Supplemental Information - 1 -
SUPPLEMENTAL INFORMATION
Genomic Analysis Reveals Few Genetic Alterations in Pediatric Acute
Myeloid Leukemia
Ina Radtkea, Charles G. Mullighana, Masami Ishiia , Xiaoping Sua, Jinjun Chenga, Jing Mab, Ramapriya Gantia,
Zhongling Caia, Salil Goorhaa, Stanley B. Poundsc, Xueyuan Caoc, Caroline Obertb, Jianling Armstrongb, Jinghui
Zhangd, Guangchun Songa, Raul C. Ribeiroe, Jeffrey E. Rubnitze, Susana C. Raimondia, Sheila A. Shurtleffa, and
James R. Downinga
Departments of aPathology, cBiostatistics, and eOncology, and the bHartwell Center for Bioinformatics and
Biotechnology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105; dCenter for
Biomedical Informatics and Information Technology, National Cancer Institute, National Institutes of Health,
Bethesda, MD, 2115 E Jefferson Street, Rockville, MD 20892.
Corresponding Author: James R. Downing, M.D. Scientific Director St. Jude Children’s Research Hospital 262 Danny Thomas Place Memphis, TN 38105 Phone: +1-901-595-3510
Supplemental Information - 2 -
Supplementary Experimental Procedures:
Patients and SNP array analysis
DNA extracted from leukemic blasts and remission bone marrow or blood samples was genotyped with
Affymetrix GeneChip Human Mapping 50K Hind 240, 50K Xba 240, 250K Sty and 250K Nsp arrays according to manufacturer’s instructions (Affymetrix, Santa Clara, CA). Copy number and genotype data of all four arrays were combined, resulting in higher resolution than achieved when analyzing each array (50K, 250K) or array platform
(100K, 500K) separately. Combination of arrays resulted in an inter-marker distance of less than 5Kb. DNA copy number and LOH analyses were performed using a reference normalization algorithm normalization (1), dChipSNP
(2) and circular binary segmentation (3), as described previously (4). Diagnostic samples were characterized by karyotyping, and by RT-PCR for PML-RARA, RUNX1-RUNX1T1 (AML1-ETO) and CBFB-MYH11.
To filter raw circular binary segmentation results and obtain candidate CNA regions, we used the following criteria: [1] mean log2 ratios of the segment ≥ 0.2 or ≤ -0.2; [2] at least 3 SNPs within a segment; [3] segment size >
0 (this was to exclude the cases where one genomic location may have more than one SNPs due to using combined data from multiple sub-arrays). All calls passing this filter were visually inspected using DChipSNP to eliminate false calls due to batch effects and noise. Calls from samples lacking matched remission materials were crosschecked against our own pool of 300+ reference samples (4, 5) and against the Database of Genomic Variants
(http://projects.tcag.ca/variation/). Alterations matching any inherited copy number variations in our pool of reference samples or the database were eliminated. The quality control date for the SNP array analysis included median SNP call percentages (and range) as follows: 50K Hind 240 of 92.2 (79.8-99.3); 50K Xba 240 96.3 (88.8-
99.5); 250K Sty 94.1 (82.0-98.0); and 250K Nsp 91.8 (76.3-97.2).
The characteristics of the patients analyzed in this study included a mean age of 9.3 yrs, WBC count of
34.75, and included 50.96% female and 49.04% male. The patients were treated a St. Jude led AML therapeutic protocols including AML80 (1 patient), AML83 (1), AML87 (18), AML91 (16), AML97 (32), AML02 (30), or off protocol (13).
Fluorescent In situ hybridization (FISH)
Archived bone marrow cells were fixed and stored in Carnoy’s fixative and dried onto slides. Bacterial artificial chromosomes (Children’s Hospital Oakland Research Institute, Oakland, CA; Open Biosystems, Supplemental Information - 3 -
Huntsville, AL) were used as probes. Their identity was validated by T7 and SP6 BAC-end sequencing. The BACs were labeled with fluorescein isothiacyanate or rhodamine fluorochromes. Hybridization and analysis was performed as described previously (4). BAC clones used are listed in Figure legends.
RT-PCR for cryptic fusions and MLL partial tandem duplications (PTD)
Total RNA was isolated using TRIZOL (Invitrogen, Carlsbad, CA) and cDNA was transcript using
Superscript III (Invitrogen, Carlsbad, CA) standard protocols. For PCR amplification and sequencing of the product of NUP98-NSD1 and the MLL-MLLT4/AF6 fusions and of MLL PTD, primers and PCR conditions previously published were used (6-8).
Genomic Taqman for CCDC26
Primers for genomic quantitative PCR were designed using PrimerExpress 3.0 (Applied Biosystems, Foster
City, CA). RNAse P primers were used for control amplification. 200ng of leukemic blast DNA or control human
DNA was amplified using the same conditions as those described for RNA real-time PCR. Standard curves for
CCDC26 and RNase P were generated using normal human DNA. Assays were performed in duplicate. Copy number values for CCDC26 were normalized by dividing the value obtained for CCDC26 by the value obtained for
RNase P for each sample. A cutoff of greater than 1.3 was used to identify gain of one copy.
Genomic DNA sequencing
Genomic sequencing was performed on exons and splice sites of following genes: FLT3, NRAS, KRAS,
PTPN11, BRAF, RUNX1/AML1, BTG1, CCDC26, ERG, CEPBA, ETV6, FAM20C, FBXW7, GATA1, IKZF1/Ikaros,
LYL1, CDKN2A/B, PAXIP1, TUSC1, TP53, XRCC2, KIT, NPM1, PTEN and MLL-PTD (see above). Primers were generated by Primer3 (9) (http://frodo.wi.mit.edu/primer3/input.htm). For the majority of exons, primers with universal tails were used (SP6, M13). Amplicon-specific primers were used for the few amplicons for which tailed primers did not yield sufficient sequencing results. DNA extracted from leukemic cells or remission material was amplified by whole genome amplification (Qiagen, Valencia, CA). PCR amplification was performed using standard protocols, Accuprime GC rich (Invitrogen) and Eppendorf Mastercyclers (Eppendorf, Westbury, NY). PCR products were purified using the Wizard SV Gel and PCR Clean-up Kit (Promega, Madison, WI) and directly sequenced Supplemental Information - 4 - using Big Dye Terminator (v3.1) chemistry on 3730xl DNA Analyzers (Applied Biosystems, Foster City, CA).
Primer sequences and PCR conditions are available on request.
Sequence analysis was performed by comparison against the reference sequence using Sequencher (Gene
Codes Cooperation, Ann Arbor, MI) and through an updated version of The Cancer Genome WorkBench (10).
Coding exons with 11bp flanking intronic sequence (to cover splice sites) were analyzed. All identified non- synonymous sequence variations not previously identified as SNPs according to dbSNP (11)
(http://www.ncbi.nlm.nih.gov/projects/SNP/) were visually inspected and confirmed by repeating PCR and sequencing of unamplified DNA.
Sequencing of remission DNA was also performed to distinguish inherited from somatic variants.
Mutations were called homozygous if the height of the secondary wild-type peak was less than 30% of the primary mutant peak. Mutations were described using the nomenclature of the human genome variation society (12)
(http://www.hgvs.org/mutnomen/).
Deposition of micro-array data
The primary SNP data was deposited on GEO.
Genomic Identification of significant targets in cancer (GISTIC)
We applied the GISTIC method describe by Beroukhim R. et al. (2007) (13) to the curated segmentation results. GISTIC identifies significantly amplified or deleted regions of the genome across a set of samples. Each aberration is assigned a G-score that considers the amplitude of the aberration as well as the frequency of its occurrence across samples. GISTIC then assesses the statistical significance of each aberration by comparing the observed G-scores to the results that would be expected by chance, using a permutation test that is based on the overall pattern of aberrations seen across the genome. The method accounts for multiple-hypothesis testing using the false-discovery rate (FDR) framework to assign a q value to each aberration. We used the default q-value cutoff of
0.25 here, as shown by the green vertical line in Figure 1. Significant regions are listed in Table S3. For each significant region, a peak region was identified, which is the part of the aberrant region with greatest amplitude and frequency of alteration and with minimal q-value. In addition, a wide peak was determined using a leave-one-out Supplemental Information - 5 - algorithm to allow for errors in the boundaries in a single sample. The wide peak boundaries are more robust for identifying the most likely gene targets in the region.
Statistical Analysis
The exact Kruskal-Wallis test was used to compare the average number of copy number gains, copy number losses, copy number alterations, and sequence mutations across subtypes. The exact Pearson chi-square test was used to compare the frequency of specific mutations across subtypes. The exact log-rank test, stratified by treatment protocol, was used to explore the association of the number of copy number abnormalities (gains, losses, or either) with event-free and overall survival. For log-rank analysis, patients were categorized as having zero, 1-2, or three or more lesions. Event-free survival was defined as the time elapsed from initiation of therapy to remission induction failure, relapse, second malignancy, or death, with those living and free of these events censored at last follow-up. Overall survival was defined as the time elapsed from initiation of therapy to death, with surviving patients censored at last follow-up. The p-values for each exact test are based on a Monte Carlo approximation using
10,000 permutations. SAS software, Windows version 9.1.3, was used to perform these analyses. Supplemental Information - 6 -
References
1. Pounds S, et al. (2008) Reference Alignment of SNP Microarray Signals for Copy Number Analysis of Tumors. Bioinformatics. 2. Lin M, et al. (2004) dChipSNP: significance curve and clustering of SNP-array-based loss-of- heterozygosity data. Bioinformatics 20:1233-1240. 3. Olshen AB, Venkatraman ES, Lucito R, & Wigler M (2004) Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics 5:557-572. 4. Mullighan CG, et al. (2007) Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 446:758-764. 5. Mullighan CG, et al. (2008) BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 453:110-114. 6. Jaju RJ, et al. (2001) A novel gene, NSD1, is fused to NUP98 in the t(5;11)(q35;p15.5) in de novo childhood acute myeloid leukemia. Blood 98:1264-1267. 7. Mitterbauer G, et al. (2000) Monitoring of minimal residual disease in patients with MLL-AF6-positive acute myeloid leukaemia by reverse transcriptase polymerase chain reaction. Br J Haematol 109:622-628. 8. Ross ME, et al. (2004) Gene expression profiling of pediatric acute myelogenous leukemia. Blood 104:3679-3687. 9. Rozen S & Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365-386. 10. Zhang J, et al. (2007) Systematic analysis of genetic alterations in tumors using Cancer Genome WorkBench (CGWB). Genome Res 17:1111-1117. 11. Sherry ST, et al. (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308-311. 12. den Dunnen JT & Antonarakis SE (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15:7-12. 13. Beroukhim R, et al. (2007) Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proc Natl Acad Sci U S A 104:20007-20012.
