRB@), 14q11.2 (TRA@), 14q32.33 (IGH@) and/or 22q11.22 (IGL@). c, Heatmap showing SNP 6.0 microarray data for the area covering TRG@ (the T cell receptor gamma locus) on chromosome 7p14.1. Light blue of the entire region shown indicates either 7p loss (in near diploid cases) or whole chromosome 7 loss. Focal deletions (light or dark blue) indicate a rearrangement at the TRG@ locus. AgR, antigen receptor rearrangement; LH, low hypodiploid. d, Gene set enrichment analysis demonstrates enrichment for Hardy Panel fraction B (pro-B cell stage) in low hypodiploid ALL compared with near haploid ALL. The gene set HARDYWTB_500UP includes the top 500 probesets upregulated in Hardy Panel B compared with the other Hardy Panel fractions, identified using limma.
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Supplementary Figure 13: RB1 alterations in pediatric hypodiploid ALL and adult ALL. a, Protein domain plot of RB1 with alterations identified in pediatric hypodiploid ALL. b-c, SNP 6.0 microarray heatmaps showing focal deletions of RB1 in pediatric hypodiploid ALL (b) and the adult ALL cohort (c). A case with a simultaneous RB1 deletion and sequence mutation is indicated by a Y in b. Blue indicates DNA loss. mNH, masked near haploid; LH, low hypodiploid; NH, near haploid; PH+, Philadelphia chromosome (BCR-ABL1) positive ALL.
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Supplementary Figure 14: Tumor suppressor gene pathway alterations in hypodiploid ALL. CDKN2A/CDKN2B are tumor suppressor genes functioning upstream of TP53 and RB1. The Total percentages indicate cases with both CDKN2A/B deletions and either TP53 (left) or RB1 (right) alterations for each hypodiploid subgroup. Genes in boxes have been subject of targeted resequencing, while genes in ovals have not been sequenced. Genes shaded in gray do not harbor any known alterations.
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Supplementary Figure 15: Deletions and sequence mutations in genes encoding histones and histone modifiers. a, SNP 6.0 microarray heatmap showing focal deletions in the histone cluster at chromosome 6p22 in hypodiploid ALL. The minimal region of deletion involved genes HIST1H2BE, HIST1H4D, HIST1H3D, HIST1H2AD and HIST1H2BF. Blue indicates DNA loss. Masked hypodiploid cases do here refer to cases with either a pure doubled hypodiploid clone or cases harboring a doubled clone constituting at least 30%. mNH, masked near haploid; LH, low hypodiploid; ND, near diploid. b, Protein domain and alteration plot of CREBBP.
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Supplementary Figure 16: GEP restricted to probes on chromosomes showing identical patterns of aneuploidy.
a-b, Unsupervised principal component analysis (PCA) of gene expression data from all hypodiploid ALL cases with available high quality RNA (N=94). Near haploid/masked near haploid, low hypodiploid/masked low hypodiploid and near diploid cases form three distinct clusters by PCA also when restricting the analysis to commonly aneuploid chromosomes (a) or only chromosome 21 (b), which always retains both the maternal and paternal chromosomal copies.
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Supplementary Figure 17: Flow cytometric analysis of signaling pathways in hypodiploid ALL. Spleen cells from mice transplanted with primary human hypodiploid ALL samples or the hypodiploid ALL cell line NALM-16 (non-transplanted) were analyzed for the presence of the indicated proteins. Healthy donor is a control, and indicates cells from a peripheral blood sample from a non-cancerous individual.
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Supplementary Figure 18: Ikzf2 and Ikzf3 knockdown efficiency assessed by immunoblot analysis. a-b, Immunoblot analysis on the murine cell lines Ba/F3 (a) and Arf-/- pre-B (b) using the antibody sc-9866 detecting Helios (a), and sc-101982 detecting Aiolos (b). Luc-1309 indicates a control shRNA specific for Firefly luciferase mRNA. The cells expressing shRNAs Ikzf2-4422, Ikzf2-8315, Ikzf3-449 and Ikzf3-1586 were used for downstream analyses. Knockdown did not influence cell viability, cell cycle distribution or proliferation (data not shown).
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Supplementary Figure 19: Flow cytometric analysis of signaling pathways in hematopoietic cell lines.
Flow cytometric analyses detecting levels of pERK (a) and pS6 (b) in murine cell lines after knockdown of Ikzf2 (in Ba/F3, left panel) and Ikzf3 (in Arf-/- pre-B cells, right panel) and stimulation with PMA (50nM, 15 minutes). Two independent shRNAs per gene were employed. Luc-1309 indicates a control shRNA specific for Firefly luciferase mRNA.
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Supplementary Figure 20: The importance of optimal normalization of SNP microarray data.
a-c, SNP 6.0 microarray data for 10 hypodiploid ALL diagnosis (D) and matched remission (G)
samples are presented from left to right in each of the three panels. Chromosomes are shown
from 1-22, X and Y from top to bottom. a, Normalization is performed using quantile
normalization in dChip, a median centering approach that borrows information across arrays,
and the hypodiploid genomes are erroneously normalized. b, The same data set presented after
performance of reference normalization96 in which only chromosomes known or predicted to be
diploid are used as reference chromosomes to guide normalization of the entire array, and in
which normalization of each array is performed independently of other samples. HYPO053,
HYPO055 and HYPO084 contains substantial proportions of a doubled clone and are
normalized as masked low hypodiploid cases. a-b; Red indicates gain of genetic material, and
blue indicates loss. c, Loss-of-heterozygosity (indicated in dark blue) visualization for the same
samples in dChip. Each tumor sample was directly compared to its matched remission sample.
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Supplementary Figure 21: Immunohistochemistry and FACS analyses of tissue from mice xenografted with human primary hypodiploid ALL cells.
a-f, Selected tissues stained for human CD45 (left) and with hematoxylin and eosin (HE) stain (right) in each panel. Scale bar corresponds to 50 microns. Spleen, meninges and sternal marrow from SJHYPO072-X2 (a-c) and SJHYPO120-X1 (d-f) are shown. g-h, FACS analysis of bone marrow from SJHYPO072-X2 (g) and -X3 (h). Cells are stained with DAPI, for mouse CD45 (PE-Cy7 conjugated antibody), and human CD45 (FITC conjugated), CD19 (APC) and CD3 (PE).
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Supplementary Figure 22: Copy number analysis of primary hypodiploid ALL samples versus xenografted leukemic samples.
a-c, Copy number analysis comparing related hypodiploid ALL diagnosis (D) and xenograft (X1-3) samples. The focal deletions that were present at diagnosis were retained in the xenograft clones. a, The masked near haploid case SJHYPO072-D harbored three homozygous focal deletions (in the Histone cluster at 6p22.1, PAG1 and IKZF3). The other corresponding chromosomal copy of chromosomes 6, 8 and 17 was lost prior to reduplication of the near haploid genome, giving rise to copy neutral loss-of-heterozygosity of these chromosomes, with focal homozygous deletions of the respective gene. b, SJHYPO039-D is a near haploid case with the only focal deletion present in ETV6. c, The near haploid case SJHYPO123-D harbored a focal deletion only in NF1, with gain of 4 lesions in the xenograft. Blue indicates loss of genetic material.
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