Blood 2015 05 646240 1 PDF

SUPPLEMENTAL INFORMATION

Profiling of somatic mutations of acute myeloid leukemia, FLT3-ITD subgroup at diagnosis and relapse
Manoj Garg1,17, Yasunobu Nagata2, Deepika Kanojia1, Anand M.T.1, Kenichi Yoshida2, Sreya Haridas
Keloth1, Zang Zhi Jiang1,Yusuke Okuno3, Yuichi Shiraishi4, Kenichi Chiba4, Hiroko Tanaka5, Satoru
Miyano5, Ling-Wen Ding1, Tamara Alpermann6, Qiao-Yang Sun1, De-chen Lin1, Wenwen Chien1, Vikas
Madan1, Li-Zhen Liu1, Kar-tong Tan1, Abhishek Sampath1, Subhashree Venkatesan1,Koiti Inokuchi7, Satoshi
Wakita7, Hiroki Yamaguchi7, Wee Joo Chng1, Shirley-Kow Yin Kham8, Allen Yeoh8, Masashi Sanada2,9,
Joanna Schiller10, Karl-Anton Kreuzer10, Steven M. Kornblau11,12, Hagop M. Kantarjian11,12, Torsten
Haferlach6, Michael Lill13, Ming-Chung Kuo14, Lee-Yung Shih14, Igor-Wolfgang Blau15, Olga Blau15, Henry
Yang1, Seishi Ogawa2 and H. Phillip Koeffler1,13,16
Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore;
1
2
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan; 3Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan;
4
Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The
University of Tokyo, Tokyo, Japan; 5Laboratory of Sequence Analysis, Human Genome Center, Institute of
Medical Science, The University of Tokyo, Tokyo, Japan; 6MLL Munich Leukemia Laboratory, Munich,
Germany; 7Department of Hematology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113-
8
8603, Japan; Department of Paediatrics, National University Health System, Singapore, Singapore;
9
Department of Advanced Diagnosis, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan;
10 11
Department I of Internal Medicine, University at Cologne, 50937 Cologne, Germany; Department of
12
Leukemia - Unit 428, M.D. Anderson Cancer Center, Houston, TX; Section of Molecular Hematology &
Therapy, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX;
13
Cedars-Senai Medical Center, Division of Hematology/Oncology, University of California Los Angeles,
14
School of Medicine, Los Angeles, California, USA; Division of Hematology-Oncology, Department of
Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan; 15Department of
Hematology, Oncology and Tumorimmunology, Charite University School of Medicine, Berlin, Germany;
16
National University Cancer Institute, National University Hospital, Singapore, Singapore;
1


17
Correspondence should be addressed to Manoj Garg; csimg@nus.edu.sg or nuscsimg@gmail.com
Table of Contents
Supplemental Methods (pages 3 – 8)
Supplemental Figures (Pages 9 – 30)
2

Supplemental Methods
Exome capture and massively parallel sequencing and analysis of whole exome sequencing data
Genomic DNAs from diagnosis, complete remission and relapse were used for whole exome sequencing
(WES). Complete remission samples were used as germline control. We included only those samples whose
bone marrow blast counts in CR were < 5 percent (Supplemental Table 1 and 6). Whole exomes were
captured using the SureSelect® Human All Exon 50M and/or V4 (Agilent) as described previously21-23.
Briefly, 2 µg of human genomic DNA was sheared using Covaris instrument. The fragment-size distribution
was 150-200bp and ends were repaired using T4 DNA ligase, dNTP mix, T4 DNA polymerase Klenow
enzyme and T4 PNK by incubating samples at 200C for 30 minutes. ‘A’ bases were added to 3′ end of the
DNA fragment. Paired end adapters were ligated and amplified using paired-end PCR primers. 500ng of
fragment libraries were hybridized with biotinylated RNA using 2X hybridization buffer for 24hr at 65°C.
Captured libraries were purified and amplified by Sure-Select indexing primers. Captured libraries were
subjected to high through-put sequencing using Hiseq2000 illumina platform with 75 to 100 bp paired-end
reads.
To detect somatic mutations and short insertions and deletions (indels) from WES, we used previously
developed in-house analysis pipeline21-23. Briefly, the sequencing reads were mapped to the human reference
genome (hg19) using BWA (version 0.5.9-r16) with default parameter settings. SAM tools (version 0.1.17)
were applied to pile up the aligned sequence data (.bam files) for each nucleotide position in the genome.
Based on pile-up data, variant calling was performed by comparing the piled-up coverage data to the reference
genome. Information about read counts, sequencing/mapping qualities and genome strands for each variant
site were retrieved for subsequent detection of somatic mutations and short indels.
Before summarizing base call data, low quality reads were filtered, including those having more than
5 mismatches to the reference sequences or whose mapping quality was less than 30. The significance of each
candidate mutation was evaluated by Fisher’s exact test by enumerating the number of the reference base and
the candidate SNV in both leukemia and control. Candidate mutations having P-values of less than 0.01 were
adopted as provisional candidates for somatic mutations. In addition, the following nucleotide positions were
3