Supplemental Information - 7 -
Table S1: AML cases studied by SNP array analysis
Case ID FAB Blast Gender Remission Cytogenetics % material AML-AE-#1 M2 89 Female PN-AE-#1 46,XX,t(1;21;8)(q42;q22;q22)[20] §,║ AML-AE-#2 M2 90 Male PN-AE-#2 45,X,-Y,ins(8;21)(q22;q22q22)[30] AML-AE-#3 M1 91 Female PN-AE-#3 46,XX,t(8;21)(q22;q22)[17]/ 46,XX[3] AML-AE-#4 M2 85 Male PN-AE-#4 46,XY,t(8;21)(q22;q22)[19]/ 46,XY[1] AML-AE-#5 M2 64 Female PN-AE-#5 46,XX,t(8;21)(q22;q22)[20] AML-AE-#6 M1 33 Male PN-AE-#6 46,XY,t(8;21)(q22;q22)[20] AML-AE-#7 M2 64 Male PN-AE-#7 46,X,-Y,t(8;21)(q22;q22),+8[20] AML-AE-#8 M2 90 Female PN-AE-#8 45,X,-X,t(8;21)(q22;q22)[20] AML-AE-#9 M2 76 Female 46,XX,t(8;21)(q22;q22)[20] AML-AE-#10 M2 74 Male 45,X,-Y,t(8;21)(q22;q22) AML-AE-#11 M2 91 Male 45,X,-Y,t(8;21)(q22;q22),del(10)(p14) AML-AE-#12 M2 72 Male 46,XY,t(8;21)(q22;q22)[32] AML-AE-#13 M2 88 Male 45,X,-Y,t(8;21)(q22;q22)[16]/ 46,XY[2] AML-AE-#14 M2 99 Male 46,XY,t(8;12;21)(q22;q13;q22)[14]/ 46,XY[5] AML-AE-#15 M2 76 Male 46,XY,t(8;21)(q22;q22)[20] AML-AE-#16 M2 83 Female 45,X,-Y, t(8;21)(q22;q22)[3]/ 45,idem,del(9)(q22q32)[11] ║ AML-AE-#17 M2 92 Male PN-AE-#17 45,X,-Y, t(8;21)(q22;q22)[20] AML-AE-#18 M2 84 Male PN-AE-#18 45,X,-Y, t(8;21)(q22;q22), i(12)(q10)[11]/46,XY[9] AML-AE-#19 M1 91 Female PN-AE-#19 46,XX,der(2)t(2;11)(p11.2;q21), t(8;21)(q22;q22), der(11)t(2;11)del(11)(p13), i(12)(q10)[20] ** AML-AE-#20 N/A 25 Female PN-AE-#20 45,X,-X,t(8;21)(q22;q22)[20]
AML-i16-#1 M1 88 Female PN-i16-#1 48,XX,+8,ins(16)(q22p13.1p13.3),+22 [20] AML-i16-#2 M4 59 Male PN-i16-#2 46,XY,inv(16)(p13.1q22)[20] AML-i16-#3 M4e 63 Female PN-i16-#3 46,XX,inv(16)(p13.1q22)[18]/ 46,XX[2] AML-i16-#4 M4e 65 Male PN-i16-#4 46,XY,der(16)ins(16)(q22p13.1p13.1)del(16)(q22)[2]/ 92,idemx2[8]/46,XY[10] ** AML-i16-#5 M4e 39 Male PN-i16-#5 48,XY,+8,inv(16)(p13.1q22),+22[12]/ 46,XY[8] AML-i16-#6 M4e 63 Male PN-i16-#6 46,XY,inv(16)(p13.1q22)[15]/ 46,XY[5] AML-i16-#7 M4e 65 Male PN-i16-#7 46,XY,inv(16)(p13.1q22)[11]/ 46,XY[9] AML-i16-#8 M4 64 Male PN-i16-#8 46,XY,t(16;16)(p13.1;q22)[19]/ 46,XY[1] AML-i16-#9 M4 94 Male 46,XY,inv(16)(p13.1q22) AML-i16-#10 M4 98 Female 46,XX,inv(16)(p13.1q22) AML-i16-#11 M2 89 Female PN-i16-#11 48,X,add(X)(q28),+9,inv(16)(p13.1q22), +22[27] AML-i16-#12 M4 89 Female 47,XX,inv(16)(p13.1q22),+22[8]/ 46,XX[4] AML-i16-#13 M4 85 Female 46,X,t(X;22)(p22;q11.2), inv(16)(p13.1q22)[23] AML-i16-#14 M4 77 Female PN-i16-#14 46,XX,inv(16)(p13.1q22)[10]/ 46,XX[11] AML-i16-#15 M4 88 Male 46,XY,inv(16)(p13.1q22)[7] ║ AML-i16-#16 M1 79 Male PN-i16-#16 46,XY,inv(16)(p13.1q22)[17]/ 47,idem,+22[1]/46,XY[2]
AML-PML-#1 M3 68 Male PN-PML-#1 46,XY,t(15;17)(q22;q12)[7]/ 46,XY[3] AML-PML-#2 M3 94 Female PN-PML-#2 46,XX,t(15;17)(q22;q12)[20] AML-PML-#3 M3 93 Male PN-PML-#3 46,XY,t(15;17)(q22;q12)[17]/ 46,XY[3] AML-PML-#4 M3 95 Female 46,XX,t(15;17)(q22;q12)[13]/ 46,XX[9] AML-PML-#5 M3 89 Male 46,XY,t(15;17)(q22;q12)[11]/ 46,XY[2] Supplemental Information - 8 -
Case ID FAB Blast Gender Remission Cytogenetics % material ║ AML-PML-#6 M3 95 Male PN-PML-#6 46,XY,t(15;17)(q22;q12)[20] AML-PML-#7 M3 83 Female PN-PML-#7 46,XX,t(15;17)(q22;q12)[16]/ 46,XX[4]
AML-MLL-#1 M1 94 Female PN-MLL-#1 47,XX,t(9;11)(p22;q23),+21[20] AML-MLL-#2 M1 76 Female 46,XX,t(11;19)(q23;p13.1)[20] AML-MLL-#3 M5 93 Female PN-MLL-#3 46,XX,t(9;11)(p22;q23)[17]/ 46,XX[3] AML-MLL-#4 M0 95 Female 46,XX,t(11;19)(q23;p13.1)[20] AML-MLL-#5 M5 50 Male PN-MLL-#5 46,XY,t(1;11)(q21;q23)[14]/ 46,XY[6] AML-MLL-#6 M5 97 Male PN-MLL- 47,XY,ins(10;11)(p12;q23q12), +mar[20] #17 AML-MLL-#7 M5 97 Female 46,XX,t(10;11)(p11.2;q23)[19]/ 46,XX[1] AML-MLL-#8 M5 73 Female PN-MLL-#8 46,XX,t(11;19)(q23;p13.3)[18]/ 46,XX[2] AML-MLL-#9 M4 91 Female 46,XX,t(11;19)(q23;p13.1)[24]/ 46,XX[6] AML-MLL-#10 M5 96 Female 46,XX,t(6;11)(q27;q23)[20] AML-MLL-#11 M5 96 Female PN-MLL- 47,XX,der(1)t(1;9)(q10;q10), der(9)t(1;9)t(9;11)(p22;p23), #11 der(11)t(9;11),+21[23] AML-MLL-#12 M5 88 Female 47,XX,+8,t(9;11)(p22;q23), inv(12)(p13q13)[20] AML-MLL-#13 M5 80 Female PN-MLL- 46,XX,t(9;11)(p22;q23)[20] ¶ #13 AML-MLL-#14 M5 90 Female 46,XX,t(11;19)(q23;p13.1)[14]/ 46,XX[2] AML-MLL-#15 M5 98 Male 47,XY,t(1;11)(q21;q23),+mar[10]/ 46,XY[2]
AML-M7-#1 M7 84 Female PN-M7-#1 47,XX,der(6)t(6;19)(p23;?),-10, der(19)t(10;19)(q11.2;p13.1), +add(19)(p or q), +mar[6]/ 46,idem, -mar[4]/46,XX[10] AML-M7-#2 M7 68 Female PN-M7-#2 46,XX,add(18)(p11.2)[13]/ 46,XX[7] ** AML-M7-#3 M7 42 Male PN-M7-#3 46,XY[24] AML-M7-#4 M7 78 Male PN-M7-#4 47,XY,add(5)(p15.3), i(7)(q10), add(11)(p11.2), +21 [20/100%] AML-M7-#5 M7 N/A Male PN-M7-#5 46,XY,-19,der(21)t(19;21)(21pter-->21q22.3::19q12q13.4::21q11.2-- ** >21q22.3::19q12-->19qter), +der(21)t(19;21)(21pter-- >21q22.3::21q11.2-->21q22.3::19q12-->19q13.4::19q12-->19qter)[18]/ 46,XY[2] AML-M7-#6 M7 82 Female 46,XX,der(16)t(1;16)(q21;p13.3)[16] AML-M7-#7 M7 78 Male PN-M7-#7 47,XY,t(3;22;5)(q21;q13;q31), +8, ins[der(22);11](q13;q13q23)[19] AML-M7-#8 M7 80 Female 49,XX,del(5)(p13p14), der(5)t(5;9)(q11.2;q13), r(7)(p22q11.2), del(9)(q34), +18, +19, +21[5] / 46,XX[15] AML-M7-#9 M7 68 Male 46,XY,dup(3)(q21q29)[24]
AML-misc-#1 M4 85 Female PN-misc-#1 47,XX,+8[15] /49,idem,+6,+19[4]/ 46,XX[1] [MLL] AML-misc-#2 M1 92 Female PN-misc-#2 47,XX,t(5;6)(q33;q21),der(7)t(7;13)(p22;q14), +20[18]/ 46,XX[2]
AML-misc-#3 M1 96 Male 46,XY,del(9)(q21q34)[11] AML-misc-#4 M2 92 Female PN-misc-#4 46,XX,?inv(5)(q33q35)[10]/ 46,XX[15] AML-misc-#5 M4 93 Male PN-misc-#5 46,XY,t(16;21)(p11.2;q22)[19]/ 46,XY[1] ** AML-misc-#6 M5 30 Female PN-misc-#6 47,XX,ins(12;7)(p13;q32q36), +19[11]/ 47,idem, der(7)t(6;7)(p11.2;p11.2)[9] ║ AML-misc-#7 M0 72 Male PN-misc-#7 48,XY, +8, +8[7]/ 46,XY[5] AML-misc-#8 M2 51 Male PN-misc-#8 45,X,-Y[4]/ 46,XY[10] AML-misc-#9 M1 80 Male PN-misc-#9 46,XY,inv(5)(q33q35)[13]/ 46,XY[10] AML-misc-#10 M5 92 Male 48,XY,+8,+13 AML-misc-#11 M5 93 Male 45,XY,-7[9]/ 46,XY[22] AML-misc-#12 M4 92 Male 47,XY,+8 AML-misc-#13 M2 94 Male PN-misc-#13 47,XY,+4[18]/ 46,XY[2] AML-misc-#14 M4 83 Female 46,XX,t(1;5)(p35;q35)[15] Supplemental Information - 9 -
Case ID FAB Blast Gender Remission Cytogenetics % material AML-misc-#15 M1 87 Female PN-misc-#15 46,XX,t(4;11)(q21;p15)[20] AML-misc-#16 M1 79 Female 47,XX,der(2)t(1;2)(q21;q35),?t(5;12)(p13;p13), del(8)(p21), +22[19]/ AML-misc-#17 M1 98 Female 46,XX,t(6;9)(p23;q34) [9] AML-misc-#18 M0 95 Male PN-misc-#18 45,XY,-16, der(21)t(16;21)(?p11.2;q22) [16]/ 46,XY [1]
AML-misc-#19 M4 87 Male PN-misc-#19 45,XY,-7[10]/ 46,XY[6] AML-misc-#20 M4 93 Male PN-misc-#20 47,XY,+8,del(9)(q13q22)[20] AML-misc-#21 M2 89 Female 46,XX,t(6;9)(p23;q34)[31] ** AML-misc-#22 M4 33 Female PN-misc-#22 46,XX,add(17)(p13)[8]/ 46,XX[12] ** AML-misc-#23 M6 26 Female PN-misc-#23 47,XX,+8[17]/46,XX[3]
AML-N-#1 M5 90 Female PN-N-#1 46,XX[30] AML-N-#2 M4 82 Female PN-N-#2 46,XX[35] AML-N-#3 M4 50 Male PN-N-#3 46,XY[31] AML-N-#4 M4 93 Male 46,XY[20] [misc] AML-N-#5 M1 95 Female PN-N-#5 46,XX[24] [MLL] AML-N-#6 M1 83 Female PN-N-#6 46,XX[24] AML-N-#7 M4 90 Female PN-N-#7 46,XX[25] AML-N-#8 M4 57 Male PN-N-#8 46,XY[25] [misc] AML-N-#9 M4 80 Male 46,XY AML-N-#10 M1 93 Male 46,XY [misc] AML-N-#11 M2 98 Female 46,XX AML-N-#12 M1 88 Male 46,XY AML-N-#13 M2 85 Male 47,XY,+4[2]/ 46,XY[18] AML-N-#14 M1 83 Male 46,XY[22] AML-N-#15 M1 93 Male 46,XY[12] AML-N-#16 M1 89 Female 46,XX[50] AML-N-#17 M2 81 Female 46,XX AML-N-#18 M1 87 Female 46,XX[20] AML-N-#19 M2 75 Female PN-N-#19 46,XX[30] ** AML-N-#20 M4 39 Male PN-N-#20 46,XY[35] ** AML-N-#21 M2 36 Female PN-N-#21 46,XX[20] ‡Subgroup in square brackets indicates cases for which subgroup was changed. §No Nsp chip available for this case. ¶No Hind and Xba available for this case. ║LOH was analyzed against the pool of reference samples. **Blast % is lower than 50% or not available (N/A).
Supplemental Information - 10 -
Supplementary Results:
Table S2a: Observed Copy Number Alterations for Each Case Copy number alterations (CNAs) are listed for each case with start and end positions indicated. regions of CN-LOH are described in Table S7 in this Supplemental Information. Mutations were heterozygous unless otherwise noted. CN, copy number; CN-LOH, copy-neutral loss of heterozygosity; homo, homozygous; GL, germ line; mt, mutant; wt, wild type.
Case ID Chromosomal Start* End* Size (kb) CN Sequence Mutation CN-LOH location Gene: type AML-AE-#1 8 q21.3 93,079,000 93,295,000 216.000 1.50 NRAS: G13D, G12 11pter-p12 AML-AE-#2 AML-AE-#3 6 q16.3--q27 102,736,905 170,823,609 68086.704 3.11 NRAS: Q61R 7 q31.1--q36.3 109,772,499 158,626,250 48853.751 0.98 8 q21.3;q22.1 93,145,729 93,540,016 394.287 1.08 11 q13.1 63,958,215 63,959,900 1.685 4.94 AML-AE-#4 AML-AE-#5 7 q36.1;q36.2 150,995,960 154,256,932 3260.972 1.15 NRAS: G12C 8 p23.2 2,291,741 2,293,385 1.644 0.75 KIT: N822K 8 q24.23 138,529,800 138,530,400 0.600 0.59 9 p24.3 1,027,639 1,028,010 0.371 6.68 AML-AE-#6 8 q21.3;q22.1 93,170,040 94,160,400 990.360 1.21 NRAS: G12D 9 q22.33 96,984,012 97,915,462 931.450 1.32 MLL-PTD 21 q22.11;q22.12 30,635,091 35,109,560 4474.469 1.17 AML-AE-#7 8 p23.3--q24.3 180,568 146,264,218 146083.650 2.98 15 q25.2;q25.3 82,904,075 83,526,153 622.078 3.00 AML-AE-#8 4 q28.2;q28.3 130,857,619 132,030,165 1172.546 0.90 8 q24.21 130,538,594 130,790,058 251.464 2.83 AML-AE-#9 AML-AE-#10 NRAS: Q61K AML-AE-#11 AML-AE-#12 8 q21.3;q22.1 93,170,040 94,425,195 1255.155 0.97 NRAS: G12D KIT: D816V AML-AE-#13 AML-AE-#14 16 p13.2 6,867,386 6,965,029 97.643 0.95 NRAS: Q61R AML-AE-#15 AML-AE-#16 3 q26.1;q26.2 168,602,647 169,576,357 973.710 1.14 17q11.2-qter AML-AE-#17 NRAS: G12D AML-AE-#18 12 p13.33--p12.1 61,880 22,380,523 22318.643 1.00 12 q13.12--q24.33 47,806,916 132,387,995 84581.079 3.00 AML-AE-#19 2 p12 81,037,265 81,193,080 155.815 0.67 CEBPA: S28RfsX130 11 p14.3--p11.12 23,153,846 50,128,453 26974.607 0.93 11 q14.1--q22.1 83,352,361 100,546,100 17193.739 0.92 12 p13.33--p11.1 36,594 34,651,598 34615.004 1.00 12 q11--q24.33 36,144,018 132,387,995 96243.977 3.00 AML-AE-#20 8 q24.21 130,646,400 130,725,004 78.604 3.16 NRAS: Q61K X p22.33--q28 34,000 154,480,000 154446.000 1.00
AML-i16-#1 5 q33.3 156,389,535 157,533,587 1144.052 0.96 NRAS: G12S 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.2 22 q11.1--q13.33 14,433,758 49,519,949 35086.191 3.21 AML-i16-#2 KRAS: G13N AML-i16-#3 AML-i16-#4 7 q36.1 148,777,415 150,128,821 1351.406 1.01 NRAS: Q61R 16 p13.11 15,725,642 16,260,667 535.025 1.02 16 p11.2 28,745,574 30,647,643 1902.069 1.06 16 q22.1 65,704,203 66,769,625 1065.422 1.00 AML-i16-#5 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.07 NRAS: G12C 16 p13.11 15,715,180 15,824,822 109.642 1.04 16 q22.1 65,704,203 65,822,130 117.927 0.90 22 q11.1--q13.33 14,433,758 49,519,949 35086.191 3.00 AML-i16-#6 5 p15.33--p13.3 81,949 30,644,260 30562.311 1.31 NRAS: Q61K 8q11.1-q11.21 11 q22.3--q25 104,757,300 134,449,982 29692.682 3.00 AML-i16-#7 16 p13.11 15,688,555 16,197,996 509.441 1.21 NRAS: Q61K Supplemental Information - 11 -
Case ID Chromosomal Start* End* Size (kb) CN Sequence Mutation CN-LOH location Gene: type AML-i16-#8 AML-i16-#9 3 p14.1 66,888,096 66,966,799 78.703 3.15 KIT: T417_L421delinsMRP AML-i16-#10 8 q24.13 124,722,755 124,991,917 269.162 3.27 16 p13.11 15,737,037 16,134,194 397.157 0.99 AML-i16-#11 9 p24.3--q34.3 30,910 138,363,203 138332.293 3.00 TP53: R156S 13 q14.3--q34 53,784,473 114,092,980 60308.507 3.00 16 q22.1 65,704,203 65,822,130 117.927 0.95 22 q11.1--q13.33 14,433,758 49,519,949 35086.191 3.00 X q26.1--q28 130,190,000 154,480,000 24290.000 1.00 AML-i16-#12 22 q11.1--q13.33 14,433,758 49,519,949 35086.191 3.00 AML-i16-#13 16 p13.11 15,738,236 16,127,627 389.391 0.98 NRAS: Q61H CDKN2A: R144C AML-i16-#14 16 p13.11 15,737,037 15,875,743 138.706 0.98 AML-i16-#15 NRAS: Q61K CEBPA: A29RfsX129 AML-i16-#16
AML-M7-#1 6 p25.3--p24.3 126,000 8,076,124 7950.124 1.00 10 p15.3;p15.2 101,955 3,235,325 3133.370 1.31 10 p14 10,443,000 10,819,693 376.693 3.00 10 q11.21--q21.3 43,366,000 70,125,600 26759.600 1.00 10 q22.1 70,815,492 72,144,000 1328.508 1.46 10 q22.1 72,147,192 73,557,621 1410.429 3.24 10 q22.1 73,570,580 73,911,125 340.545 7.49 10 q22.1 73,911,130 74,517,640 606.51 4.33 10 q22.1;q22.2 74,527,553 75,209,820 682.267 2.62 10 q22.2 75,247,849 76,533,362 1285.513 3.84 19 p13.2 8,240,000 11,295,510 3055.510 8.65 19 p13.2;p13.13 11,298,882 12,670,726 1371.844 6.79 19 p13.13 12,688,923 12,895,045 206.122 5.33 19 p13.13;p13.12 12,945,626 13,991,215 1045.589 3.81 19 p13.12 15,114,411 15,670,750 556.339 4.43 19 p13.12;p13.11 15,685,070 16,213,400 528.33 7.04 19 p13.11 16,232,818 19,227,643 2994.825 4.65 19 p13.11;p12 19,274,092 20,386,516 1112.424 4.03 AML-M7-#2 18 p11.32--p11.21 149,885 12,437,303 12287.418 1.32 21 q22.12--q22.3 35,085,383 46,924,580 11839.197 2.88 AML-M7-#3 AML-M7-#4 1 q31.3--q44 195,545,407 245,378,187 49832.780 3.13 GATA1: D65AfsX75 4 q34.3 182,171,180 182,348,000 176.820 0.93 homo 5 p15.33 81,949 1,322,000 1240.051 1.00 5 p15.33 1,353,000 1,497,000 144.000 0.00 5 p15.33 1,501,000 3,903,437 2402.437 1.00 5 p15.31 7,056,920 7,098,934 42.014 0.83 5 p15.31 7,269,226 7,689,756 420.530 0.95 5 q22.2;q22.3 111,875,200 113,856,600 1981.400 1.57 7 p22.3--p11.1 141,322 57,730,637 57589.315 0.96 7 q11.1--q36.3 61,076,556 158,626,250 97549.694 3.00 11 p14.1--p12 27,204,392 41,251,030 14046.638 1.00 21 p11.2--q22.3 9,887,804 46,924,580 37036.776 3.00 X p11.4 39,318,000 40,089,000 771.000 0.00 AML-M7-#5 19 p13.3 4,014,067 4,686,663 672.596 3.98 19 p13.3 4,706,178 5,139,880 433.702 2.84 19 p13.3--p13.12 6,750,677 14,562,093 7811.416 2.84 19 p12 21,470,638 22,799,870 1329.232 2.44 21 q22.13--q22.3 38,248,322 45,412,532 7164.210 3.00 AML-M7-#6 1 q24.3--q44 169,262,000 244,505,000 75243.000 3.00 13 q31.3 91,414,118 91,523,833 109.715 0.90 17 q24.3 66,236,699 66,441,030 204.331 3.48 20 p11.21--q11.21 25,614,807 31,177,153 5562.346 2.58 AML-M7-#7 8 p23.3--q24.3 180,568 146,264,218 146083.650 2.87 15 q21.1 42,910,906 43,055,474 144.568 3.85 AML-M7-#8 5 q14.3--q35.3 86,041,120 180,629,495 94588.375 1.51 Supplemental Information - 12 -
Case ID Chromosomal Start* End* Size (kb) CN Sequence Mutation CN-LOH location Gene: type 7 p22.3--p15.2 141,322 25,721,480 25580.158 1.50 7 p15.2--p14.1 25,724,452 39,435,510 13711.058 2.44 7 p14.1;p13 42,333,704 44,461,640 2127.936 2.50 7 p13;p12.3 45,403,085 49,370,399 3967.314 2.51 7 q22.1 97,961,455 99,366,917 1405.462 2.54 7 q22.1--q36.3 99,391,781 158,626,250 59234.469 1.51 9 p24.3--p13.2 30,910 37,153,530 37122.620 2.50 AML-M7-#9 3 q12.1--q26.1 99,591,060 166,265,867 66674.807 3.00
AML-MLL-#1 21 p11.2--q22.3 9,887,804 46,924,580 37036.776 3.00 NRAS: G12D AML-MLL-#2 AML-MLL-#3 AML-MLL-#4 10 q11.22 48,037,000 48,280,710 243.710 3.00 12 q21.1 71,540,830 71,650,830 110.000 0.79 AML-MLL-#5 AML-MLL-#6 3 q25.2--q29 153,644,700 199,322,068 45677.368 4.69 NRAS: Q61R
AML-MLL-#7 4 q13.1 63,414,725 63,960,004 545.279 3.66 11 q23.3 117,874,000 117,886,000 12.000 1.00 15 q25.3 85,120,222 85,214,000 93.778 3.00 AML-MLL-#8 10 q22.2 76,150,456 77,123,948 973.492 3.06 21pter--qter 19 p13.2 7,080,086 7,330,528 250.442 1.11 AML-MLL-#9 1 q31.1 185,716,000 185,754,256 38.256 0.69 PTPN11: A72V 18 p11.21 11,551,254 11,557,557 6.303 0.65 AML-MLL-#10 10 q22.3 79,930,594 80,046,429 115.835 0.78 KRAS: G13N AML-MLL-#11 21 p11.2--q22.3 9,887,804 46,924,580 37036.776 3.00 AML-MLL-#12 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.00 AML-MLL-#13 AML-MLL-#14 AML-MLL-#15 13 q21.33--q34 68,981,327 114,092,980 45111.653 4.62 NRAS: G12D