eliminated from further analysis; those positions at a depth of less than 10 in either tumor or control, or the
most frequent SNV or indel accounts for less than 7% of all reads in tumor. Finally, a list of candidate somatic
mutations was generated by excluding synonymous SNVs and other variants registered in dbSNP131, 1000
genomes, or our in-house SNP database constructed from 180 normal samples as well as subtraction of the
sequence variants of germline control of each of our patients 21-23. All the somatic mutations were validated by
using Sanger sequencing and the primer sequences were detailed in Supplemental Table 4 and 9.
Clustering and clonal analysis
To study the emergence of disease clones and their progression from diagnosis to relapse disease, somatic
mutations detected in both phases by WES were clustered according to their mutant variant allele frequencies
(VAFs). For clonal analysis along with the validated mutations, we also used high quality (min depth 14 in
tumor, supported by at least 5 reads for variant allele, < 2% allele frequency in matched normal) tier2
mutations (mutations in observed in conserved genomic regions with phastConsElements46way score greater
than 280). Read counts for these variants were obtained for both diagnosis tumor and relapse tumor and
Mutant Variant Allele Frequencies (VAFs) were calculated. VAF allows an estimate of the number and size of
tumor clonal populations in each AML sample. Copy number–adjusted deep sequencing data, in which the
VAFs of genes on the X chromosome in male cases or in regions of uniparental disomy were halved, were
subjected to unsupervised clustering. Long indels of >3 bp, except for those affecting key genes such as
FLT3-ITD, NPM1and WT1, and mutations in repetitive regions were excluded from the analysis because their
VAFs tended to be underestimated. These VAFs were later clustered using 'Bayesian mixture modelling of
beta distributions' (bmm method) implemented in sciClone R package16, 61-64. The coverage provided by whole
exome sequencing limited the power to detect clones with VAF of less than 5%.
Mutational Signature
The six classes of transition and transversion mutations were analyzed by integrating the information on the
bases immediately 5’ and 3’ to each mutated base (16 possible combination for each class), creating a total of
96 possible mutated tri-nucleotides. We then used EMu, a probabilistic method which uses Expectation
39-41
Maximization (EM) algorithm to identify Mutational Signatures . EMu also infers tumor specific
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“opportunity” for all mutations types, from the sequence composition thus increasing the accuracy of
deciphered signatures.
Frequency analysis using targeted deep sequencing (TDS)
Genomic DNA (1-1.5 μg) samples from all 80 cases were subjected to targeted deep sequencing of 299 genes
(Supplemental Table 5) using multiplex SureSelect custom kit (Agilent Technology) designed to capture all
of the coding regions of 299 target genes. We also included 14 cases from the discovery cohort to ensure
capture efficiency. Deep sequencing was performed on multiplexed library pools using Hiseq2000 illumina
platform with 75 to 100 bp paired-end reads. We used more stringent pipeline for TDS. We removed the
following nucleotide positions: those whose supporting depth from both directions was less than 5 in either
tumor or control, or where the most frequent SNV or indel accounted for less than 8% of all reads in the AML
samples.
To identify possible somatic mutations of FLT3-ITD cell lines, where no paired germline controls
were available, nucleotide positions were removed if: #1, supporting depth from both directions was less than
5; #2, most frequent SNV or indel accounted for less than 8% of all reads; #3, frequency of the SNV or indel
was between 45% and 55% in the region in question without copy number abnormalities (most likely a rare
SNP).
Significantly mutated gene (SMG) analysis and pathway analysis
Validated list of Single Nucleotide Polymorphisms (SNVs) and InDels across the cohort were used to identify
significantly altered genes. We applied Mutational Significance in Cancer (MuSiC) package to determine
significantly mutated genes (SMG) and pathways. The SMG test in MuSiC used seven categories of mutations
(AT transition, AT transversion, CG transition, CG transversion, CpG transition, CpG transversion, and indel)
and then used statistical tests including convolution, Fisher’s test, and a likelihood test to combine the
category-specific binomials to obtain an overall p value. All P-values were combined using the same
methods as described in Dees et al35. For Pathway analysis, we used PathScan algorithm integrated into MuSic
pipeline to identify significantly affected pathways among known cellular pathways defined in KEGG
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pathway database36,37,38. We also used Mutation Relation Test (MRT) from the same MuSic pipeline to
identify co-occurring and exclusively occurring genes across the cohort.
Single nucleotide polymorphism array (SNP-array) analysis
For SNP-array, genomic DNA was subjected to the Affymetrix GeneChip Human Mapping 250K SNP arrays
(Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocols. SNP-array results were
analyzed for determination of both total and allelic-specific copy numbers (AsCNs) using the copy number
analysis tool for genechip (CNAG) software, as previously described25,26.
Cell culture and antibodies
Myeloid leukemia cell lines; MOLM13 and MV4-11 were kind gifts by Dr. Martin Grundy (Department of
Academic Haematology, University of Nottingham). MOLM14 cells were generously provided by Dr. Didier
Bouscary (Department of Hematology-Immunology, Institut Cochin, Paris, France). 32D cell line stably
expressing wild type FLT3 and FLT3-ITD were provided by Drs. J. Mueller and H. Serve (Department of
Medicine, Frankfurt, Germany). Cell lines were cultured and maintained in RPMI medium containing 10%
fetal bovine serum (FBS), 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 370C in a humidified
atmosphere with 5% CO2. 293T cell line was purchased from ATCC and grown in Dulbecco modified Eagle
medium (DMEM) with 10% FBS. All cell lines were regularly screened for mycoplasma. Antibodies were
against FAT1 (HPA023882, Sigma-Aldrich), GAPDH (Cell Signalling), MLL3 (Santa Cruz).
RNA interference
Human MLL3, FAT1 gene specific siRNA duplexes including scramble siRNA duplexes were purchased from
Integrated DNA Technologies (IDT) (Coralville, USA). Murine MLL3 gene specific siRNA was obtained
from Santa Cruz Biotechnology, INC. (Heidelberg, Germany) and IDT. MV4-11 and MOLM14 cells were
transfected with 100nM siRNA duplex using Nucleofector Kit L (Lonza, Cologene, Germany) according to
the manufacturer’s protocol. 2ug of pmaxGFP vector was used to ensure transfection efficiency. Average
transfection efficiency was approximately 60-70 percent. Cells were incubated for at least 48 hours after
nucleofection before performing experiments. Each experiment was performed at least in triplicate on three
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different occasions. To obtain stable knockdown of MLL3 in either MV4-11 or MOLM14 cells for in vivo
studies, a set of 4 human MLL3 gene specific shRNAs and a scramble shRNA cloned in a lentiviral vector
were obtained from Origene (Rockville, MD, USA). Lentiviral particles were generated according to the
manufacturer’s protocol. Cells were spin-infected with lentivirus particles at a MOI of 25 with 5 ug/ml
polybrene (Sigma-Aldrich) at 2000 rpm for 1hr; stable cells were selected using 0.5-1 ug/ml puromycin for 1-
2 weeks. shRNA3 and shRNA4 confirmed best knockdown compared to the scrambled shRNA. Further, we
used these two shRNAs for biological study. MLL3 shRNA and siRNA sequences targeting to MLL3 or FAT1
are listed in Supplemental Table 14A, B and C.
RT-PCR analysis and quantitative real-time PCR (qRT-PCR)
Total RNA was reverse transcribed to cDNA with Superscript III (Invitrogen) according to the manufacturer’s
protocol. qRT-PCR experiments were performed as described earlier27. Primer sequences were detailed in
Supplemental Table 15.
Gel electrophoresis and Immunoblotting
Immunoblotting experiments were performed as described27. Briefly, 60 micrograms of protein per lane were
loaded for immunoblotting. Precise Tris-Glycine 4-20% gradient gels (Thermo Scientific) were used for
MLL3 and FAT1 analyses. Gels were run according to the manufacturer’s protocol. Proteins were transfer to
PVDF membrane using methanol-free transfer buffer ( 25 mM TRIS pH 8.3, 192 mM glycine, 03% SDS)
overnight in the cold room using a constant current (200 mAmp). Membranes were incubated with primary
antibody (MLL3, FAT1 and GAPDH) at 1:1000 dilutions in blocking buffer (5% milk) overnight at 4 °C.
Membranes were washed with PBS-Tween 20 and incubated with HRP conjugated secondary antibody for 1
hr. Blots were developed using ECL reagent (Thermo Scientific).
Xenografts model
The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of National
University of Singapore; 5 weeks old NOD/SCID mice were used. 2 X 106 of MV4:11 cells (stably expressing
either scrambled shRNA or MLL3 shRNA) were mixed with Matrigel solution (1:1 ratio) (BD Biosciences) in
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200 μl of total volume which was injected subcutaneously on the upper flanks of the NOD/SCID mice27. At
defined time points, all mice were sacrificed and weights of resected tumors were measured. Tumors from
both groups were immediately stored at -80oC for western blotting experiments.
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Supplemental Figures
Supplemental Figure 1. Mean coverage of whole exome sequencing (WES) in 13 trios (DX, CR and
REL) from discovery cohort samples. Coverage of the target regions was calculated and indicated depth
plotted for each patient at diagnosis (DX), complete remission (CR) and relapse (REL). Mean depth for all the
DX, CR and REL is indicated.
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Supplemental Figure 2. Somatic mutations identified by whole-exome sequencing (WES), targeted deep
sequencing and their distribution in FLT3-ITD AML. (A) Number of mutations discovered in 50
individual at diagnosis and relapse (light grey), 25 individual at diagnosis (dark grey) and 5 individual at
relapse (black). Individuals examined by either WES or TDS are color coded in light or dark brown,
respectively. Bar graph displays frequency of mutations per patient sample (s). Types of mutations are color-
coded. (B) Pie chart represents distribution of somatic mutations verified by Sanger sequencing in the 14
paired DX and REL genomes. (C) Somatic mutations for each cases sorted into three categories: shared
(mutations present in both DX and REL samples), specific for diagnosis and specific for relapse.
Supplemental Figure 3. Mean coverage of targeted deep sequencing (TDS) in samples from 80 patients
(50 individual at diagnosis, remission and relapse; 25 individual at diagnosis and remission; 5
individual at relapse and remission). Mean coverage of SureSelect XT2 captured targets for the 210
samples. Mean depth of the coding regions was analyzed and plotted for each sample.
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Supplemental Figure 4. Hotspot mutations identified in FLT3-ITD AML. Schematic representation of
known or predicted functional domains and locations of alterations in DNMT3A, NPM1 and FLT3, detected by
either by whole exome or targeted deep sequencing (using protein paint tool developed by St. Jude Children’s
Research Hospital and Washington University in St. Louis). Each patient with a DNMT3A, NPM1 and FLT3,
mutation is represented by blue (missense) or green (frameshift) color circle. Alterations at the same amino
acid in different cases are depicted.
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Supplemental Figure 5. Hotspot mutation of SF3B1, IDH1 and IDH2 genes were identified in FLT3-
ITD AML. Schematic of type and location of alterations of SF3B1, IDH1 and IDH2detected by either whole
exome or targeted deep sequencing (using protein paint tool developed by St. Jude Children’s Research
Hospital and Washington University in St. Louis). Boxes represent functional domains of SF3B1, IDH1 and
IDH2. Mutations are represented by blue (missense) or green (frameshift) color circle.
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Supplemental Figure 6. Somatic mutation pattern identified in matched diagnosis (D) and relapse (R)
samples of 50 trios FLT3-ITD AML patients. Each column displays an individual sample (UPN numbers
are indicated in the top row). Colored bars represents the presence of a mutation, blank bars represent wild-
type for the respective gene. Light orange color displayed different ITD insertion sequence at relapse. The
“Stability” of mutation was calculated by the number of mutations that persisted in relapse divided by all the
mutations present at diagnosis. The mutation “Gained at Relapse” was calculated by the number of cases that
gained a new mutation at relapse and absent at diagnosis divided by the number of cases that did not have the
same mutation at diagnosis.
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14

15

16

17

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Supplemental Figure 7. Recurrent mutations in DNMT3A were validated by Sanger sequencing in
matched diagnosis and relapse samples of FLT3-ITD AML (trios). Sample ID and location of DNMT3A
mutation is indicated by orange text and arrows. The horizontal black text represents the status of disease and
variant allele frequency (VAF) calculated from next generation sequencing data for each patient. The Vertical
black text shows the direction of the primer used for Sanger sequencing of PCR product. Sequencing trace
chromatograms shows a clear heterozygous mutation.
19

20

21

22

Supplemental Figure 8. Stability of NPM1 mutations were confirmed using Sanger sequencing in
matched diagnosis and relapse samples of FLT3-ITD AML (trios). Sample ID and type of NPM1 mutation
is indicated by red text. Location of NPM1 mutation is indicated by orange rectangle. The horizontal black
text represents the status of disease for each patient. The Vertical black text shows the direction of the primer
used for Sanger sequencing of PCR product. Sequencing trace chromatograms shows a clear heterozygous
mutation.
23

Supplemental Figure 9. Mutual exclusivity between genes. (A) Mutually-exclusive gene sets identified
using MuSic algorithm with P < 0.05 (NPM1 and RUNX1), P < 0.001 (NPM1 and MLL3), P < 0.05 (WT1 and
MLL3), P< 0.05 (MLL3 and RUNX1), P < 0.05 (NPM1 and WT1). (B) Mutually-exclusivity is observed within
the genes coding for spliceosome and cohesin complex.
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Supplemental Figure 10. Mutation matrix of chromatin modifier and histone methyltransferase genes.
(A-B) Mutational matrix of chromatin modifier and histone methyltransferase genes as noted in 25 and 24
patient samples, respectively. Data show that at least one mutation of these gene sets is present in each patient
sample.
25

Supplemental Figure 11. Potential therapeutic targets in AML FLT3-ITD. Graphic representation of
several actionable genomic lesions in 52 ITD patient samples. Patients are listed on the X-axis. Target genes
identified in one or more patients and the drugs that target these genes are listed on the y axis (gene symbols
on the left side, corresponding drug names on the right side). A box representing each gene-drug combination
for each patient is colored according to the class of gene alteration: blue for SNVs, green for indels and red for
RNA overexpression. Drugs in phase 1, 2 and 3 clinical trials are marked in sky blue, purple and brown,
respectively (see, URLs). FDA approved drugs are marked with dark green color (see, URLs).
26