AML-PML-#1 AML-PML-#2 AML-PML-#3 7 q32.1--q36.3 127,360,351 158,605,053 31244.702 1.01 8 q22.3--q24.3 104,884,548 146,264,218 41379.670 3.17 AML-PML-#4 9 p21.3;p21.2 25,476,469 25,669,024 192.555 0.85 FLT3:R595-N609dup; 13 q32.1 94,738,000 94,816,547 78.547 4.59 (14aa, mut=wt) AML-PML-#5 17 p12 12,704,473 12,862,370 157.897 3.27 CDKN2A: A127S (p16 only) AML-PML-#6 1 q12;q21.1 141,484,300 143,571,366 2087.066 2.78 FLT3: Y591-L601dup 1 q21.1;q21.2 144,327,670 144,930,000 602.330 2.66 (10aa, mut AML-misc-#1 6 q27 168,081,288 168,174,708 93.420 3.11 NRAS: Q61K [MLL] 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.31 [MLL-MLLT4] 11 q23.3 117,874,000 117,886,000 12.000 1.00 AML-misc-#2 7 p22.3 141,322 294,586 153.264 1.05 CEBPA: G35RfsX123 13 q21.1--q34 57,344,180 114,092,980 56748.800 3.00 (GL), S191-H192dup (GL) 20 p13--q13.33 17,408 62,376,960 62359.552 2.68 AML-misc-#3 2 p22.3 35,711,937 35,719,183 7.246 0.61 FLT3: D601_L602insMP; 13pter-qter 3 q13.11 106,783,800 106,856,500 72.700 0.73 S585-D601dup 9 q13--q22.2 67,730,415 116,000,881 48270.466 1.00 (15aa, mut>wt) 11 p13 31,731,972 32,752,668 1020.696 0.91 AML-misc-#4 NPM: INScctg Supplemental Information - 13 - Case ID Chromosomal Start* End* Size (kb) CN Sequence Mutation CN-LOH location Gene: type FLT3: K603N;K603_W604insN; D586-K603dup (17aa, mut=wt) MLL-PTD AML-misc-#5 8 q24.21 130,610,600 130,801,854 191.254 2.93 AML-misc-#6 6 p25.3--p12.1 99,536 57,458,180 57358.644 2.77 [HLXB9-ETV6] 7 p22.3--p11.2 141,322 55,108,211 54966.889 1.32 19 p13.3--q13.43 212,033 63,785,051 63573.018 3.00 AML-misc-#7 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.00 AML-misc-#8 CEBPA: A111GfsX247 11pter-p11.2 (GL) K304del (not in GL) AML-misc-#9 5 q35.1;q35.2;q3 169,538,580 177,831,914 8293.334 1.30 CEBPA: P23QfsX80 (GL), 5.3 M310TfsX38 (not in GL) AML-misc-#10 11 q24.2;q24.3 127,066,538 128,598,668 1532.130 3.09 FLT: D586-G669dup 13pter-qter (83aa, mut>wt) RUNX1: S212RfsX17 MLL-PTD AML-misc-#11 7 p22.3--q36.3 141,322 158,626,250 158484.928 1.24 RUNX1: D96GfsX11 PTPN11: A72R AML-misc-#12 7 p12.3;p12.2 49,473,728 50,228,460 754.732 3.31 FLT: R595_E596insG; 8 p23.3--q24.3 180,568 146,264,218 146083.650 3.00 R595-L602dup; KW603- 604DG (8aa, mut AML-N-#1 10 q25.1 109,064,716 109,347,816 283.100 1.12 21 p11.2;p11.1 9,887,804 10,105,718 217.914 0.96 AML-N-#2 RUNX1: D184RfsX2 MLL-PTD AML-N-#3 NPM1: INScatg KIT: D816Y AML-N-#4 5 q35.2;q35.3 176,421,862 177,874,223 1452.361 3.07 FLT3: Y597-E604dup [Misc] 7 q21.11 85,643,878 85,867,223 223.345 3.56 (7aa, mut=wt) [NUP98-NSD1] 11 p15.5;p15.4 2,589,728 3,705,407 1115.679 0.96 Supplemental Information - 14 - Case ID Chromosomal Start* End* Size (kb) CN Sequence Mutation CN-LOH location Gene: type 17 q24.2;q24.3 63,153,353 64,993,580 1840.227 3.16 AML-N-#5 6 q27 168,088,794 168,174,708 85.914 3.87 CEBPA: M15IfsX138 (not [MLL] 11 q23.3 117,856,311 118,081,700 225.389 0.94 in GL) [MLL-MLLT4] AML-N-#6 CEBPA: H24AfsX103, 15q15.1-qter S28N , R306_N307insL (all not in GL) NRAS: G12D AML-N-#7 NRAS: G12V NPM1: INStctg AML-N-#8 11 p15.4 3,722,768 3,957,904 235.136 3.63 [Misc] [NUP98-NSD1] AML-N-#9 4 q28.1 127,757,370 128,189,184 431.814 0.85 NRAS: G13D 9 p24.2;p24.1 3,765,583 5,066,691 1301.108 0.95 9 p21.3 20,935,207 22,880,200 1944.993 1.00 9 q33.1;q33.2 118,357,800 120,246,473 1888.673 0.93 11 p14.1 27,216,990 29,986,520 2769.530 0.88 16 q22.1 65,497,239 66,413,740 916.501 0.97 20 q11.22 32,325,220 32,978,126 652.906 0.93 AML-N-#10 CEBPA: V314STOP [Misc] FLT3: V592-R607dup [NUP98-NSD1] (15aa, mut *hg17 (Build 35) Supplemental Information - 15 - Table S2b: Mean Number of CNA per Case Changes resulting from immunoglobulin or T cell antigen gene rearrangements were excluded. p value was computed using Kruskal- Wallis rank sum test (Monte Carlo approximation with 10,000 permutations). Subtype N Gains Losses Total (range) (range) (range) t(8;21) 20 0.75 (0-5) 0.95 (0-4) 1.70 (0-8) inv(16) 16 0.69 (0-3) 0.88 (0-4) 1.56 (0-7) t(15;17) 7 1.14 (0-3) 2.00 (0-12) 3.14 (0-15) MLL 15 0.93 (0-3) 0.40 (0-2) 1.33 (0-4) FAB-M7 9 7.00 (0-41) 2.33 (0-12) 9.33 (0-45) Misc. 23 0.78 (0-2) 0.96 (0-4) 1.74 (0-5) Normal 21 0.81 (0-4) 1.05 (0-7) 1.86 (0-7) All cases 111 1.32 1.06 2.38 (0-41) (0-12) (0-45) p 0.013 0.86 0.24 Supplemental Information - 16 - Table S3: Genomic Identification of Significant Targets in Cancer (GISTIC) Genomic Identification of significant targets in cancer (GISTIC) (13) was performed as described in Experimental Procedures in this Supplemental Information. GISTIC identified 11 deletion peaks and 5 amplification peaks with highest significance scores as listed in this table. Wider peaks are included in Table S4 in this Supplemental Information Appendix. The regions were ranked by their size and the number of genes contained: broad lesions, focal lesions on broad and focal lesions. Cancer Gene Census genes are listed in red (www.sanger.ac.uk/genetics/CGP/Census), translocation associated genes in blue. Max Freq %, Maximum frequency in %; CGC, Cancer Gene Census; Amp, amplification; Del, deletion. Type of Region Peak Center in Peak type Start End Max # Gene CGC genes Cytoband Freq genes symbols if % <6 Broad: 1 region Amp Peak 5 22q12.3 Peak Limits 1 49,554,710 4.5 410 focal on broad: Amp Peak 1 8q24.21 Peak Limits 130,642,820 130,733,018 13.5 0 [CCDC26] 4 regions Wide Peak Limits 130,597,260 130,773,734 0 [CCDC26] Chrom. 8 Contributing Broad 1 146,264,217 event Deletion Peak 7p21.3 Peak Limits 8,669,362 12,192,099 5.4 4 NDUFA4, 2 PHF14, THSD7A, TMEM106 B Wide Peak Limits 1 25,724,451 120 CARD, PMS2, ETV1 7p Contributing Broad 1 62,745,428 event Deletion Peak 7q36.1 Peak Limits 148,767,769 150,139,978 5.4 22 3 Wide Peak Limits 127,342,952 158,628,139 220 SMO, CREB3L2, KIAA1549, BRAF 7q Contributing Broad 109,754,676 158,076,752 event Deletion Peak 9q22.33 Peak Limits 96,978,350 97,921,521 2.7 13 XPA 7 Wide Peak Limits 38,449,764 103,934,147 186 SYK, OMD, FANCC, XPA, NR4A3 9q Contributing Broad 38,449,764 103,934,147 event Focal >25 Amp Peak 3 19p13.2 Peak Limits 7,906,002 11,298,881 2.7 88 genes: Wide Peak Limits 6,642,632 14,603,919 215 LYL1 3 regions Amp Peak 4 21q22.2 Peak Limits 38,247,119 45,415,210 4.5 81 ERG, TMPRSS2 Wide Peak Limits 35,069,945 46,944,323 114 RUNX1, ERG, TMPRRS2 Deletion Peak 12p13.31 Peak Limits 7,665,296 10,013,470 2.7 31 10 Wide Peak Limits 1 22,640,669 227 CCND2, ZNF384, ETV6 Focal 6-25 Deletion Peak 9p21.3 Peak Limits 21,225,443 22,324,144 3.6 12 CDKN2A genes 5 Wide Peak Limits 20,996,931 22,324,144 21 CDKN2A 3 regions Deletion Peak 11p14.1 Peak Limits 27,208,555 29,999,147 2.7 7 8 Wide Peak Limits 27,197,654 41,299,760 51 WT1, LMO2 Deletion Peak 16q22.1 Peak Limits 65,678,217 65,886,827 4.5 17 CBFB 12 Wide Peak Limits 65,678,217 65,886,827 17 CBFB -translocation- Focal 2-5 genes Deletion Peak 5p15.33 Peak Limits 1,322,366 1,500,840 1.8 3 TERT, 3 regions 1 CLPTM1L, SLC6A3 Wide Peak Limits 1 30,665,557 64 Deletion Peak 11q23.3 Peak Limits 117,856,312 117,930,628 2.7 4 MLL, MLL 9 Wide Peak Limits 117,856,312 117,930,628 4 HBP21, -translocation- TMEM25, C11orf60 Deletion Peak 16p13.11 Peak Limits 15,597,221 15,983,108 5.4 5 KIAA0430, MYH11 11 Wide Peak Limits 15,597,221 15,983,108 5 NDE1, -translocation- MYH11, C16orf63, Supplemental Information - 17 - Type of Region Peak Center in Peak type Start End Max # Gene CGC genes Cytoband Freq genes symbols if % <6 ABCC1 Focal - Single Amp Peak 2 13q32.1 Peak Limits 94,732,707 94,838,029 3.6 1 ABCC4 Genes: Wide Peak Limits 68,980,224 114,142,980 110 ERCC5 3 regions Deletion Peak 8q21.3 Peak Limits 93,145,730 93,551,555 2.7 1 RUNX1T1 4 Wide Peak Limits 93,145,730 94,160,749 1 -translocation- Deletion Peak 9p21.2 Peak Limits 25,474,432 25,679,953 1.8 1 TUSC1 6 Wide Peak Limits 22,041,671 25,745,951 3 DMRTA1, ELAVL2, TUSC1 Focal no gene: Deletion Peak 18p11.21 Peak Limits 11,543,981 11,592,052 1.8 0 1 region 13 Wide Peak Limits 1 12,506,081 51 Supplemental Information - 18 - Table S4: Minimally Altered Recurrent Regions (MAR) not identified by GISTIC, rank ordered by frequency Recurring regions not identified within distinct peaks in GISTIC are listed in these tables. They were identified by determination of overlap between two or more lesions with the overlap of lesions being <20Mb (to exclude regions defined by whole chromosome or chromosomal arm changes). The number of affected cases was assessed for focal lesions defining the locus (<20Mb) and broad lesions including the locus (>20Mb). Segments 1-8 are within significant wider peak limits or broader events identified by GISTIC. "wide" represents the wide peak limits which allow more robust identification of target genes. "broad" marks regions which were identified as broad contributing events. Segments 9-24 were not within significant regions identified by GISTIC. Within each group of segments (1- 8 and 9-24), regions were rank ordered by frequency of total lesions at locus. Lesions adjacent to translocation breakpoints are marked by "translocation" in the "comments" column. For lesions with 10 or less genes gene symbols were listed, for others only the number of genes and genes from the Cancer Gene census (in red) (www.sanger.ac.uk/genetics/CGP/Census). In MAR defined by complex lesions and exceeding beyond peak limits, green color marks the Peak limits and genes within and black color represent the wide peak limits MAR, minimally altered region; APeak: Amplification Peak; DPeak: Deletion Peak. Supplemental Information - 19 - Segment