Supplemental Figure 12. MLL3 deletion in AML FLT3-ITD. SNP Chip analysis of chromosome 7 showed
amplifications (red) and deletions (blue). Green line at chromosome 7q36.1 demarks MLL3 gene.
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Supplemental Figure 13. Tumor suppressive function of MLL3 in FLT3-ITD AML cells. (A) Integrative
Genome Viewer (IGV) segmentation map of 7q36.1 showing loss of copy number (blue) of MLL3 gene in 200
AML cases from TCGA data set (see, URLs). (B) Real time and western blot analysis show reduced MLL3
mRNA and protein levels in MLL3 siRNA transduced cells (MV4-11) compared to scrambled siRNA treated
cells. GAPDH was used as an internal control. MV4-11 cells transfected with either MLL3 siRNA or
scramble siRNA were examined using both short-term proliferation assay (MTT). (C) Methylcellulose colony
formation assay (D). Data in A-D represent mean ± s.d.; n = 3. ** P ≤ 0.01.
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Supplemental Figure 14. Copy number alterations in relapse FLT3-ITD AML samples. Copy number
changes were identified using SNP array data. Patients are grouped based on hierarchical clustering. Recurrent
focal loss of chromosomes 4, 5 and 7 is observed. Red and blue color represents amplification and deletion,
respectively.
Supplemental Figure 15. FAT4 gene is deleted and mutual exclusivity. (A) Integrative Genome Viewer
(IGV) segmentation map of 4q28.1 showing deletion (blue) of FAT4 gene in the TCGA data (200 AML
cases). (B) Individuals with either FAT1 or FAT4 gene mutation (mutual exclusivity).
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Supplemental Figure 16. Increased XPO1 (CRM1) expression in relapse samples; and inhibitor of XPO1
(KPT330) enhances doxorubicin cell killing of FLT3-ITD positive AML cells with. (A) RNA sequencing
analysis of four paired diagnosis and relapse samples show increased expression of XPO1 in relapse cases. (B)
Proliferation study of the combination of KPT330 with doxorubicin. Data represent means ± s.d.; n = 3. (C)
Heat plot shows growth inhibition of FLT3-ITD AML cells (MV4-11) by KPT300 in combination with
doxorubicin. (D) Combinational effect of KPT330 and doxorubicin on MV4-11 cells displayed as the
combination index (CI). CI defines the interaction between KPT-330 and doxorubicin as plotted against
fraction of cell viability. CI < 1, CI = 1 and CI > 1 represent synergism, additive, and antagonism of the two
compounds, respectively.
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Supplemental Table 1: Clinical parameters of 13 FLT3-ITD AML cases subjected to whole exome sequencing (Discovery Cohort)
MLL
Sample Age/ Cytogenetic Cytogenetic DX-BM CR- BM REL-BM Hb WBC PLT Dead/ Survival RFS Induction ITD ITD ITD
FAB PTD (DX
ID Sex (DX) (REL) Blast (%) Blast (%) Blast (%) (g/dl) (*109/L) (*109/L) Alive (Months) (Months) Chemotherapy (DX) (CR) (REL)
and REL
Cytarabine and
UPN001 17/M M1 46,XY 46 XY 98.0 3.9 90.1 7.0 11.7 41 Dead 100.1 61 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN002 61/M M4 46,XY 46 XY 53.4 3.2 59.6 8.4 19.6 31 Dead 8.4 2.5 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN003 56/M M2 46,XY 46 XY 58.8 0.0 43.8 8.3 2.5 64 Dead 53.7 22 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
46,XY,+14,der
UPN004 65/M M1 47,XY,+8 75.0 3.5 37.5 7.7 9.7 15 Alive 71.9 16 daunomycine Pos Neg Pos Neg
(14;18) (q10;q10)
(7+3)
Cytarabine and
46,XY,del(3)
UPN005 75/M M1 46,XY 88.0 4.0 79.5 10.5 31.7 73 Dead 7.3 3 daunomycine Pos Neg Pos Neg
(q21;q26)
(7+3)
Cytarabine and
UPN006 75/F M4 46,XX 46,XX 83.5 3.0 76.4 5.1 60.0 172 Dead 17.4 5 daunomycine Pos Neg Pos Neg
(7+3)
45,XX,der Cytarabine and
UPN007 53/F M4 (7;13) 46,XX 76.5 4.7 NA 8.6 2.4 39 Dead 19.2 5 daunomycine Pos Neg Pos Neg
(p10;q10) (7+3)
Cytarabine and
UPN008 46/F M1 46,XX 46,XX 80.5 4.5 NA 8.9 NA NA Dead 8.7 1 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN009 66/M M4 46,XY 46,XY 78.0 2.0 68.0 7.8 190.0 190 Dead 5.9 2 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
46,XX,del(3)
UPN010 69/F M2 46,XX 67.0 3.5 42.5 8.9 27.0 30 Dead 7.8 4 daunomycine Pos Neg Pos Neg
(q21;q26)
(7+3)
Cytarabine and
UPN011 83/M M2 46,XY 46,XY 76.0 0.0 30.0 10.9 20.0 63 Dead 4.0 2 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN012 57/F M1 46,XX 46,XX 81.4 2.2 NA 6.8 24.6 15 Alive 139 29 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN013 61/F M1 46,XX 46,XX 87.5 3.0 NA 13.8 70.0 143 Dead 37 16 daunomycine Pos Neg Pos Neg
(7+3)
1
FAB : French American and British classification scheme for leukemia
M : Male
F : Female
BM : Bone Marrow
Hb : Hemoglobin
WBC : White blood cell
PLT : Platelets
RFS : Relapse-free survival
DX : Diagnosis
CR : Complete remission
REL : Relapse
NA : No data available
Pos : Positive
Neg : Negative
2
Supplemental Table 2: Coverage analysis of whole exome sequencing of FLT3-ITD AML cases from
Discovery Cohort (13 trio consisting of DX/CR/REL samples)
Target
Mean Target Target Target
Sample ID Coverage Captured Region Bait Set
Coverage Coverage >2X Coverage >10X
>20X
UPN001 DX 135.352824 94.48 86.97 81.05 Whole exome SureSelect
UPN001 CR 111.308110 92.85 82.77 73.98 Whole exome SureSelect
UPN001 REL 130.495242 94.24 86.04 78.69 Whole exome SureSelect
1
Average 119.602128 95.32 88.34 79.48
DX : Diagnosis
REL : Relapse
2
Supplemental Table 3: Summary of somatic mutations in Discovery Cohort
Annotated Mutation Amino Acid Allele Status in Sample

Position RefSeq
Gene Type Change Change Disease ID
GJB5 Missense chr1:35223047 T39M C>T NM_005268 DX and REL UPN001
RP1L1 Missense chr8:10470739 G290D C>T NM_178857 DX and REL UPN001
BOC Missense chr3:112969659 P119S C>T NM_033254 DX UPN001
SLC34A1 Missense chr5:176824856 P497S C>T NM_003052 DX UPN001
EPHB1 Missense chr3:134670756 G223R G>C NM_004441 REL UPN001
COL4A6 Missense chrX:107412727 R1231Q C>T NM_001847 REL UPN001
TNRC6C Missense chr17:76046753 W537L G>T NM_001142640 REL UPN001
EFCAB5 Missense chr17:28409995 W1171C G>T NM_198529 REL UPN001
C12orf51 Missense chr12:112608143 V3882L C>A NM_001109662 REL UPN001
USP31 Missense chr16:23079996 P1144T G>T NM_020718 REL UPN001
PCDHB9 Unknown chr5:140568533 Unknown C>T Unknown REL UPN001
GRK7 Missense chr3:141497529 Q135K C>A NM_139209 REL UPN001
DIRC2 Missense chr3:122598139 G451R G>A NM_032839 REL UPN001
PRAM1 Missense chr19:8564178 E172K C>T NM_032152 REL UPN001
VCPIP1 Missense chr8:67546752 P1218Q G>T NM_025054 REL UPN001

Frameshift
*NPM1 chr5:170837545 W288fs */+TCTG NM_199185 REL UPN001
insertion
Frameshift
TCF7 chr5:133473764 Q37fs */+C NM_213648 REL UPN001
insertion
NBPF1 Unknown chr1:6902856 Unknown C>T Unknown DX UPN002
OAZ3 Unknown chr1:151743613 Unknown C>T Unknown DX UPN002
CNGA4 Missense chr11:6265317 R469H G>A NM_001037329 DX and REL UPN002
GYG1 Missense chr3:148711998 L26Q T>A NM_001184720 DX and REL UPN002
SLC7A8 Missense chr14:23612387 S179G T>C NM_012244 DX and REL UPN002
*DNMT3A Missense chr2:25457242 R882H C>T NM_175629 DX and REL UPN002
ARID2 Missense chr12:46244845 T980S C>G NM_152641 DX and REL UPN002
STK40 Missense chr1:36809750 K285N C>A NM_032017 DX and REL UPN002
IDH3B Stoploss chr20:2639090 X384Q A>G NM_174855 DX and REL UPN002
KIF26A Missense chr14:104639410 R506H G>A NM_015656 DX and REL UPN002
YLPM1 Missense chr14:75264700 E900D G>T NM_019589 DX and REL UPN002

Frameshift */+
*NPM1 chr5:170837545 W288fs NM_199185 DX and REL UPN002
insertion TCTG
POTEE Missense chr2:131976330 A119T G>A NM_001083538 DX UPN002
OAZ3 Unknown chr1:151743613 Unknown C>T Unknown DX UPN002
ANKRD24 Missense chr19:4219666 R1028W C>T NM_133475 REL UPN002
OR10G7 Missense chr11:123909483 V76L C>G NM_001004463 REL UPN002
*CEBPA Missense chr19:33793211 A30V C>T NM_004364 DX UPN002
ESRRA Missense chr11:64074838 E63K G>A NM_004451 REL UPN002
1
FRG1 Stopgain chr4:190878622 K168X A>T NM_004477 DX UPN003
Frameshift
*BCORL1 chrX:129148584 612 fs */- CT NM_021946 DX and REL UPN003
deletion
GPR156 Missense chr3:119886721 P531S G>A NM_001168271 DX and REL UPN003
GPBAR1 Missense chr2:219128429 D328Y G>T NM_001077191 REL UPN003
ASAH2B Missense chr10:52502716 T11M C>T NM_001079516 DX and REL UPN003
CDT1 Splice site chr16:88870951 exon2:c.229-2A A>G NM_030928 DX and REL UPN003
KCNG1 Missense chr20:49626637 T80M G>A NM_002237 DX and REL UPN003
*RUNX1 Splice site chr21:36206706 exon5 724+1G G>A NM_001754 DX and REL UPN003
CRHR1 Missense chr17:43893837 C44G T>G NM_001145146 DX and REL UPN003
*BCORL1 Missense chrX:129148585 S613P T>C NM_021946 DX and REL UPN003
WDR33 Missense chr2:128480877 R414Q C>T NM_018383 DX and REL UPN003
KCNH6 Missense chr17:61613189 A421T G>A NM_030779 REL UPN003
PAM16 Missense chr16:4393223 R25W G>A NM_016069 REL UPN003
SCAMP5 Unknown chr15:73097785 Unknown C>T Unknown REL UPN003
MUC17 Missense chr7:100677974 T1093A A>G NM_001040105 REL UPN003
*DOT1L Missense chr19:2185892 M55T T>C NM_032482 DX and REL UPN004
*SMC1A Missense chrX:53432580 R586W G>A NM_006306 DX and REL UPN004
*RUNX1 Missense chr21:36252877 R135K C>T NM_001001890 DX and REL UPN004
TTN Missense chr2:179611997 E5044K C>T NM_133379 DX and REL UPN004

Frameshift
WT1 chr11:32417941 V354fs */+A NM_000378 DX and REL UPN004
insertion
Frameshift
PFAS chr17:8167584 A616fs */+ C NM_012393 DX UPN004
insertion
RNF213 Missense chr17:78355494 L4649F C>T NM_001256071 DX UPN004
ACTN2 Missense chr1:236906244 E386K G>A NM_001103 DX UPN004