Cytoband Gene symbols (Number; CGC genes) † Loss/ Focal lesions Broader #total Comments GISTIC‡ (Mb) Gain defining the lesions lesions at locus including the locus locus 1 8q24.13 C8ORFK36, ANXA13, FAM91A1, FERIL6 (4; none) Gain 1 11 12 APeak1broad (124) 2 7q36.1-q36.2 PRKAG2, GALNTL5, GALNT11, MLL3, XRCC2, Loss 1 5 6 DPeak3wide (150-154) ACTR3B, FLJ42291, DPP6, PAXIP1 (9; none) 3 7p22.3 FAM20C (1; none) Loss 1 5 6 DPeak2wide (0) 4 21q22.12- (114; RUNX1, ERG, TMPRSS2) Gain 4 4 APeak4wide q22.3 (35-46) 5 11p13 ELP4, PAX6, RCN1, WT1, WIT1, PCID1, CCDC73 (7; Loss 1 2 3 DPeak8wide (31-32) WT1) 6 12p13.2 (32; ETV6) Loss 1 2 3 DPeak9wide (10-12) 7 11p14.1-p12 (51; WT1, LMO2) Loss 2 2 DPeak8wide (27-41) 8 *19p13.13- (450; 4-7Mb: SH3GL1, MLLT1; 7-11Mb: 88 genes, none; Gain 1-2 2 2-3 Complex APeak3 (7-11) p12 11-22Mb: LYL1, BRD4, TMP4, JAK3, ELL) amplifications of APeak3wide (6-14) (4-22) AML-M7-#1 and #5 Segment Cytoband Gene symbols (Number; CGC genes) † Loss/ Focal lesions Broader #total Comments GISTIC‡ (Mb) Gain defining the lesions lesions at locus including the locus locus 9 6q27 MLLT4 (1; MLLT4) Gain 2 1 3 translocation no (168) 10 9p24.3 (0; none) Gain 1 2 3 no (1) 11 Xp11.4 BCOR (1; none) Loss 1 2 3 no (39-40) 12 1q23.3-q24.3 (55; PBX1) Gain 1 1 2 Overlap of two broad no (160-168) 13 2q33.3 (12; none) Loss 1 1 2 Overlap of focal with no (206-207) broad 14 3q25.2-26.1 (41; GMPS; MLF1) Gain 2 2 Overlap of two broad no (153-166) 15 4q13.1 (0; none) Gain 1 1 2 no (63) 16 4q27 PRDM5 (1; none) Gain 1 1 2 no (121-122) 17 *5q22.2— APC, SRP19, REEP5, DCP2, MCC, TSSK1B, YTHDC2, Loss 1 1 2 Defined by complex no q22.3 KCNN2 (8; APC) deletion of AML- (111-113) M7-#4 18 5q33.3 (13; ITK) Loss 1 1 2 no (156-157) 19 5q35.1-q35.3 (78; RANBP17, TLX3, NPM1, NSD1) Loss 1 1 2 Translocation no Supplemental Information - 20 - Segment Cytoband Gene symbols (Number; CGC genes) † Loss/ Focal lesions Broader #total Comments GISTIC‡ (Mb) Gain defining the lesions lesions at locus including the locus locus (169-177) 20 7q21.11 (0; none) Gain 1 1 2 no (85) 21 7q22.1 (32; none) Gain 1 1 2 no (97-99) 22 10q22.2 MYST4, DUPD1, DUSP13 (3; MYST4) Gain 2 2 Overlap of 8 and 9 no (76) gene amplification 23 11q24.2- ETS1, FLI1, KCNJ1, KCNJ5, C11orf45, P53AIP1, RICS (7; Gain 1 1 2 no q24.3 FLI1) (127-128) 24 20p11.21- (36; none) Gain 1 1 2 no q11.21 (25-31) *Segments defined by complex lesions were combined. Supplemental Information - 21 - Table S5a-g: Copy Number Alterations Tabulated by Size These Tables list copy number alterations sorted by size and number of genes. In total we found 26 whole chromosome losses or gains, 34 subchromosomal regions >20Mb, 27 regions <20Mb and >25 genes, 49 regions with 6-25 genes, 20 with 2-5 genes, 23 single gene loci and 24 loci without annotated RefSeq gene. Results from validation are listed (including used BAC clones). Genes in red are listed in the cancer gene census (http://www.sanger.ac.uk/genetics/CGP/Census/). CN, copy number. Chrom, chromosome; CGC, cancer gene census. Table S5a: Whole chromosome losses and gains (n=26) Case ID Chrom CN AML-misc-#13 4 3.29 AML-misc-#11 7 1.24 AML-misc-#19 7 1.44 AML-i16-#1 8 3.20 AML-misc-#1 8 3.31 AML-AE-#7 8 2.98 AML-misc-#12 8 3.00 AML-MLL-#12 8 3.00 AML-M7-#7 8 2.87 AML-misc-#20 8 3.00 AML-misc-#7 8 3.70 AML-i16-#5 8 3.07 AML-misc-#23 8 3.00 AML-i16-#11 9 3.00 AML-misc-#6 19 3.00 AML-misc-#2 20 2.68 AML-MLL-#1 21 3.00 AML-M7-#4 21 3.00 AML-MLL-#11 21 3.00 AML-i16-#1 22 3.21 AML-i16-#11 22 3.00 AML-i16-#12 22 3.00 AML-misc-#16 22 3.05 AML-i16-#5 22 3.00 AML-AE-#16 23 1.00 AML-AE-#20 23 1.00 Table S5b: Sub-chromosomal losses and gains > 20Mb (n=34) Case ID Chrom Cyto- Start* Size (kb) CN Validation/Comments # genes band >20Mb AML-M7-#4 1 q31.3-- 195,545,407 49832.780 3.13 358 q44 AML-M7-#6 1 q24.3-- 169,262,000 75243.000 3.00 447 q44 AML-misc-#16 1 q23.3-- 160,880,500 84497.687 3.21 532 q44 AML-N-#18 2 q33.3-- 206,907,500 24141.98 1.20 132 q37.1 AML-M7-#9 3 q12.1-- 99,591,060 66674.807 3.00 336 q26.1 AML-MLL-#17 3 q25.2-- 153,644,700 45677.368 4.69 204 q29 AML-i16-#6 5 p15.33-- 81,949 30562.311 1.31 62 p13.3 AML-M7-#8 5 q14.3-- 86,041,120 94588.375 1.51 514 q35.3 AML-AE-#3 6 q16.3-- 102,736,905 68086.704 3.11 298 q27 AML-misc-#6 6 p25.3-- 99,536 57358.644 2.77 583 p12.1 AML-PML-#3 7 q32.1-- 127,360,351 31244.702 1.01 210 q36.3 AML-AE-#3 7 q31.1-- 109,772,499 48853.751 0.98 268 q36.3 AML-misc-#6 7 p22.3-- 141,322 54966.889 1.32 247 p11.2 AML-M7-#4 7 p22.3-- 141,322 57589.315 0.96 261 p11.1 AML-M7-#4 7 q11.1-- 61,076,556 97549.694 3.00 565 q36.3 Supplemental Information - 22 - Case ID Chrom Cyto- Start* Size (kb) CN Validation/Comments # genes band >20Mb AML-M7-#8 7 p22.3-- 141,322 25580.158 1.50 112 p15.2 AML-M7-#8 7 q22.1-- 99,391,781 59234.469 1.51 365 q36.3 AML-PML-#3 8 q22.3-- 104,884,548 41379.670 3.17 FISH (CCDC26; RP11- 181 q24.3 259L23) shows gain+1 AML-misc-#16 8 p22-- 15,917,540 25963.424 0.95 151 p11.21 AML-misc-#3 9 q13-- 67,730,415 48270.466 1.00 FISH (FOXE1/9q22.33,) 245 q22.2 confirms deletion AML-misc-#20 9 q12-- 67,354,917 36579.183 0.86 FISH (FOXE1/9q22.3, 166 q31.1 RP11-23B15) confirms deletion AML-M7-#8 9 p24.3-- 30,910 37122.620 2.50 FISH 174 p13.2 (CDKN2A+B/9p21.3) (RP11-149I2) confirms gain+1 in 62% of cells AML-M7-#1 10 q11.21-- 43,366,000 26759.600 1.00 82 q21.3 AML-i16-#6 11 q22.3-- 104,757,300 29692.682 3.00 FISH (ETS1/11q24.3, 236 q25 RP11-272C3) confirms gain AML-AE-#19 11 p14.3-- 23,153,846 26974.607 0.93 Bi-allelic loss of WT1 121 p11.12 (RP11-74J1) + monosomy 11 confirmed by FISH AML-AE-#18 12 p13.33-- 61,880 22318.643 1.00 226 p12.1 AML-AE-#18 12 q13.12-- 47,806,916 84581.079 3.00 633 q24.33 AML-AE-#19 12 p13.33-- 36,594 34615.004 1.00 271 p11.1 AML-AE-#19 12 q11-- 36,144,018 96243.977 3.00 697 q24.33 AML-misc-#2 13 q21.1-- 57,344,180 56748.800 3.00 115 q34 AML-i16-#11 13 q14.3-- 53,784,473 60308.507 3.00 117 q34 AML-MLL-#15 13 q21.33-- 68,981,327 45111.653 4.62 111 q34 AML-misc-#18 16 p11.2-- 31,111,030 57579.746 1.00 368 q24.3 AML-i16-#11 X q26.1-- 130,190,000 24290.000 1.00 177 q28 Table S5c: Focal losses and gains <20Mb and >25genes (n=27) Case ID Chrom Cyto- Start* Size (kb) CN Validation # First 20 genes (CGC genes) band <20Mb, genes >25 genes AML-N-#12 1 p36.31;p 5,990,434 5895.566 1.16 71 KCNAB2, CHD5, RPL22, 36.23;p3 RNF207, C1orf188, ICMT, 6.22 C1orf211, HES3, GPR153,ACOT7,HES2,ESPN, TNFRSF25, PLEKHG5, NOL9, TAS1R1, HKR3, KLHL21, PHF13, THAP3 AML-PML-#6 1 q23.1-- 154,102,792 14586.684 2.91 170 FCRL5, FCRL4, FCRL3, FCRL2, q24.3 FCRL1, CD5L, KIRREL, CD1D, CD1A, CD1C, CD1B, CD1E, OR10T2, OR10K2, OR10K1, OR10R2, OR6Y1, OR10X1, OR10Z1, SPTA1 AML-misc- 2 q37.1;q3 233,760,000 8970.382 0.91 77 INPP5D, ATG16L1, SAG, DGKD, #16 7.2;q37. USP40, UGT1A8, UGT1A10, 3 UGT1A9, UGT1A7, UGT1A6, UGT1A5, UGT1A4, UGT1A3, HCG3, UGT1A1, DKFZp762E1312, TRPM8, SPP2, ARL4C, SH3BP4 AML-N-#4 5 q35.2;q3 176,421,862 1452.361 3.07 t(5;11)[NUP98-NSD1], 30 ZNF346, FGFR4, NSD1, RAB24, 5.3 confirmed by FISH (RP11- PX19, MXD3, LMAN2, RGS14, 348A20, RP11-99N22) SLC34A1, F12, GRK6, PRR7, DBN1, PDLIM7, DOK3, DDX41, FLJ10404, TMED9, B4GALT7, NY-REN-7 Supplemental Information - 23 - Case ID Chrom Cyto- Start* Size (kb) CN Validation # First 20 genes (CGC genes) band <20Mb, genes >25 genes AML-misc-#9 5 q35.1;q3 169,538,580 8293.334 1.30 This case has WT NPM1. 