Frameshift
DDI2 chr1:15970053 E266fs */+ C NM_032341 DX UPN004
insertion
PIK3R6 Unknown chr17:8725217 Unknown A>G Unknown DX UPN004
LRP2 Stopgain chr2:170038679 Y3332X A>C NM_004525 DX UPN004
ZNF844 Stopgain chr19:12187601 E556X G>T NM_001136501 REL UPN004
USP36 Missense chr17:76798564 K955T T>G NM_025090 REL UPN004
CORO1A Missense chr16:30199318 Y306C A>G NM_007074 DX and REL UPN005
ANKMY1 Missense chr2:241452239 D479N C>T NM_017844 DX and REL UPN005
RTL1 Missense chr14:101349079 E683K C>T NM_001134888 DX and REL UPN005
APOB Missense chr2:21233084 R2219H C>T NM_000384 DX and REL UPN005
SUV39H2 Splice site chr10:14941685 456+1G>A G>A NM_001193426 DX and REL UPN005
*DNMT3A Missense chr2:25457243 R882C G>A NM_175629 DX and REL UPN005
PEG10 Unknown chr7:94294152 Unknown G>A Unknown DX and REL UPN005
CELSR2 Missense chr1:109801179 L1146I C>A NM_001408 DX UPN005
CCDC33 Missense chr15:74628374 A750T G>A NM_025055 DX UPN005
*IDH1 Missense chr2:209113113 R132C G>A NM_005896 DX UPN005
2
Frameshift
LSP1 chr11:1902785 E43fs */+ C NM_001013253 DX UPN005
insertion
CDH18 Missense chr5:19591228 M313V T>C NM_001167667 DX UPN005
Frameshift
PNPLA7 chr9:140356678 L1175fs */+ C NM_152286 REL UPN005
insertion
PXK Missense chr3:58381469 G269R G>A NM_017771 REL UPN005
Frameshift
*DNMT3A chr2:25457192 R899fs */- G NM_175629 DX and REL UPN006
deletion
*DNMT3A Missense chr2:25469543 W409R A>G NM_175629 DX and REL UPN006
EML5 Missense chr14:89087241 H1744Q A>T NM_183387 DX and REL UPN006
INSC Missense chr11:15198680 K189N A>C NM_001031853 DX and REL UPN006
STEAP4 Missense chr7:87913436 R50Q C>T NM_001205316 DX and REL UPN006
PCSK2 Missense chr20:17417442 A232T G>A NM_001201529 DX UPN006
NECAB2 Missense chr16:84028280 R261T G>C NM_019065 DX UPN006
FBXO34 Missense chr14:55819210 K701T A>C NM_017943 DX UPN006
B3GAT3 Missense chr11:62388030 A66S C>A NM_012200 DX UPN006
OR5I1 Missense chr11:55703354 V175I C>T NM_006637 DX UPN006
INO80 Missense chr15:41276399 S1358N C>T NM_017553 DX UPN006
FLNC Missense chr7:128478828 R461H G>A NM_001127487 DX UPN006
SRGAP2 Unknown chr1:206628260 Unknown T>C Unknown REL UPN006
MN1 Stopgain chr22:28192754 E1260X C>A NM_002430 DX UPN007
SUSD2 Missense chr22:24584219 I790V A>G NM_019601 DX and REL UPN007
SEMA4G Missense chr10:102732670 M1V A>G NM_001203244 DX UPN007
ZFHX4 Missense chr8:77767850 R2898H G>A NM_024721 DX UPN007

Frameshift
DNAJB6 chr7:157151323 I19fs */+ A NM_005494 DX UPN007
insertion
PROKR1 Missense chr2:68873414 A154V C>T NM_138964 DX UPN007
CNN3 Missense chr1:95363318 D324N C>T NM_001839 DX UPN007
SPDYE6 Missense chr7:101989035 R280C G>A NM_001146210 DX UPN007
BMP7 Missense chr20:55746095 V406I C>T NM_001719 DX UPN007
ALAD Missense chr9:116155790 R17Q C>T NM_000031 DX UPN007
GPR139 Missense chr16:20043317 D268N C>T NM_001002911 DX UPN007
TTK Missense chr6:80736201 S454N G>A NM_001166691 DX UPN007
POLD1 Stopgain chr19:50921124 G1082X C>T NM_002691 DX UPN007
BAIAP2L2 Missense chr22:38483155 N412T T>G NM_025045 DX UPN007
FRG1 Splice site chr4:190874281 exon4:c.317+1G G>T NM_004477 DX UPN007
CELSR3 Missense chr3:48698497 P524L G>A NM_001407 DX and REL UPN008
NFIX Stopgain chr19:13135982 E59X G>T NM_002501 DX UPN008
MUC16 Missense chr19:8976274 S14185N C>T NM_024690 DX UPN008
ABR Splice site chr17:959273 1450+2T>G A>C NM_001092 DX UPN008
ZBTB7A Missense chr19:4054027 R402C G>A NM_015898 DX UPN008
3
NSMCE1 Missense chr16:27238083 D186E G>C NM_145080 DX UPN008
Frameshift
NSMCE1 chr16:27238082 A187fs */+ T NM_145080 DX UPN008
insertion
C22orf15 Missense chr22:24107032 K126N G>T NM_182520 DX UPN008
TRPM6 Missense chr9:77454976 T165A T>C NM_001177310 DX UPN008
CYP4A11 Missense chr1:47400177 K282M T>A NM_000778 DX UPN008
SPPL2C Missense chr17:43922621 G117R G>A NM_175882 DX UPN008
LRP2 Missense chr2:170068562 V2066F C>A NM_004525 DX UPN008
TSPAN10 Unknown chr17:79614938 Unknown A>G Unknown DX UPN008
ASB2 Missense chr14:94404103 S523L G>A NM_016150 DX UPN008
DCHS2 Missense chr4:155156962 L2493F G>A NM_017639 DX UPN008
BAZ2B Stopgain chr2:160243034 R1101X G>A NM_013450 DX UPN008

Frameshift
*WT1 chr11:32417941 V354fs */+ A NM_000378 DX UPN008
insertion
CADM2 Missense chr3:85008777 A7S G>T NM_001167674 DX UPN008
KCNH7 Missense chr2:163241333 D943N C>T NM_033272 DX UPN008
MUC4 Missense chr3:195511918 P2178H G>T NM_018406 REL UPN008
SETD1B Missense chr12:122260824 V1404I G>A NM_015048 DX and REL UPN009
MYOM3 Missense chr1:24433697 S90P A>G NM_152372 DX and REL UPN009
CCDC89 Missense chr11:85397124 P17L G>A NM_152723 DX and REL UPN009
TMEM179 Missense chr14:105063347 E141D C>A NM_207379 DX and REL UPN009
XKR4 Missense chr8:56436340 V503I G>A NM_052898 DX and REL UPN009
SEC22B Unknown chr1:145115815 Unknown A>C Unknown DX and REL UPN009
FBXO38 Missense chr5:147819992 L984P T>C NM_030793 DX and REL UPN009

Frameshift
CTCF chr6:67645228 Q165fs */+ C NM_006565 DX and REL UPN009
deletion
Frameshift */+
insertion TGCA
Frameshift
PYGO2 chr1:145115815 Q150fs */+ C NM_138300 DX UPN009
insertion
EXOC6 Missense chr10:94715416 N568S A>G NM_001013848 DX UPN009
*FLT3 Missense chr13:28602340 N676K G>T NM_004119 REL UPN009
TRIB1 Missense chr8:126448540 R316C C>T NM_025195 REL UPN009
DCTN2 Unknown chr12:57927805 Unknown Unknown Unknown REL UPN009
NSDHL Missense chrX:152037380 R281H G>A NM_015922 DX and REL UPN010
FLNA Missense chrX:153586879 Y1511F T>A NM_001110556 DX and REL UPN010

Frameshift
*DNMT3A chr2:25470993 P257fs */-G NM_175629 DX and REL UPN010
deletion
ODZ2 Missense chr5:167674669 R2233H G>A NM_001122679 DX and REL UPN010
B3GNT3 Missense chr19:17922435 N208S A>G NM_014256 DX and REL UPN010
RBM42 Missense chr19:36125182 K348E A>G NM_024321 DX and REL UPN010
IGF2BP3 Missense chr7:23353265 G468E C>T NM_006547 DX and REL UPN010
DMD Missense chrX:31187647 R408H C>T NM_004017 DX and REL UPN010
4
Frameshift */+
insertion CATG
EVPLL Missense chr17:18286382 H185Q C>A NM_001145127 DX UPN010
Frameshift
CLCA4 chr1:87025674 F73fs */+A NM_012128 REL UPN010
insertion
COL4A5 Missense chrX:107908806 P1148L C>T NM_000495 DX UPN010
BAHCC1 Unknown chr17:79423461 Unknown A>C Unknown REL UPN010
HYAL1 Missense chr3:50339502 H296Y G>A NM_007312 DX and REL UPN011
RYR3 Missense chr15:33928610 G1139W G>T NM_001036 DX and REL UPN011

Frameshift */+
insertion TGCA
ARID5A Missense chr2:97217073 V270M G>A NM_212481:c.G808A:p. DX UPN011
SRCAP Missense chr16:30744990 G2122V G>T NM_006662 DX UPN011
CCDC84 Missense chr11:118886056 V282A T>C NM_198489 DX UPN011
ACTG2 Missense chr2:74143816 T261I C>T NM_001199893 DX UPN011
DAPK1 Missense chr9:90273025 V636F G>T NM_004938 DX UPN011
ZNF80 Missense chr3:113955333 T197P T>G NM_007136 DX UPN011
METTL13 Missense chr1:171753364 R127K G>A NM_014955 DX UPN011
*TET2 Missense chr4:106190816 G1365V G>T NM_001127208 DX UPN011
NPHS1 Splice site chr19:36339537 1170+2T>A A>T NM_004646 DX UPN011

Frameshift
ADAM8 chr8:135085034 E389fs */+G NM_001164490 DX UPN011
insertion
JAG2 Missense chr14:105614483 P702T G>T NM_145159 DX UPN011
LAMA2 Stopgain chr6:129634114 R1095X C>T NM_000426 REL UPN011
COG7 Missense chr16:23446015 V210E A>T NM_153603 DX UPN011
GOLGA6L1 Missense chr15:22743174 R520K G>A NM_001001413 REL UPN011
EPPK1 Missense chr8:144940706 R2239H C>T NM_031308 REL UPN011