78 LCP2, LOC257358, KCNMB1, 5.2;q35. KCNIP1, GABRP, RANBP17, 3 TLX3, NPM1, FGF18, FBXW11, STK10, DC-UbP, FLJ40453, SH3PXD2B, DUSP1, ERGIC1, RPL26L1, ATP6V0E, LOC153222, BNIP1 AML-M7-#1 6 p25.3-- 126,000 7950.124 1.00 44 DUSP22, IRF4, EXOC2, HUS1B, p24.3 FOXQ1, FOXF2, FOXC1, GMDS, C6orf195, RP11-145H9.1, WRNIP1, SERPINB1, SERPINB9, SERPINB6, DKFZp686I15217, NQO2, LOC401233, RIPK1, BPHL, TUBB2A AML-misc- 6 p24.2-- 11,038,360 7294.792 0.87 t(6;9)[DEK-NUP214], 32 LOC221711, ELOVL2, HERV- #21 p22.3 confirmed by FISH (CTD- FRD, NEDD9, C6orf105, HIVEP1, 3027D6, RP11-235F20) EDN1, PHACTR1, TBC1D7, GFOD1, C6orf114, SIRT5, NOL7, RANBP9, C6orf79, RNF182, CD83, JARID2, DTNBP1, MYLIP AML-M7-#8 7 p15.2-- 25,724,452 13711.058 2.44 73 NFE2L3, HNRPA2B1, CBX3, p14.1 SNX10, SCAP2, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, EVX1, HIBADH, TAX1BP1, tcag7.981 AML-M7-#8 7 q22.1 97,961,455 1405.462 2.54 32 TMEM130, TRRAP, SMURF1, ARPC1A, ARPC1B, PDAP1, BUD31, PTCD1, CPSF4, ATP5J2, ZNF789, ZNF394, ZFP95, C7orf38, ZNF655, ZNF498, CYP3A5, CYP3A7, CYP3A4, CYP3A43 AML-PML-#6 9 p24.1-- 6,212,553 13854.990 1.42 31 C9orf26, TPD52L3, UHRF2, p21.3 GLDC, JMJD2C, C9orf123, PTPRD, TYRP1, C9orf150, MPDZ, NFIB, ZDHHC21, CER1, FREM1, C9orf52, SNAPC3, PSIP1, C9orf93, BNC2, C9orf39 AML-misc- 9 q34.11;q 128,874,915 2377.830 0.89 t(6;9)[DEK-NUP214], 30 FAM73B, DOLPP1, CRAT, #21 34.12;q3 confirmed by FISH (CTD- PPP2R4, IER5L, C9orf106, 4.13 3027D6, RP11-235F20) C9orf50, C9orf32, ASB6, PRRX2, PTGES, TOR1B, TOR1A, C9orf78, USP20, FNBP1, GPR107, FLJ46836, FREQ, ASS1 AML-M7-#4 11 p14.1-- 27,204,392 14046.638 1.00 mono-allelic loss of WT1 53 CCDC34, LGR4, LIN7C, BDNF, p12 (RP11-74J1) confirmed by KIF18A, METT5D1, KCNA4, FISH FSHB, C11orf46, MPPED2, FLJ46154, DCDC1, ZCSL3, IMMP1L, ELP4, PAX6, RCN1, WT1, WIT1, PCID1 AML-AE-#19 11 q14.1-- 83,352,361 17193.739 0.92 52 DLG2, TMEM126B, TMEM126A, q22.1 ZF, CCDC89, SYTL2, CCDC83, PICALM, EED, C11orf73, CCDC81, ME3, PRSS23, FZD4, TMEM135, RAB38, CTSC, GRM5, TYR, NOX4 AML-N-#12 12 p13.2 10,665,965 2113.740 0.98 This lesion contains ETV6. 32 STYK1, CSDA, TAS2R7, TAS2R8, This case has a TAS2R9, TAS2R10, PRR4, homozyogous ETV6 LOC440082, PRH1, TAS2R13, PRH2, TAS2R14, TAS2R50, mutation. TAS2R49, TAS2R48, TAS2R44, TAS2R46, TAS2R43, TAS2R42, PRB3 AML-i16-#4 16 p11.2 28,745,574 1902.069 1.06 58 LOC440348, ATXN2L, TUFM, SH2B1, ATP2A1, RABEP2, CD19, NFATC2IP, SPIN1, LAT, LAT1- 3TM, GIYD1, GIYD2, SULT1A4, SULT1A3, SPN, QPRT, C16orf54, KIF22, MAZ AML-i16-#4 16 q22.1 65,704,203 1065.422 1.00 45 C16orf70, MGC4655, TRADD, FBXL8, HSF4, NOL3, EXOC3L, E2F4, ELMO3, LRRC29, HSPC171, FHOD1, SLC9A5, PLEKHG4, LRRC36, CGI-38, ZDHHC1, HSD11B2, ATP6V0D1, AGRP AML-N-#9 16 q22.1 65,497,239 916.501 0.97 37 CDH16, RRAD, FAM96B, CES2, FLJ21736, FLJ37464, CBFB, C16orf70, MGC4655, TRADD, FBXL8, HSF4, NOL3, EXOC3L, E2F4, ELMO3, LRRC29, HSPC171, FHOD1, SLC9A5 Supplemental Information - 24 - Case ID Chrom Cyto- Start* Size (kb) CN Validation # First 20 genes (CGC genes) band <20Mb, genes >25 genes AML-M7-#2 18 p11.32-- 149,885 12287.418 1.32 49 USP14, THOC1, COLEC12, p11.21 CETN1, CLUL1, C18orf56, TYMS, ENOSF1, YES1, ADCYAP1, C18orf2, METTL4, KNTC2, EMILIN2, LPIN2, MYOM1, MRCL3, MRLC2, TGIF, DLGAP1 AML-M7-#1 19 p13.2 8,240,000 3055.510 8.65 76 CD320, NDUFA7, RPS28, ANKRD47, ANGPTL4, RAB11B, MARCH2, HNRPM, PRAM1, ZNF414, MYO1F, ADAMTS10, MGC33407, OR2Z1, ZNF558, MBD3L1, ZNF560, OR1M1, OR7G2, OR7G1 AML-M7-#1 19 p13.2;p1 11,298,882 1371.844 6.79 41 RAB3D, UNQ501, LPPR2, 3.13 C19orf39, EPOR, MGC20983, PRKCSH, ELAVL3, ZNF653, ECSIT, CNN1, ELOF1, ACP5, ZNF627, LOC401898, ZNF441, ZNF491, ZNF440, ZNF439, ZNF69 AML-M7-#1 19 p13.11 16,232,818 2994.825 4.65 83 KLF2, EPS15L1, CALR3, C19orf44, CHERP, SLC35E1, CRSP7, C19orf42, TMEM38A, LOC284434, SIN3B, F2RL3, CPAMD8, NY-SAR-48, MYO9B, MDS032, OCEL1, NR2F6, USHBP1, HSPC142 AML-M7-#5 19 p13.3-- 6,750,677 7811.416 2.84 VAV1 amplification 196 VAV1, EMR1, FLJ25758, p13.12 confirmed by FISH (28% of MBD3L2, ZNF557, INSR, cells have multiple copies) ARHGEF18, PEX11G, C19orf45, ZNF358, MCOLN1, PNPLA6, (RP11-815E8) XAB2, PCP2, STXBP2, RETN, MCEMP1, FCER2, CLEC4G, CD209 AML-M7-#6 20 p11.21-- 25,614,807 5562.346 2.58 40 ZNF337, C20orf91, FLJ45832, q11.21 MGC72104, DEFB118, DEFB119, DEFB121, DEFB123, REM1, HM13, ID1, COX4I2, BCL2L1, TPX2, MYLK2, FKHL18, DUSP15, TTLL9, PDRG1, XKR7 AML-M7-#2 21 q22.12-- 35,085,383 11839.197 2.88 FISH (ERG/21q22.3: 132 RUNX1, SETD4, CBR1, CBR3, q22.3 tttccc/ttcc [90/10%]) DOPEY2, MORC3, CHAF1B, (RP11-95I21) CLDN14, SIM2, HLCS, DSCR6, PIGP, TTC3, DSCR9, DSCR3, DYRK1A, KCNJ6, DSCR4, DSCR8, DSCR10 AML-AE-#6 21 q22.11;q 30,635,091 4474.469 1.17 61 KRTAP23-1, KRTAP13-2, 22.12 KRTAP13-1, KRTAP13-3, KRTAP13-4, KRTAP15-1, KRTAP19-1, KRTAP19-2, KRTAP19-3, KRTAP19-4, KRTAP19-5, KRTAP19-6, KRTAP19-7, KRTAP6-3, KRTAP6-2, KRTAP22-1, KRTAP6-1, KRTAP20-1, KRTAP20-2, KRTAP21-2 AML-M7-#5 21 q22.13-- 38,248,322 7164.210 3.00 FISH (ERG/21q22.3) 96 DSCR4, DSCR8, DSCR10, q22.3 shows tttttccc in 18% of KCNJ15, ERG, C21orf24, ETS2, cells (RP11-95I21) FLJ45139, DSCR2, BRWD1, HMGN1, WRB, C21orf13, SH3BGR, C21orf88, B3GALT5, PCP4, DSCAM, BACE2, PLAC4 AML-misc- 21 q22.2;q2 38,690,144 8234.436 0.95 FISH (ERG/21q22.3) 111 ERG, C21orf24, ETS2, FLJ45139, #18 2.3 confirms deletion (RP11- DSCR2, BRWD1, HMGN1, WRB, 95I21) C21orf13, SH3BGR, C21orf88, B3GALT5, PCP4, DSCAM, BACE2, PLAC4, FAM3B, MX2, MX1, TMPRSS2 Supplemental Information - 25 - Table S5d: Losses and gains of loci with 6-25 genes (n=49) Case ID Chrom Cyto- Start* Size (kb) CN Validation # first 20 genes (CGC genes) band genes (6-25) AML-PML-#6 1 q12;q21.1 141,484,300 2087.066 2.78 19 PPIAL4, PDE4DIP, SEC22B, NOTCH2NL, HFE2, TXNIP, POLR3GL, LIX1L, RBM8A, PEX11B, ITGA10, ANKRD35, PIAS3, NUDT17, POLR3C, ZNF364, CD160, PDZK1, NBPF11 AML-N-#12 2 q33.3 206,519,469 1164.131 0.95 8 FLJ20309, NDUFS1, EEF1B2, GPR1, ADAM23, MDH1B, FASTKD2, CPO AML-N-#12 4 q31.3 152,506,822 2504.880 0.93 mono-allelic loss of 12 ESSPL, PET112L, FBXW7, FBXW7 confirmed by TMEM154, TIGD4, ARFIP1, FISH (RP11-316E17) KIAA1727, TRIM2, MND1, KIAA0922, TLR2, RNF175 AML-i16-#1 5 q33.3 156,389,535 1144.052 0.96 12 HAVCR1, HAVCR2, CRSP9, FAM71B, ITK, CYFIP2, MGC27121, ADAM19, SOX30, ICF45, LSM11, CLINT1 AML-M7-#4 5 p15.33 81,949 1240.051 1.00 17 KIAA1909, CCDC127, SDHA, PDCD6, AHRR, EXOC3, SLC9A3, CEP72, TPPP, ZDHHC11, BRD9, TRIP13, NKD2, SLC12A7, SLC6A19, SLC6A18, TERT AML-M7-#4 5 p15.33 1,501,000 2402.437 1.00 7 AYTL2, MRPL36, NDUFS6, IRX4, IRX2, CEI, IRX1 AML-M7-#4 5 q22.2;q22 111,875,200 1981.400 1.57 8 APC, SRP19, REEP5, DCP2, MCC, .3 TSSK1, YTHDC2, KCNN2 AML-PML-#6 5 q14.1 77,664,850 2092.463 1.00 15 SCAMP1, LHFPL2, ARSB, DMGDH, BHMT2, BHMT, JMY, HOMER1, PAPD4, CMYA5, MTX3, THBS4, SERINC5, SPZ1, ZFYVE16 AML-M7-#8 7 p14.1;p13 42,333,704 2127.936 2.50 25 C7orf25, PSMA2, MRPL32, HECW1, STK17A, C7orf44, BLVRA, MRPS24, URG4, UBE2D4, WBSCR19, DBNL, PGAM2, POLM, AEBP1, POLD2, MYL7, GCK, YKT6, CAMK2B AML-M7-#8 7 p13;p12.3 45,403,085 3967.314 2.51 10 ADCY1, IGFBP1, IGFBP3, TNS3, PKD1L1, FLJ21075, HUS1, SUNC1, UPP1, ABCA13 AML-i16-#4 7 q36.1 148,777,415 1351.406 1.01 22 KRBA1, ZNF467, LOC401431, ATP6V0E2L, LRRC61, C7orf29, RARRES2, REPIN1, ZNF775, GIMAP8, GIMAP7, GIMAP4, GIMAP6, GIMAP2, GIMAP1, GIMAP5, LR8, HCA112, ABP1, KCNH2 AML-AE-#5 7 q36.1;q36 150,995,960 3260.972 1.15 9 PRKAG2, GALNTL5, GALNT11, .2 MLL3, XRCC2, ACTR3B, FLJ42291, DPP6, PAXIP1 AML-N-#9 9 p24.2;p24 3,765,583 1301.108 0.95 8 GLIS3, SLC1A1, C9orf68, .1 PPAPDC2, CDC37L1, AK3, RCL1, JAK2 AML-PML-#6 9 p21.3 20,083,557 1815.443 0.00 21 MLLT3, KIAA1797, PTPLAD2, IFNB1, IFNW1, IFNA21, IFNA4, IFNA14, IFNA7, IFNA10, IFNA16, IFNA17, IFNA5, KLHL9, IFNA6, IFNA13, IFNA2, IFNA8, IFNA1, IFNE1 AML-N-#9 9 p21.3 20,935,207 1944.993 1.00 23 KIAA1797, PTPLAD2, IFNB1, IFNW1, IFNA21, IFNA4, IFNA14, IFNA7, IFNA10, IFNA16, IFNA17, IFNA5, KLHL9, IFNA6, IFNA13, IFNA2, IFNA8, IFNA1, IFNE1, MTAP AML-N-#11 9 p21.3 20,998,380 1396.300 0.00 21 PTPLAD2, IFNB1, IFNW1, IFNA21, IFNA4, IFNA14, IFNA7, IFNA10, IFNA16, IFNA17, IFNA5, KLHL9, IFNA6, IFNA13, IFNA2, IFNA8, IFNA1, IFNE1, MTAP, CDKN2A AML-misc- 9 p21.3 21,228,127 1090.931 0.20 FISH 12 IFNA14, IFNA5, KLHL9, IFNA6, #18 (CDKN2A+B/9p21.3) IFNA13, IFNA2, IFNA8, IFNA1, (RP11-149I2) confirms IFNE1, MTAP, CDKN2A, CDKN2B homozygous deletion AML-PML-#6 9 p21.2;p21 25,745,952 5737.315 1.46 10 C9orf82, PLAA, IFT74, LRRC19, .1 TEK, C9orf11, MOBKL2B, IFNK, C9orf72, LRRN6C AML-AE-#6 9 q22.33 96,984,012 931.450 1.32 FISH (FOXE1/9q22.33) 13 ZNF322B, KIAA1529, TDRD7, confirms deletion (RP11- TMOD1, C9orf97, NCBP1, XPA, 23B15) FOXE1, C9orf156, HEMGN, ANP32B, NANS, TRIM14 Supplemental Information - 26 - Case ID Chrom Cyto- Start* Size (kb) CN Validation # first 20 genes (CGC genes) band genes (6-25) AML-M7-#1 10 p15.3;p15 101,955 3133.370 1.31 13 ZMYND11, DIP2C, C10orf108, .2 LARP5, GTPBP4, IDI2, C10orf110, IDI1, WDR37, LOC399706, ADARB2, PFKP, PITRM1 AML-M7-#1 10 q22.1 70,815,492 1328.508 1.46 18 HK1, TACR2, TSPAN15, NEUROG3, C10orf35, COL13A1, H2AFY2, AMID, TYSND1, SAR1A, PPA1, NPFFR1, LRRC20, EIF4EBP2, NODAL, KIAA1274, PRF1, ADAMTS14 AML-M7-#1 10 q22.1 72,147,192 1410.429 3.24 12 ADAMTS14, C10orf27, SGPL1, PCBD1, UNC5B, SLC29A3, CDH23, C10orf54, PSAP, CHST3, SPOCK2, ASCC1 AML-M7-#1 10 q22.1;q22 74,527,553 682.267 2.62 14 NUDT13, ECD, DNAJC9, .2 MRPS16, TTC18, ANXA7, ZMYND17, PPP3CB, USP54, MYOZ1, SYNPO2L, FLJ32658, SEC24C, FUT11 AML-M7-#1 10 q22.2 75,247,849 1285.513 3.84 FISH (MYST4/10q22.2) 9 CAMK2G, C10orf55, PLAU, VCL, confirms gain of 3-10 AP3M1, ADK, MYST4, DUPD1, signals (RP11-1030K8) DUSP13 AML-MLL-#8 10 q22.2 76,150,456 973.492 3.06 FISH (MYST4/10q22.2) 8 MYST4, DUPD1, DUSP13, confirms gain of 3-8 signals SAMD8, VDAC2, COMTD1, (RP11-1030K8) LOC439985, ZNF503 AML-N-#12 10 q23.2;q23 87,847,802 2859.018 0.97 21 GRID1, WAPAL, OPN4, LDB3, .31 BMPR1A, MMRN2, SNCG, C10orf116, GLUD1, FAM35A, FAM22A, MINPP1, PAPSS2, ATAD1, PTEN, C10orf59, LIPL1, LIPF, ANKRD22, STAMBPL1 AML-misc-#3 11 p13 31,731,972 1020.696 0.91 7 ELP4, PAX6, RCN1, WT1, WIT1, PCID1, CCDC73 AML-N-#4 11 p15.5;p15 2,589,728 1115.679 0.96 Translocation: 15 KCNQ1, KCNQ1DN, CDKN1C, .4 t(5;11)[NUP98-NSD1], SLC22A18AS, SLC22A18, confirmed by FISH (RP11- PHLDA2, NAP1L4, CARS, OSBPL5, C11orf36, ZNF195, 348A20, RP11-99N22) ART5, ART1, CHRNA10, NUP98 AML-N-#9 11 p14.1 27,216,990 2769.530 0.88 6 CCDC34, LGR4, LIN7C, BDNF, KIF18A, METT5D1 AML-misc- 11 q24.2;q24 127,066,538 1532.130 3.09 FISH (ETS1/11q24.3) 7 ETS1, FLI1, KCNJ1, KCNJ5, #10 .3 confirms amplification (1 C11orf45, P53AIP1, RICS larger signal) (RP11- 272C3) AML-N-#5 11 q23.3 117,856,311 225.389 0.94 t(6;11)[MLL-MLLT4] 6 MLL, TMEM25, C11orf60, unbalanced, confirmed by ARCN1, PHLDB1, TREH FISH+RT-PCR (RP11- 91A14, CTD2501M11) AML-misc- 12 p13.31 8,493,691 1518.987 0.96 20 CLEC6A, CLEC4D, CLEC4E, #16 AICDA, MFAP5, FAM80B, A2ML1, PHC1, M6PR, KLRG1, A2M, PZP, KLRB1, CLEC2D, DCAL1, CD69, KLRF1, CLEC2B, CLEC2A, FLJ46363 AML-AE-#7 15 q25.2;q25 82,904,075 622.078 3.00 10 FLJ43276, ZSCAN2, SCAND2, .3 WDR73, NMB, SEC11L1, ZNF592, ALPK3, SLC28A1, PDE8A AML-i16-#4 16 p13.11 15,725,642 535.025 1.02 6 NDE1, MYH11, C16orf63, ABCC1, ABCC6, NOMO3 AML-i16-#11 16 q22.1 65,704,203 117.927 0.95 12 C16orf70, MGC4655, TRADD, FBXL8, HSF4, NOL3, EXOC3L, E2F4, ELMO3, LRRC29, HSPC171, FHOD1 AML-i16-#5 16 q22.1 65,704,203 117.927 0.90 12 C16orf70, MGC4655, TRADD, FBXL8, HSF4, NOL3, EXOC3L, E2F4, ELMO3, LRRC29, HSPC171, FHOD1 AML-N-#4 17 q24.2;q24 63,153,353 1840.227 3.16 16 NOL11, FALZ, C17orf58, KPNA2, .3 AMZ2, SLC16A6, ARSG, WIPI1, PRKAR1A, FAM20A, ABCA8, ABCA9, ABCA6, ABCA10, ABCA5, MAP2K6 AML-N-#12 17 q11.2 25,889,787 1465.545 0.98 mono-allelic loss of NF1 15 DKFZP434O047, confirmed by FISH (CTD- DKFZp667M2411, CRLF3, 2503N11) C17orf41, C17orf42, CENTA2, RNF135, NF1, OMG, EVI2B, EVI2A, RAB11FIP4, C17orf79, UTP6, SUZ12 AML-M7-#5 19 p13.3 4,014,067 672.596 3.98 21 ZBTB7A, MAP2K2, CREB3L3, SIRT6, EBI3, CCDC94, SHD, TMIGD2, FSD1, STAP2, FLJ14981, SH3GL1, CHAF1A, UBXD1, HDGF2, LOC440503, LRG1, SEMA6B, TNFAIP8L1, C19orf10 Supplemental Information - 27 - Case ID Chrom Cyto- Start* Size (kb) CN Validation # first 20 genes (CGC genes) band genes (6-25) AML-M7-#5 19 p13.3 4,706,178 433.702 2.84 7 C19orf30, FEM1A, TICAM1, M6PRBP1, C19orf31, UHRF1, JMJD2B AML-M7-#1 19 p13.13 12,688,923 206.122 5.33 14 TNPO2, C19orf43, ASNA1, VMD2L1, HOOK2, JUNB, PRDX2, RNASEH2A, RTBDN, MAST1, DNASE2, KLF1, GCDH, FARSLA AML-M7-#1 19 p13.13;p1 12,945,626 1045.589 3.81 16 DAND5, NFIX, LYL1, TRMT1, 3.12 BTBD14B, STX10, IER2, CACNA1A, CCDC130, MGC3207, C19orf53, ZSWIM4, C19orf57, CC2D1A, PODNL1, RFX1 AML-M7-#1 19 p13.12 15,114,411 556.339 4.43 10 NOTCH3, ABHD9, BRD4, AKAP8, AKAP8L, PGLYRP2, FLJ39501, CYP4F8, CYP4F3, CYP4F12 AML-M7-#1 19 p13.12;p1 15,685,070 528.33 7.04 14 OR10H2, OR10H3, OR10H5, 3.11 OR10H1, CYP4F2, CYP4F11, OR10H4, FLJ25328, TPM4, RAB8A, HSH2D, CIB3, FAM32A, AP1M1 AML-M7-#1 19 p13.11;p1 19,274,092 1112.424 4.03 16 SF4, KIAA0892, GATAD2A, 2 TSSK6, NDUFA13, FLJ44968, CILP2, PBX4, EDG4, GMIP, ATP13A1, ZNF101, ZNF14, ZNF253, ZNF93, ZNF682 AML-M7-#5 19 p12 21,470,638 1329.232 2.44 6 ZNF429, ZNF100, ZNF43, ZNF208, ZNF257, ZNF676 AML-N-#18 19 q13.11 37,494,099 1391.511 0.93 This case has mt>wt 19 ZNF507, DPY19L3, PDCD5, CEBPA mutation. ANKRD27, RGS9BP, NUDT19, TDRD12, SLC7A9, CCDC123, C19orf40, RHPN2, GPATCH1, WDR88, LRP3, SLC7A10, CEBPA, CEBPG, PEPD, CHST8 AML-N-#9 20 q11.22 32,325,220 652.906 0.93 9 AHCY, ITCH, DYNLRB1, MAP1LC3A, CDC91L1, TP53INP2, NCOA6, GGTL3, ACSS2 AML-N-#1 21 p11.2;p11 9,887,804 217.914 0.96 6 TPTE, BAGE2, BAGE3, BAGE4, .1 BAGE5, BAGE † Table S5e: Losses and Gains of loci with 2-5 genes (n=20, 6 of which are adjacent to translocation breakpoints) Case ID Chrom Cyto- Start* Size (kb) CN Validation/comment # Genes (CGC genes) band genes (2-5) AML-PML-#6 1 q21.1;q2 144,327,670 602.330 2.66 4 ACP6, GJA5, GJA8, 1.2 GPR89A AML-AE-#16 3 q26.1;q2 168,602,647 973.710 1.14 5 SERPINI2, WDR49, 6.2 PDCD10, SERPINI1, GOLPH4 AML-M7-#4 5 p15.33 1,353,000 144.000 0.00 2 CLPTM1L, SLC6A3 AML-M7-#4 5 p15.31 7,269,226 420.530 0.95 2 FLJ25439, ADCY2 AML-PML-#6 5 q14.2;q1 81,729,650 3758.916 1.50 5 TMEM167, XRCC4, 4.3 CSPG2, HAPLN1, EDIL3 AML-PML-#6 7 p21.3 8,673,266 3510.681 1.48 3 NDUFA4, PHF14, TMEM106B AML-N-#18 7 p21.1 16,687,806 484.414 3.63 2 BCMP11, AHR AML-misc-#12 7 p12.3;p1 49,473,728 754.732 3.31 3 VWC2, ZPBP, IKZF1 2.2 AML-i16-#10 8 q24.13 124,722,755 269.162 3.27 2 ANXA13, FAM91A1 AML-PML-#6 9 p21.3 22,366,805 1907.154 1.58 2 DMRTA1, ELAVL2 AML-N-#9 9 q33.1;q3 118,357,800 1888.673 0.93 2 DBC1, CDK5RAP2 3.2 AML-M7-#1 10 q22.1 73,570,580 340.545 7.49 5 ASCC1, C10orf104, DDIT4, DNAJB12, CBARA1 AML-M7-#1 10 q22.1 73,911,130 606.51 4.33 5 CBARA1, CCDC109A, OIT3, PLA2G12B, P4HA1 AML-N-#8 11 p15.4 3,722,768 235.136 3.63 Translocation, 4 NUP98, FRAG1, RHOG, t(5;11)[NUP98-NSD1], STIM1 validated by RT-PCR AML-i16-#7 16 p13.11 15,688,555 509.441 1.21 Translocation 5 NDE1, MYH11, C16orf63, ABCC1, ABCC6 AML-i16-#10 16 p13.11 15,737,037 397.157 0.99 Translocation 3 MYH11, C16orf63, ABCC1 Supplemental Information - 28 - Case ID Chrom Cyto- Start* Size (kb) CN Validation/comment # Genes (CGC genes) band genes (2-5) AML-i16-#13 16 p13.11 15,738,236 389.391 0.98 Translocation 3 MYH11, C16orf63, ABCC1 AML-i16-#14 16 p13.11 15,737,037 138.706 0.98 Translocation 2 MYH11, C16orf63 AML-i16-#5 16 p13.11 15,715,180 109.642 1.04 Translocation 2 NDE1, MYH11 AML-PML-#5 17 p12 12,704,473 157.897 3.27 2 KIAA0672, ELAC2 Table S5f: Losses and gains of loci with a single gene (n=23, 7 of which are adjacent to translocation breakpoints) Case ID Chrom Cytoband Start* Size (kb) CN Validation/comment #gene Genes (CGC genes) AML-N-#17 4 q27 121,931,400 224.600 3.00 1 PRDM5 AML-misc- 5 q33.3 158,084,826 310.670 3.46 1 EBF #18 AML-misc-#1 6 q27 168,081,288 93.420 3.11 Translocation: MLL- 1 MLLT4 MLLT4 was shown by FISH AML-N-#5 6 q27 168,088,794 85.914 3.87 Translocation: 1 MLLT4 t(6;11)[MLL-MLLT4] unbalanced, confirmed by FISH+RT-PCR (RP11- 91A14, CTD-2501M11) AML-misc-#2 7 p22.3 141,322 153.264 1.05 1 FAM20C AML-AE-#3 8 q21.3;q2 93,145,729 394.287 1.08 Translocation 1 RUNX1T1 2.1 AML-AE-#6 8 q21.3;q2 93,170,040 990.360 1.21 Translocation 1 RUNX1T1 2.1 AML-AE-#12 8 q21.3;q2 93,170,040 1255.155 0.97 Translocation 1 RUNX1T1 2.1 AML-N-#15 8 q24.21 130,471,754 300.239 3.77 1 CCDC26 AML-AE-#8 8 q24.21 130,538,594 251.464 2.83 FISH (CCDC26): appears 1 CCDC26 to be amplified (1 larger signal) (RP11-259L23) AML-misc-#5 8 q24.21 130,610,600 191.254 2.93 1 CCDC26 AML-AE-#20 8 q24.21 130,646,400 78.604 3.16 1 CCDC26 AML-PML-#6 9 p21.3 24,279,551 1460.946 0.00 1 TUSC1 AML-PML-#4 9 p21.3;p2 25,476,469 192.555 0.85 FISH (TUSC1) confirmed 1 TUSC1 1.2 CN=1 (RP11-580I21) AML-MLL- 10 q11.22 48,037,000 243.710 3.00 1 GDF10 #16 AML-misc-#1 11 q23.3 117,874,000 12.000 1.00 Translocation: MLL- 1 MLL MLLT4, validated by FISH (RP11-91A14, CTD- 2501M11) AML-MLL-#7 11 q23.3 117,874,000 12.000 1.00 Translocation 1 MLL AML-M7-#6 13 q31.3 91,414,118 109.715 0.90 1 GPC5 AML-PML-#4 13 q32.1 94,738,000 78.547 4.59 1 ABCC4 AML-M7-#7 15 q21.1 42,910,906 144.568 3.85 1 C15orf43 AML-AE-#14 16 p13.2 6,867,386 97.643 0.95 1 A2BP1 AML-MLL-#8 19 p13.2 7,080,086 250.442 1.11 1 INSR AML-M7-#4 23 p11.4 39,318,000 771.000 0.00 1 BCOR Table S5g: Losses and gains of loci without annotated RefSeq genes Case ID Chrom Cytoband Start* Size (kb) CN Validation #gene AML-MLL-#9 1 q31.1 185,716,00 38.256 0.69 0 0 AML-misc- 1 q31.1 184,375,00 127.700 0.82 0 #21 0 AML-misc-#3 2 p22.3 35,711,937 7.246 0.61 0 AML-N-#14 2 p12 82,106,021 114.109 3.83 0 AML-AE-#19 2 p12 81,037,265 155.815 0.67 0 AML-N-#21 2 q14.1 117,013,80 29.400 3.98 0 0 AML-i16-#9 3 p14.1 66,888,096 78.703 3.15 0 AML-M7-#4 4 q34.3 182,171,18 176.820 0.93 0 0 AML-MLL-#7 4 q13.1 63,414,725 