Frameshift
GP1BB chr22:19711808 W148fs */+G NM_000407 REL UPN011
insertion
Frameshift */+
insertion CCTG
ABCC11 Missense chr16:48201486 G1326A C>G NM_032583 DX and REL UPN012
*FLT3 Missense chr13:28608260 Y599F T>A NM_004119 DX and REL UPN012
*FLT3 Missense chr13:28608255 L601F G>T NM_004119 DX and REL UPN012
PCDHB16 Missense chr5:140563377 A415P G>C NM_020957 DX and REL UPN012
SMC1A Missense chrX:53432580 R586W G>A NM_006306 DX and REL UPN012
FAT4 Missense chr4:126336833 R2239G A>G NM_024582 DX UPN012
CYP4F2 Missense chr19:16000328 R275C G>A NM_001082 DX and REL UPN012

Frameshift
ARID1A chr1:27101611 Q1631fs */+C NM_006015 REL UPN012
insertion
ASXL1 Missense chr20:31022796 A761T G>A NM_015338 REL UPN012
JHDM1D Missense chr7:139826469 G286S C>T NM_030647 REL UPN012

Frameshift
SF3A1 chr22:30733827 P536fs */+G NM_001005409 REL UPN012
insertion
ACTRT2 Missense chr1:2938413 K55X A>T NM_080431 DX and REL UPN012
ASH1L Missense chr1:155385700 P1948R G>C NM_018489 DX and REL UPN013
5
Frameshift
*BCORL1 chrX:129190011 S1679fs */-C NM_021946 DX and REL UPN013
deletion
GATA4 Stopgain chr8:11615935 W427X G>A NM_002052 DX and REL UPN013
NSD1 Missense chr5:176721721 R2451K G>A NM_022455 DX and REL UPN013
PTGES3L Missense chr17:41131448 D51N C>T NM_001142654 DX and REL UPN013
TTN Missense chr2:179414557 N28063S T>C NM_133378 DX and REL UPN013
SEMA4G Missense chr10:102740640 R510H G>A NM_017893 DX and REL UPN013
SRCAP Missense chr16:30722106 R389H G>A NM_006662 DX and REL UPN013
EPHA2 Missense chr1:16482385 G15C C>A NM_004431 DX and REL UPN013
MYOM3 Missense chr1:24433589 R126W G>A NM_152372 DX and REL UPN013
DNAH6 Missense chr2:84928399 S2666Y C>A NM_001370 DX and REL UPN013
TET1 Missense chr10:70405836 N1117S A>G NM_030625 DX UPN013

Frameshift
JARID2 chr6:15501214 L674fs */+A NM_004973 DX UPN013
insertion
KLHL6 Missense chr3:183217586 M313I C>A NM_130446 DX UPN013
FANCI Missense chr15:89836265 I754M A>G NM_001113378 REL UPN013
FAT1 Missense chr4:187628125 D953N C>T NM_005245 DX UPN013
FAT1 Missense chr4:187541208 A2178T C>T NM_005245 REL UPN013
ZNF837 Missense chr19:58879118 R528C G>A NM_138466 REL UPN013
Color types Number
Confirmed as a valid mutation by Sanger sequencing 165
Confirmed as a false mutation by Sanger sequencing 12
Germline SNP 2
Total Sanger events 179
True positive rate 92.17
Difficulty in designing primers; PCR failure; poor signal of Sanger sequencing 23
* : Indicates that mutations of these genes have been previously reported in AML
DX : Diagnosis
REL : Relapse
RefSeq : The Reference Sequence
6
Supplemental Table 4: List of primers used for the validation of the somatic mutations detected in whole exome analysis
(Discovery Cohort)
Gene Forward Primer Reverse Primer Sample ID
GJBS 5'-CCCAGCTCCTCTAGTGATGG-3' 5'-GTAGAGGCGCCCACTGTTCT-3' UPN001