545.279 3.66 0 AML-N-#9 4 q28.1 127,757,37 431.814 0.85 0 0 Supplemental Information - 29 - Case ID Chrom Cytoband Start* Size (kb) CN Validation #gene AML-AE-#8 4 q28.2;q28.3 130,857,61 1172.546 0.90 0 9 AML-M7-#4 5 p15.31 7,056,920 42.014 0.83 0 AML-N-#4 7 q21.11 85,643,878 223.345 3.56 0 AML-AE-#5 8 p23.2 2,291,741 1.644 0.75 0 AML-AE-#5 8 q24.23 138,529,80 0.600 0.59 0 0 AML-AE-#5 9 p24.3 1,027,639 0.371 6.68 0 AML-PML-#6 9 p21.3 22,044,000 284.657 0.00 0 AML-N-#1 10 q25.1 109,064,71 283.100 1.12 0 6 AML-misc- 10 p12.31 19,551,220 28.780 1.00 0 #21 AML-AE-#3 11 q13.1 63,958,215 1.685 4.94 0 AML-MLL- 12 q21.1 71,540,830 110.000 0.79 0 #16 AML-MLL-#7 15 q25.3 85,120,222 93.778 3.00 0 AML-M7-#6 17 q24.3 66,236,699 204.331 3.48 0 AML-MLL-#9 18 p11.21 11,551,254 6.303 0.65 0 *hg17 (Build 35) Table S6a-b: Cancer Gene Census genes within CNA or MAR<20Mb Genes marked with * are target of a CNA in which this is the only gene within the altered region in at least one case. Among recurrently affected genes, genes in bold and underlined reside within peak limits in GISTIC analysis, genes in bold reside within wide peak limits. Genes in blue reside within lesion adjacent to translocation breakpoint. Recurrent Single Case Del Amp Del Amp MYH11 EXT1 IRF4 PDE4DIP XPA ERG FBXW7 JAK3 WT1 TMPRSS2 OLIG2 ELL LMO2 RUNX1* JAK2 IKZF1 MLL* MLLT4 MLLT3 NUP98 ETV6 PBX1 CFBF SDHC ITK GMPS RPL22 FCGR2B RANBP17 MLF1 BMPR1A PRKAR1A TLX3 MYST4 PTEN HNRNPA2B1 NPM1 FLI1 CARS HOXA9 NSD1 LYL1 NUP98 HOXA11 APC SH3GL1 NF1 HOXA13 MLLT1 SUZ12 JAZF1 BRD4 FNPBP1 TMP4 ABL1 NUP214 DEK PICALM MAML2 CEBPA Supplemental Information - 30 - Table S6b: CNA Involving a Single Gene in at least One Case Loci with a single gene are rank-ordered by the frequency of all lesions (broad and focal) affecting the loci. Locus Gene Loss/Gain # lesions with # broader lesions associated with known single gene including gene translocation 8q24.21 CCDC26 Gain 4 11 7p22.3 FAM20C Loss 1 5 13q32.1 ABCC4 Gain 1 3 11q23.3 MLL Loss 2 1 Yes 8q21.3-q22.1 RUNX1T1 Loss 3 Yes 6q27 MLLT4 Gain 2 Yes 9p21.3-21.2 TUSC1 Loss 2 Xp11.4 BCOR Loss 1 1 4q27 PRDM5 Gain 1 1 5q33.3 EBF Gain 1 15q21.1 C15orf43 Gain 1 10q11.22 GDF10 Gain 1 16p13.2 A2BP1 Loss 1 19p13.2 INSR Loss 1 13q31.3 GPC5 Loss 1 Supplemental Information - 31 - Table S7a-c: Copy Neutral Loss of Heterozygosity (CN-LOH) Table S7a. Number of cases with LOH in paired analysis of matched tumor and germ line samples and in unpaired analysis with unmatched pool of reference samples. Paired analysis N=60 Unpaired analysis Combined Paired N=51 andUnpaired Cases with large 5 8 13 regions of LOH (>20MB) Cases with small 1 n. a. 1 regions of LOH (<20MB, 3 contigous SNP) Total 6 (10%) 8 (16%) 14 (13%) Table S7b. Regions of CN-LOH detected in paired analysis Case ID Chromosom Start / size in CGC Genes within region of LOH Comments al location Mb* AML-AE-#1 11pter-p12 0Mb / 41.584Mb CARS, HRAS, FANCF, LMO1, NUP98, LMO2, WT1, DDB2 AML-i16-#6 8q11.1- 47.535Mb / no gene Validated by SNPshot q11.21 0.496Mb (3) AML-MLL-#8 21pter-qter 0Mb / 46.840Mb ERG, OLIG2, RUNX1, TMPRSS2 AML-misc-#8 11pter-p11.2 0Mb / 46.163Mb CARS, HRAS, FANCF, LMO1, NUP98, Adjacent to LMO2, WT1, DDB2, EXT2 homozygous deletion around CDKN2A locus AML-misc-#18 9p24.3-p13.2 0Mb / 37.428Mb JAK2, PSIP2, MLLT3, CDKN2A AML-N-#6 15q15.1-qter 40.290Mb / BLM, C15orf21, NTRK3, PML, TCF12 59.921Mb *Number of informative SNPs is given in brackets if less than 10. Table S7c. Regions of UPD detected in un-paired analysis Case ID Chromosomal Start / size in CGC Genes within region of LOH Comments location Mb AML-AE-#16 17q11/2-qter 23.247/55.153 CLTC, SUZ12, LASP1, NF1, RARA, BRCA1, ETV4, MLLT6, ERBB2, COL1A1, BCL5, BRIP1, HLF, MSI2, PRKAR1A, ASPSCR1, MSF, ALO17 AML-misc-#3 13pter—qter 0/113 Homozygous FLT3-ITD AML-misc-#10 13pter—qter 0/113 Homozygous FLT3-ITD AML-misc-#14 13pter—qter 0/113 Homozygous FLT3-ITD AML-misc-#17 13pter—qter 0/113 Homozygous FLT3-ITD AML-N-#11 9p24.3—p21.3 0/27.142 JAK2, PSIP2, MLLT3, CDKN2A Adjacent to homozygous deletion around CDKN2A/B locus AML-N-#14 6pter—p21.2 0/39.559 IRF4, DEK, CCND3, FANCE, POU5F1, HIST1H4I, PIM1, HMGA1, SFR3S, TFEB AML-N-#15 11pter—p11.2 0/47.37 CARS, HRAS, FANCF, LMO1, NUP98, LMO2, WT1, DDB2, EXT2 Supplemental Information - 32 - Figure S1: Micro Deletions Adjacent to the Genes Involved in translocations (A), shows schematic representation of micro deletions we observed in 7 of 16 cases with inv(16), along with the cytobanding on chromosome 16. (B-D), log2ratio heatmaps of region around both breakpoints. Blue represents deletion (see color scale for log ratio on the bottom). (D), Three of 20 cases with t(8;21) had deletions at the RUNX1T1/ETO locus (AE-#3, 6, 12) as shown in this log2ratio heatmap. A AML-i16-# B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 16p13.1 MYH11 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CBF 16q22 Supplemental Information - 33 - Figure S2: Focal CNA Identified Cryptic Translocations (A) shows the genomic organization of NUP98 and NSD1 with the t(5;11) translocation breakpoints depicted by black arrows. The extent of amplification (red) and deletion (green) identified in individual cases are shown by horizontal lines and labeled with the case ID number. The location of SNPs on the combined 100K and 500K Affymetrix arrays are shown by vertical blue lines. (B) shows the NUP98-NSD1 fusion product that results from the t(5;11). (C) FISH analysis confirming the presence of a translocation in case AML-N-#4, with the white arrow marking the fused green (NUP98; RP11-348A20) and red (NSD1; RP11-99N22) signals. (D) Results of RT-PCR analysis for the chimeric NUP98-NSD1 transcript confirming the presence of the fusion transcript in AML-N-#8 and detecting the chimeric transcript in two other cases, AML-N-#10 and AML-misc#20. Sequencing revealed fusion of exon 12 in NUP98 and exon 6 in NSD1 in all three cases analyzed. GAPDH was used as control. NTC, no template control. (E) shows the schematic representation of lesions in MLL and MLLT4 in AML cases AML-N-#20 and AML-misc#1 (amplifications: red solid lines, deletions: green solid lines; deletions/amplifications that stretch beyond the region shown are arrowed, vertical arrows mark putative breakpoints). The SNP coverage of MLL is sparse (each blue vertical line is a SNP), dotted lines adjacent to deletions mark the distance from the last deleted to the next SNP with normal copy number. (F) Schematic representation of the fusion product. (G) FISH confirmed fusion in both cases (MLL in red, RP11-91A14; MLLT4 in green, CTD-2501M11). (H) RT-PCR confirmed fusion products. Sequence analysis of the fusion products after RT-PCR revealed breakpoint in intron 6 for AML-N-#20 and in intron 7 of MLL for AML-misc#1. ABL was used as control. The cell line ML-2 was used as positive control. NTC, no template control. Supplemental Information - 34 - A AML-N#8 AML-N-#4 1 2 4 6 8 10a 11 12 14 16 18 20 22 24 26 28 30 32 33 NUP98 tel cent 5kb AML-N#4 1 2b 4 6 8 10 12 14 16 18 20 22 23 NSD1 cent tel B NUP98 NSD1 NH2 FG FG SET exons 2 4 6 8 10a 12 6 7 8 10 12 14 16 18 20 22 23 #20 C D - #10 #8 - - N N misc - - - AML NTC AML AML control NUP98- NSD1 - 200bp NUP98 NSD1 NSD1- AML-N-#4 NUP98 - 200bp GAPDH - 200bp Supplemental Information - 35 - Figure S3: Comparison Paired and Unpaired LOH Analysis Different approaches assessing Loss-of-Heterozygosity using DChipSNP were compared and shown for a subset of samples with paired normal germ line samples (Default DChipSNP parameters were chosen: 0.5 for Probability of LOH, regions of homozygosity consistent with 5% of reference samples were removed). (A) LOH was inferred on the tumor samples by the Hidden-Markov-Model in an unpaired analysis without reference samples. (B) LOH in the tumor samples was also inferred by the Hidden-Markov-Model using a pool of reference germ line samples. (C) LOH was also inferred by paired analysis for each pair of tumor and germ line sample. The number of false positive calls decreases significantly with the usage of a pool or paired samples. Regions exceeding 20Mb were consistently detected in both paired and unpaired analysis. Thus, for analysis of LOH in patients lacking matched germ line SNP array data in this study, only regions exceeding this threshold were deemed somatic. LOH, Loss of Heterozygosity.