RP1L 5'-GGGAGGCTCGTGACTGTTT-3' 5'-GTCTACCTCCCCCAGAACG-3' UPN001
BOC 5'-GTGGAACCTCCAAGGATGAA-3' 5'-AGGTTTTCCAAGGGCACAAG-3' UPN001
SLC34A1 5'-GCACATCACCCCTAAGTGGA-3' 5'-ACTCCGACTCTGCAGGACAT-3' UPN001
EPHB1 5'-ATTGCCACCAAGAAGTCAGC-3' 5'-AGGCCTAGCACTAGGAATGG-3' UPN001
COL4A6 5'-CTCCAATTTGAGGTCCCTTG-3' 5'-GGCTTGAGGTTAGTAAGGAGGA-3' UPN001
TNRC6C 5'-ATAGACCAAGGGCACATCCA-3' 5'-TGTCCAAGGACAGACGATGA-3' UPN001
EFCAB5 5'-GCTGGGATTACAGGCATGAG-3' 5'-TTTTGGTCACGTGAGTCAGTT-3' UPN001
C12ORF51 5'-TCTCCTGGGAATGACAAAGG-3' 5'-GAGCCAAGCTCCTTCTGCTA-3' UPN001
USP31 5'-AAGCCAGGAGTCATCCCTTT-3' 5'-AGCCCCTTGTCCTCAGACTT-3' UPN001
GRK7 5'-AAGCTGTCCCTGAACTTCCA-3' 5'-CCCCTTTCATGCTCTATGCT-3' UPN001
DIRC2 5'-TCAAGGTGGATTAAATTGATGAAA-3' 5'-TCCCCTATTATCACTCAAGTCTCC-3' UPN001
PRAM1 5'-GTCACTGACCTCCCCAAGAA-3' 5'-CTGGACTGAGGCTTCCAGAG-3' UPN001
VCPIP1 5'-TCCTCAAAGGAAAACAGAAGTTG-3' 5'-GGCCAATCACACACTTGCTA-3' UPN001
NPM1 5'-TTAACTCTCTGGTGGGTAGAATGAA-3' 5'-CATTTTGGACAACACATTCTTGGC-3' UPN001
TCF7 5'-GGCCAATCACACACTTGCTA-3' 5'-TTCTTTGGGCCCTTAGTTTG-3' UPN001
CNGA4 5'-GCCTGGTCCCCAGTATGTTA-3' 5'-CTGCCCTCTTCATCTTTGGA-3' UPN002
GYG1 5'-GTTTTCTCCCCAAAGGGCTA-3' 5'-GGATGGAGTGAAGGAAGACG-3' UPN002
SLC7A8 5'-ATTTTACCCCCAAAGCCAGT-3' 5'-GGGGAAAATGGTTTCTCCTT-3' UPN002
DNMT3A 5'-GCACATGGTTGGTGATCTGA-3' 5'-ATGATGTCCAACCCTTTTCG-3' UPN002
ARID2 5'-GGTTTCAGAACATTGCACCA-3' 5'-TTTCGGAAGCAGAAGCTGTT-3' UPN002
STK40 5'-CTGAGTGAGCCCTCTCCTTG-3' 5'-TTCTCAGAAACCCGTCCATC-3' UPN002
IDH3B 5'-TCATCAAGTCTGTCATCGGTCA-3' 5'-AATGCAAATGGACATTCTCTGG-3' UPN002
KIF26A 5'-CAGCTTCTGATGGGAAAGGA-3' 5'-AGCTCGCTTTGGTTCTGGAG-3' UPN002
YLPM1 5'-CCCCAGTACATCCCTAAGTCC-3' 5'-TACTGCTGTTCCTCCCTCCA-3' UPN002
POTEE 5'-TAGACGCGATCTGTTGGCTA-3' 5'-GGAGGGTATGTCCCCATCAT-3' UPN002
ANKRD24 5'-CGAGGTGGGAGGATCACTTA-3' 5'-GAACAGCAGCCTGTGTGTGT-3' UPN002
OR10G7 5'-TGCTATCTCCCTTCCTTTCTG-3' 5'-CGGTGCGTCACAGAAGTAGT-3' UPN002
FRG1 5'-TGATAGGTGATTTTGGTTTCTTTTC-3' 5'-AATGAGTCTATTTGAAACACCACA-3' UPN003
BCORL1 5'-GAGGGGAGAGGTCCCATACT-3' 5'-CCCTAACCACCAGCATTTGA-3' UPN003
GPR156 5'-ACTTCAGGGCCCTCCTCATA-3' 5'-GTGCAGAGCCAGGAAAGTGT-3' UPN003
GPBAR1 5'-TACCTGGAGGCAGGCAAG-3' 5'-TGTTGAGCCATTTCCTCCTG-3' UPN003
ASAH2B 5'-GCAGAGAGAACCCACTGCTT-3' 5'-CCCTAATTTCACATGATGGTTG-3' UPN003
CDT1 5'-GAAGACGAGAGCAAGGCTGT-3' 5'-GACTCTTGCCCCCAGCTC-3' UPN003
KCNG1 5'-CCCTCTTACCGGGAGACAAT-3' 5'-CGCGAACTCCTCAATCTTCT-3' UPN003
RUNX1 5'-TGATCTCTTCCCTCCCTCCT-3' 5'-ACCCAGAGCTCCTCGTATCC-3' UPN003
CRHR1 5'-TCTTCCTCCTCTGCTCTGGA-3' 5'-AATACAGCGCTGACCTTTGG-3' UPN003
WDR33 5'-ACAGGGTTTCGCTATGTTGC-3' 5'-CTGCCAGGCTATTAGGTTCG-3' UPN003
KCNH6 5'-CCGGAGGAAGTTCTCCATAAA-3' 5'-CATGACGCAGATGGAGAAGA-3' UPN003
PAM16 5'-GGGAGCTCGGTCAAGTTCA-3' 5'-GTATCCAGATGGGCCAGTGT-3' UPN003
SCAMP5 5'-CTAGCCTATGGGGCCTGAGT-3' 5'-CTTCCCAAAGCCACCTTCTC-3' UPN003
DOT1L 5'-GAGTTTGAGACCAGCCTTGC-3' 5'-CTGACTCCACACATGCCAAT-3' UPN004
SMC1A 5'-CCAGCCCACACAAAAGAAGT-3' 5'-CCTTGGCCTTCAGGTCACT-3' UPN004
RUNX1 5'-GGTTGGTGCAAATGTAACTGC-3' 5'-CGTTGCATGTTCCAAATCAG-3' UPN004
TTN 5'-GGTCGTCCACTTTCTCCTGA-3' 5'-CCGTCTTCCCTTTCTATTTTTG-3' UPN004
1
PFAS 5'-GAGACCGGAGAGTGAGTTGG-3' 5'-GCAGCCTCAGAACCCTCTC-3' UPN004
RNF213 5'-GCTTGCTAGCCTTCCTTTCA-3' 5'-CGAATACCGCAGGTCTTAGC-3' UPN004
ACTN2 5'-CAGCCCTCTACATCTTTGTCC-3' 5'-GTGCCATTTTGGGAAGAAAT-3' UPN004
DDI2 5'-TCATTTAATTATTGCATCGCTACAG-3' 5'-AACTTTGACGGAACACAGGA-3' UPN004
PIK3R6 5'-GTGGCTCATGCCTGTAATCC-3' 5'-CAGGAGGCCAACTGCAAC-3' UPN004
FAM83B 5'-TGTGTCATTTTGAGCTTCTGG-3' 5'-TGTGCTCTTGGTGGATGAAA-3' UPN004
WT1 5'-GATTGGGAGCTCTTCCAGGT-3' 5'-CCCCACAGCCTCTTTACAAC-3' UPN004
USP36 5'-GTCAACAGGCTTGGCTCAGT-3' 5'-CCAGGACACCAGCCATTC-3' UPN004
CORO1A 5'-CTACAGAAGCACCTGGAGGA-3' 5'-ATCACCTTTCGAGGCACTGT-3' UPN005
JMJD5 5'-AGGCTGCAGTGAGCTGAGAT-3' 5'-GTGAGGGATCTTGGGAGGAG-3' UPN005
ANKMY1 5'-GTCTTGCTCAGCGTGTTGG-3' 5'-CCCTGTGGACTGCCTGAATA-3' UPN005
RTL1 5'-CAGTGATCACAGCGAGACCT-3' 5'-GAGCAGTAGACGTTGTGATGG-3' UPN005
APOB 5'-CAAGTTTCACATGCCAAGGA-3' 5'-TCCCAATTGATCTAAAAGCACTC-3' UPN005
SUV39H2 5'-AGATGAATGCTTCTTTGGTTCA-3' 5'-TTTGGTAGGCACCTCAATGTT-3' UPN005
PEG10 5'-CTTGCAAGTGATGCTCCAGA-3' 5'-AATAGAGTGGCGGTTGTGGT-3' UPN005
CCDC33 5'-TGGATACTCTGAGGCCTGGT-3' 5'-GCCCAGAGTGAGGTCTAGGA-3' UPN005
IDH1 5'-AACGACCAAGTCACCAAGGA-3' 5'-TGTGTTGAGATGGACGCCTA-3' UPN005
MUC6 5'-TGATCTCTCAGGACGAGGTG-3' 5'-GCTCCCGTCCTTCCTCTAAC-3' UPN005
CDH18 5'-AAAAGTCCATTTGATGCATTACC-3' 5'-CATTGGGACATTTCAGGAAGA-3' UPN005
PXK 5'-TCAACAATGCAGACTGTCGT-3' 5'-TTGAGGCAAAATATGACAATGC-3' UPN005
DNMT3A 5'-AGGTCACAGAAACCAGCACA-3' 5'-GAGGAAGGGAAGGGAGCTT-3' UPN006
DNMT3A 5'-AGGCAGCGACTCCTTTCTCT-3' 5'-CCCCACCCTCCTTACAGTG-3' UPN006
EML5 5'-TGGGGACTAGCAACACATCC-3' 5'-TGGTGCCCAGTGGGTTTAT-3' UPN006
INSC 5'-AGCTCAAATGCCCCTTCTTT-3' 5'-CCTTTTCCTTGTTACCAACTCC-3' UPN006
STEAP4 5'-TGACAATTTTTACTCCTTCCAAA-3' 5'-GACTTGCATCCAGTGCTCCT-3' UPN006
PCSK2 5'-GCTTCAGCAAAGGTGGTCTC-3' 5'-CTTCCTTCCCACTGTCGTTT-3' UPN006
NECAB2 5'-GCACAGACCTGGTGTGGAA-3' 5'-CTGACCACCCTCTGCTCCT-3' UPN006
FBXO34 5'-GGCCCTTAAATGTACCTGCT-3' 5'-TCACAGCCAACTGAATCCAA-3' UPN006
B3GAT3 5'-GAGAGGGTCTGCTGCTTGAG-3' 5'-CTCCTGACCTCAGGTGATCC-3' UPN006
OR5I1 5'-GGTGTGCCCTGCAGTTTTAT-3' 5'-GTAGCTGGGCCGTGAGTAAA-3' UPN006
INO80 5'-TCCCTTCTTCCTTATGCAGTTT-3' 5'-CATGTCACTGCTGCTCTCGT-3' UPN006
FLNC 5'-GCAGTCCCTTTGAGGTGAAC-3' 5'-AGAACAAGTGGGGCAGTCAC-3' UPN006
SRGAP2 5'-GAATCCAGGTGTCCCCTAGC-3' 5'-AGCAATGACCACATGCAAAC-3' UPN006
MN1 5'-AGATCCACCCCCTGGAGAT-3' 5'-GGGCGGGGTACTCTACAAGA-3' UPN007
SUSD2 5'-AGGCCTGTTGTCTGCACTCT-3' 5'-CTTGGGTGTTGGAACACTGG-3' UPN007
SEMA4G 5'-AAACGGACCTCAGAAAACCA-3' 5'-CTCTTTGTGAGCCCCATCTC-3' UPN007
MUC12 5'-CAGATCACCGGACACAACAC-3' 5'-TGCTGTGGACTGTTGTCGAT-3' UPN007
ZFHX4 5'-CTGCTGAGGCTGGGTATGAT-3' 5'-TGATCATGAAAGGCTTCCCTA-3' UPN007
DNAJB6 5'-CCCCGTCTCTAGAAGGGTCT-3' 5'-GGCACTCAAGAATGCCAAGT-3' UPN007
PROKR1 5'-GTGACCAATTCCAGGACGTT-3' 5'-CATCCATAGCAAGGGAGATCA-3' UPN007
CNN3 5'-AGCACCAGGTACCAGAAGAGA-3' 5'-TTTGCATAAAAGAACTGGCTGT-3' UPN007
SPDYE1 5'-CTTTGGCCTCCCAGTGTG-3' 5'-TCTGAGAGCGGTTCTTCCTG-3' UPN007
BMP7 5'-TGCTTTGGTTCCCTTGTCTG-3' 5'-CTGCTAGGTTTTGCCTGCAC-3' UPN007
ALAD 5'-TGAACAAGAAGCAGTCACATAAAA-3' 5'-ACTGCAAGAGCTCAGGATGG-3' UPN007
GPR139 5'-CCAGCATCCCCTATTACTGG-3' 5'-CTTCCCATCTGGAAATGGAT-3' UPN007
TTK 5'-TGCCTATCTCTTTTAGTATGCCTTT-3' 5'-CATGATTATCAAGACGTTTTACAT-3' UPN007
POLD1 5'-ACGCCAGGTGACAGGTGATA-3' 5'-GCTTTTGCTGAGGATGAGG-3' UPN007
BAIAP2L2 5'-AGGGAAAAGGATAGCGTGTG-3' 5'-CCAGCTACTCAGGAGGCTCA-3' UPN007
FRG1 5'-CAAGGCCATCCCTTGAATAA-3' 5'-CCTGTTTGCCTTTGGACTTC-3' UPN007
2
NFIX 5'-CGCTGCTTTTCTCCTTCTCT-3' 5'-TCCCCTTAAACAAAATCACCA-3' UPN008
CELSR3 5'-GTTTTTGAGCAAGCGCAGTA-3' 5'-GCGCAAGGCATACTCTCTCT-3' UPN008
MUC16 5'-CTTGGGTTGCCAACTGATCT-3' 5'-CCTGAGGTCAGGAGTTCGAG-3' UPN008
ABR 5'-CGGGCAGCTCACATATAAGG-3' 5'-GTAGAGACGGGGTTTCACCA-3' UPN008
ZBTB7A 5'-ACTCTCCGGGCTTCCTGT-3' 5'-GTGTGTGCGTCTGTGTGTGT-3' UPN008
NSMCE1 5'-CTTTGCCTCAGTTTCCCTTG-3' 5'-AGATGATGCAGGTTGCACAG-3' UPN008
DNMT3A 5'-AGGTCACAGAAACCAGCACA-3' 5'-GGTATTTCCGCCTCTGTGG-3' UPN008
C22orf15 5'-GAGAGGCAGAGTAGGGACCA-3' 5'-CCCTATGCCACATCTGTGAA-3' UPN008
CYP4A11 5'-TGCCTTGGTTTATCAATGTCC-3' 5'-CCAGGAGGCTGTGGATCTC-3' UPN008
SPPL2C 5'-TTCCTCCTCCTCATCAGCAC-3' 5'-CAGCCAGGATGAAGATGACC-3' UPN008
LRP2 5'-TCCGATGTTGTTTTTAGATTGC-3' 5'-AAAATCTGGGGACGAGGTCT-3' UPN008
ASB2 5'-TGAGGCTCAGAGGTGATGTG-3' 5'-TGGAAAGATGTAGCGGAGAGA-3' UPN008
DCHS2 5'-CACAGTCCAAGCCAAAGACA-3' 5'-CACAGGGACCACCTCGTTAC-3' UPN008
BAZ2B 5'-CCATGGGGGTTACAGAAATG-3' 5'-CTTTGTGAGACATTATGTGGAAAA-3' UPN008
WT1 5'-CTCTGGGACAGCCATTTCAT-3' 5'-AACCTGGGTCCTTAGCAGTG-3' UPN008
KCNH7 5'-CCCAAGTAGCTACATTTTCAATAGG-3' 5'-TCTGAAATCTGAACGCTTCG-3' UPN008
SETD1B 5'-TGAGACCACAGATGCCTCAC-3' 5'-AGCAGGAGTTCCCTTCCAA-3' UPN009
MYOM3 5'-CTTGGCTGTCGTGAAAACAA-3' 5'-AGGCTCAGTAACAGGGATGC-3' UPN009
CCDC89 5'-GCCAGACACTGAGACAGTTGAC-3' 5'-CTCAGCCTCCTGCATCATCT-3' UPN009
TMEM179 5'-CAGGAGATGGCGTCCAGT-3' 5'-ATCTAAGCCCTGGTGGCAAG-3' UPN009
XKR4 5'-TGGGATCTTCATCGTCCTTC-3' 5'-TCCCATGCTGGGTACCTATT-3' UPN009
SEC22B 5'-AACCTGCACTTCTAACCACCTT-3' 5'-TATATGGCTTGGCACCCAGA-3' UPN009
FBXO38 5'-TGTTGCATTAAGCACCTTTTG-3' 5'-AAGCCCAAACCAGTTTGACTAA-3' UPN009
CTCF 5'-GTGATGATGGAACAGCTGGA-3' 5'-GTTGGCTTTGGAGGCTTCAT-3' UPN009
PYGO2 5'-CATGACCCTCTTTCCCTCAA-3' 5'-GGACCAGGAAAGGGACTTGT-3' UPN009
NSDHL 5'-AAATGTGCCTTTCCAAGTGC-3' 5'-CACGGTCCTCTCCATAGCAT-3' UPN010
FLNA 5'-GAGCAGATGTGGCAGGAAG-3' 5'-GCTCACCGTGATCTGGACA-3' UPN010
DNMT3A 5'-TTTCCATTTTTCACGGCAAG-3' 5'-TCCATCTGAATGAGGCAAGA-3' UPN010
ODZ2 5'-TCAGCAAACACTTCGACACC-3' 5'-TGAGGTAATCTCCGAGTTGG-3' UPN010
B3GNT3 5'-CAGCATTTCTTGGTGCTTCA-3' 5'-TAGAAGCAGGGGTCAAAGGA-3' UPN010
RBM42 5'-TGAGGTAAGCAGGGAGCCTA-3' 5'-CTGGGCGACAGAGTGGTACT-3' UPN010
IGF2BP3 5'-TTCACCAAGCATTCAGTTGC-3' 5'-TAGCTGGCACCAACACTCAG-3' UPN010
DMD 5'-TCCGTGTAAATAGGCCAATCA-3' 5'-TGCATCCATTGTCTTCTTTCAG-3' UPN010
EVPLL 5'-GGAGCCAATACCGAGACCTA-3' 5'-CTCCAGCTGGTTTACGCTCT-3' UPN010
CLCA4 5'-TCACGTAAATGACTCCAAAGTCC-3' 5'-GAAGGTCAGGGGTGAAGTGA-3' UPN010
BAHCC1 5'-CACAACCCACAGCCACTG-3' 5'-GACCTGTCACACCCCACAA-3' UPN010
HYAL1 5'-TCTATGGCTTCCCTGACTGC-3' 5'-CGCCTGTATCCCAGCTACTC-3' UPN011
RYR3 5'-AGACCACTCCTCCTTGTTGG-3' 5'-ATGGGATCGTCTTCACCATT-3' UPN011
ARID5A 5'-CACCTCTTGCTCCTCAGGTC-3' 5'-TTCACTGACAGCCCCTCTTT-3' UPN011
SRCAP 5'-ACCATGGCCATCTCTACCTG-3' 5'-TCCCCCAACATTCTCTTCTG-3' UPN011
CCDC84 5'-AAATGGTTTCCTTGCCTGTG-3' 5'-TTGCTGCAGCTTCAGTTTTG-3' UPN011
ACTG2 5'-TGACAAGGAAGGGGGTATGA-3' 5'-GGCTCCAGTCCAGGATGATA-3' UPN011
DAPK1 5'-GATGGCTCCTTCCTCCTTTC-3' 5'-AGAGCAGGGTGTGGTGAGAT-3' UPN011
ZNF80 5'-CTGGGGTGAAGCCTTATGAA-3' 5'-GCAAAAGCAGAGTGGTAGCC-3' UPN011
METTL13 5'-TGCTGTCCTGACAGATGAGG-3' 5'-TCGCACAAGTCCAGAGACAC-3' UPN011
TET2 5'-TGTCATTCCATTTTGTTTCTGG-3' 5'-ACAGCCATGTGGAACTGTGA-3' UPN011
NPHS1 5'-TCCAGCCCAGCTTAGTGTCT-3' 5'-TGTCAGGTTGGACATGGAGA-3' UPN011
JAG2 5'-GCTGCTACGACCTGGTCAAT-3' 5'-TGGCTGTAAGGAGAGGAGGA-3' UPN011
LAMA2 5'-TCCATAAATGCAGGAAGCAA-3' 5'-TGGCAATGAGATTTTGTCCA-3' UPN011
3
COG7 5'-TGTGAGGAACAGTGGGGAAT-3' 5'-TAGACCAAGAGCACCCCCTA-3' UPN011
ABCB1 5'-TCTCATGAAGGTGAGTTTTCAGA-3' 5'-TGGTTATAAACACATTCTTAGAGC-3' UPN012
ABCC11 5'-AAATGGAGACCTGGAGAGGAA-3' 5'-GGCCGATCAAATTCTACCAC-3' UPN012
FLT3 5'-CATTGTCGTTTTAACCCTGCT-3' 5'-TTGCGTTCATCACTTTTCCA-3' UPN012
SMC1A 5'-TTTTGGGCAAGAACATGGAT-3' 5'-CAAGGGAATCCACACCTTGT-3' UPN012
FAT4 5'-ACAAGTGTTCGCAGCAGATG-3' 5'-GAGGAGAGAACAGGGGAAGC-3' UPN012
ARID1A 5'-AGATCAGAGGGCCAACCAC-3' 5'-GACCAGATTCAACCTCATCCA-3' UPN012
ASXL1 5'-GAGCACCCCTGGAAAGTGTA-3' 5'-AGAATGGGACCATTGTCTGC-3' UPN012
JHDM1D 5'-TTTGGTTTGGGAATAGATGAGAA-3' 5'-AAGTGACAGTTCCTGGTTAAAAA-3' UPN012
ASH1L 5'-AACATGGCCAGATAAGGAAGTC-3' 5'-CAAAGTTTGAGGGAGGCGTA-3' UPN013
GATA4 5'-CAGCCTAGACCTCCCAAGC-3' 5'-TTCCCCTAACCAGATTGTCG-3' UPN013
NSD1 5'-CGCCAGACAGACTGCTCAT-3' 5'-GCCTGGGTGAGTGATTTAACA-3' UPN013
PTGES3L 5'-CAACTTCTCTTCAGGCAGCA-3' 5'-GCCACCTTTTCCTTCCATTT-3' UPN013
TTN 5'-TTCCTGGCCTTGTGATATGG-3' 5'-TGCTTTTGCAGAACCAGATG-3' UPN013
SEMA4G 5'-TTCAGGGAGTCCCAGTCTGT-3' 5'-GAACAAGGGGAACTGGGTTT-3' UPN013
SRCAP 5'-TGCTGAGAGAAAACCCTTGA-3' 5'-CTCAGCTCCATGGCATGAT-3' UPN013
EPHA2 5'-GGTTCTCACCCAACTTCCAT-3' 5'-CGACACCAGGTAGGTTCCAA-3' UPN013
TET1 5'-CATCAGTTGCCACCAAGAAA-3' 5'-TCGATGCTATCCAAATGTCG-3' UPN013
JARID2 5'-CCAGAGCCTGTCAGAGTCCT-3' 5'-CAGAGGGTGGAACTTGTGGT-3' UPN013
FANCI 5'-GAGGAGGAGGAAGAGGCATT-3' 5'-CTTCAGCGCTCTCTAGTGGAA-3' UPN013
FAT1 5'-TGCAGCATGAGCACTCCTTA-3' 5'-TTGTCTTTGGCCCTCACAGT-3' UPN013
FAT1 5'-CGTTGTCGTGAAGGTCATTG-3' 5'-TGAACTGGCTGAAAGGGTCT-3' UPN013
4
Supplemental Table 5: Genes for targeted capture in the Frequency Cohort study
Gene List Discovery Cohort TGCA (AML) Cancer Gene Census Method
AARSD1 No No Yes Sure Select

ABR Yes Yes Yes Sure Select
ACTG2 Yes No No Sure Select
ACTN2 Yes No No Sure Select
AGAP2 No No Yes Sure Select
AGR3 No No Yes Sure Select
AHNAK Yes Yes Yes Sure Select
ALAD Yes No No Sure Select
ANKMY1 Yes No No Sure Select
ANKRD24 Yes Yes Yes Sure Select
APOB Yes Yes Yes Sure Select
ARHGAP39 No Yes Yes Sure Select
ARHGEF3 No Yes Yes Sure Select
ARID1A Yes No Yes Sure Select
ARID2 Yes Yes Yes Sure Select
ARID5A Yes No Yes Sure Select
ASAH2B Yes Yes Yes Sure Select
ASB2 Yes Yes Yes Sure Select
ASH1L Yes No Yes Sure Select
ASXL1 Yes Yes Yes Sure Select
ASXL2 Yes Yes Yes Sure Select
ATP2A2 No Yes Yes Sure Select
ATRX No Yes Yes Sure Select
B3GAT3 Yes No No Sure Select
B3GNT3 Yes No No Sure Select
BAHCC1 Yes No No Sure Select
BAIAP2L2 Yes No No Sure Select
BAZ2B Yes No No Sure Select
BCLAF1 No Yes Yes Sure Select
BCOR No Yes Yes Sure Select
BCORL1 Yes Yes Yes Sure Select
BMP7 Yes No No Sure Select
BOC Yes Yes Yes Sure Select
BPGM No No Yes Sure Select
BRIP1 No No Yes Sure Select
BRIP1 No No Yes Sure Select
BTBD8 No Yes Yes Sure Select
C12ORF51 Yes Yes Yes Sure Select
C22ORF15 No Yes Yes Sure Select
C5ORF60 No Yes Yes Sure Select
CACNA1E No Yes Yes Sure Select
1
CASP5 No No Yes Sure Select
CCDC33 Yes No No Sure Select
CCDC84 Yes Yes Yes Sure Select
CCDC89 Yes Yes Yes Sure Select
CDH18 Yes No No Sure Select
CDT1 Yes No No Sure Select
CEBPA Yes Yes Yes Sure Select, PCR
CEBPB No No Yes Sure Select
CEBPE No No Yes Sure Select
CELSR3 Yes Yes Yes Sure Select
CENPF No No Yes Sure Select
CHST5 No No Yes Sure Select
CLCA4 Yes No No Sure Select
CNGA4 Yes No No Sure Select
CNN3 Yes No No Sure Select
CNTNAP4 No Yes Yes Sure Select
COG7 Yes No No Sure Select
COL12A1 No Yes No Sure Select
COL4A6 Yes Yes No Sure Select
CORO1A No Yes No Sure Select
CRHR1 Yes No No Sure Select
CSMD1 Yes Yes Yes Sure Select
CTCF Yes Yes Yes Sure Select
CYP11B2 No No Yes Sure Select
CYP4A11 No No Yes Sure Select
DAAM2 No Yes No Sure Select
DAPK1 Yes No No Sure Select
DAXX No Yes No Sure Select
DCHS2 Yes Yes No Sure Select
DDI2 Yes Yes No Sure Select
DDX4 No Yes No Sure Select
DDX41 No Yes Yes Sure Select
DIRC2 Yes Yes No Sure Select
DIS3 No Yes No Sure Select
DMD Yes Yes No Sure Select
DNAH9 No Yes Yes Sure Select
DNAJB6 Yes Yes Yes Sure Select
DNAJC12 No Yes Yes Sure Select
DNMT3A Yes Yes Yes Sure Select
DOT1L Yes Yes Yes Sure Select
DUSP14 No Yes Yes Sure Select
EFCAB5 Yes No No Sure Select
EGR1 No No Yes Sure Select
EHD2 No Yes No Sure Select
EML5 Yes No No Sure Select
2
EP300 No No Yes Sure Select
EPHA2 No Yes Yes Sure Select
EPHB1 Yes Yes No Sure Select
EPPK1 Yes No No Sure Select
ETV6 No Yes Yes Sure Select
EVPLL Yes Yes Yes Sure Select
EXOC6 Yes No No Sure Select
EZH2 No No Yes Sure Select
FAM83B No No Yes Sure Select
FANCI No Yes Yes Sure Select
FAT1 Yes No Yes Sure Select
FAT4 Yes No Yes Sure Select
FBXO34 Yes No No Sure Select
FBXO38 Yes No No Sure Select
FLNA Yes No No Sure Select
FLNB No No Yes Sure Select
FLNC Yes Yes No Sure Select
FLT3 Yes Yes Yes Sure Select
FMNL2 No Yes Yes Sure Select
FRG1 Yes Yes Yes Sure Select
GATA1 No No Yes Sure Select
GATA2 No No Yes Sure Select
GJB5 Yes No No Sure Select
GLUD2 No Yes No Sure Select
GP1BB Yes Yes No Sure Select
GPBAR1 Yes Yes No Sure Select
GPR139 Yes Yes No Sure Select
GPR156 Yes Yes No Sure Select
GPR183 No Yes No Sure Select
GRIA2 No No Yes Sure Select
GRIA4 No No Yes Sure Select
GRK7 No No Yes Sure Select
GYG1 Yes No No Sure Select
HLA-DRB1 No Yes Yes Sure Select
HUWE1 No Yes No Sure Select
HYAL1 Yes No No Sure Select
IDH1 Yes Yes Yes Sure Select
IDH2 No Yes Yes Sure Select
IDH3B Yes No No Sure Select
IDO2 No No Yes Sure Select
IGF2BP3 Yes No No Sure Select
IL17RD No Yes Yes Sure Select
INO80 Yes No No Sure Select
INSC Yes Yes Yes Sure Select
JAG2 Yes No No Sure Select
3
JAK1 No No No Sure Select
JAK2 No Yes No Sure Select
JAK3 No No No Sure Select
JARID2 Yes No No Sure Select
JHDM1D Yes No No Sure Select
JMJD5 No Yes No Sure Select
KCNG1 Yes Yes Yes Sure Select
KCNH6 Yes Yes Yes Sure Select
KCNH7 Yes Yes Yes Sure Select
KDM5C No Yes No Sure Select
KDM6A No Yes No Sure Select
KIF26A Yes No No Sure Select
KIT No Yes Yes Sure Select
KLHL7 Yes Yes No Sure Select
LAMA2 Yes No No Sure Select
LNK No No Yes Sure Select
LRBA No No Yes Sure Select
LRP2 Yes No No Sure Select
LTK No No Yes Sure Select
MAP2K3 No Yes No Sure Select
MCM10 No Yes No Sure Select
METTL13 Yes No No Sure Select
MGA No Yes Yes Sure Select
MLL No No Yes Sure Select
MLL2 No Yes Yes Sure Select
MLL3 No Yes Yes Sure Select
MLPH No No Yes Sure Select
MN1 Yes No No Sure Select
MSH4 No No Yes Sure Select
MTDH No No Yes Sure Select
MUC16 Yes Yes Yes Sure Select
MYO18B No Yes No Sure Select
MYOM3 Yes Yes No Sure Select
NBPF1 Yes Yes No Sure Select
NBPF14 Yes Yes No Sure Select
NECAB2 Yes No No Sure Select
NFIX Yes No No Sure Select
NOTCH1 No Yes No Sure Select
NPHS1 Yes No No Sure Select
NPM1 Yes Yes No Sure Select
NRAS No Yes No Sure Select
4
NSD1 Yes No No Sure Select
NSDHL No No Yes Sure Select
NSMCE1 Yes No No Sure Select
NUBP2 Yes Yes No Sure Select
ODZ2 Yes Yes No Sure Select
OR10G7 No Yes No Sure Select
OR5L1 Yes Yes No Sure Select
TP53 Yes Yes No Sure Select
PAM16 Yes Yes No Sure Select
PAX3 No No Yes Sure Select
PAX5 No No Yes Sure Select
PCDHB16 Yes Yes No Sure Select
PCSK2 Yes No No Sure Select
PDXDC1 No Yes No Sure Select
PEG10 Yes No No Sure Select
PHF6 No Yes No Sure Select
PHF8 No No Yes Sure Select
PIK3R6 Yes No No Sure Select
PIRT No Yes No Sure Select
PKD1L2 No Yes No Sure Select
PLEKHH1 No Yes No Sure Select
POLD1 Yes No No Sure Select
POM121C Yes Yes No Sure Select
POTEE No Yes No Sure Select
PPP1R12B No Yes No Sure Select
PRAM1 Yes No No Sure Select
PRDM15 No No Yes Sure Select
PRDM7 No Yes No Sure Select
PROKR1 Yes No No Sure Select
PRPF40B No Yes Yes Sure Select
PRSS16 No Yes Yes Sure Select
PSMD3 No No Yes Sure Select
PTPN11 No Yes No Sure Select
PTPRB No Yes No Sure Select
PTPRT No Yes No Sure Select
PXK Yes No No Sure Select
PYGO2 Yes No No Sure Select
RAB20 No Yes No Sure Select
RAD21 No Yes No Sure Select
RBM39 No Yes No Sure Select
RBM42 Yes Yes No Sure Select
RBMX No Yes No Sure Select
RGN No No Yes Sure Select
RNF213 Yes Yes No Sure Select
RP1L1 Yes Yes No Sure Select
5
RTL1 Yes Yes No Sure Select
RUNX1 Yes Yes Yes Sure Select
RYR3 No Yes No Sure Select
SCAMP5 Yes No No Sure Select
SCF No No Yes Sure Select
SEC22B Yes Yes No Sure Select
SEMA4G Yes Yes No Sure Select
SEMA6D No Yes No Sure Select
SETBP1 No No Yes Sure Select
SETD1A No Yes No Sure Select
SETD1B Yes Yes No Sure Select
SETDB1 No Yes No Sure Select
SF3A1 Yes Yes No Sure Select
SF3B1 Yes Yes No Sure Select
SGK2 No No Yes Sure Select
SGK3 No No Yes Sure Select
SLC26A8 No Yes No Sure Select
SLC34A1 Yes Yes No Sure Select
SLC7A8 Yes Yes No Sure Select
SMC1A Yes Yes No Sure Select
SMC3 No Yes No Sure Select
SPDYE6 Yes Yes No Sure Select
SPPL2C Yes No No Sure Select
SRCAP Yes No No Sure Select
SRGAP2 No Yes Yes Sure Select
SRSF2 No Yes Yes Sure Select
STAG1 No Yes Yes Sure Select
STEAP4 Yes Yes No Sure Select
STK4 No No Yes Sure Select
STK40 Yes No No Sure Select
STOX2 No No Yes Sure Select
SUN2 No No Yes Sure Select
SUSD2 Yes Yes Yes Sure Select
SUV39H1 No No Yes Sure Select
SUV39H2 Yes No Yes Sure Select
SUZ12 No Yes No Sure Select
SVIL No No Yes Sure Select
TCF7 Yes No No Sure Select
TET1 Yes Yes Yes Sure Select
TET2 Yes Yes Yes Sure Select
6
THNSL2 No No Yes Sure Select
TMEM179 Yes Yes No Sure Select
TNR No Yes No Sure Select
TNRC6C No Yes No Sure Select
TPO No No Yes Sure Select
TRIM69 No Yes No Sure Select
TTK Yes Yes No Sure Select
TTN Yes Yes No Sure Select
TYRP1 No Yes No Sure Select
U2AF35 No Yes Yes Sure Select
U2AF65 No Yes Yes Sure Select
UNC5B No Yes No Sure Select
USP31 Yes Yes No Sure Select
USP36 Yes Yes No Sure Select
USP44 No Yes No Sure Select
USP9X Yes Yes No Sure Select
VCPIP1 Yes No No Sure Select
VPS13C No Yes No Sure Select
WAC No Yes No Sure Select
WDR33 Yes No No Sure Select
WRAP53 No No Yes Sure Select
WT1 Yes Yes No Sure Select
XIRP1 No Yes No Sure Select
XIRP2 No Yes No Sure Select
XKR4 Yes No No Sure Select
XPO1 No No Yes Sure Select
XPO7 No No Yes Sure Select
YLPM1 Yes No No Sure Select
YTHDC1 No No Yes Sure Select
ZBTB7A Yes Yes No Sure Select
ZFHX4 Yes Yes No Sure Select
ZNF80 Yes No No Sure Select
ZRSR2 No No Yes Sure Select
7
Supplemental Table 6: Clinical Parameters of 67 FLT3-ITD AML cases subjected to targeted deep sequencing (Frequency Cohort)
DX- MLL
CR-BM
Sample Age/ Cytogenetic Cytogenetic BM REL-BM Hb WBC PLT Dead/ OS RFS Induction ITD ITD ITD PTD
FAB Blast
ID Sex (DX) (REL) Blast Blast (%) (g/dl) (*109/L) (*109/L) Alive (Mon) (Mon) Chemotherapy (DX) (CR) (REL) (DX and
(%)
(%) REL)
Cytarabine and
UPN014 45/F M4 46,XX 46,XX 95 0 80 9.7 85 37 Dead 11.0 5 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN015 66/F M2 46,XX 46,XX 80 <5 95 9.0 126 63 Dead 8 5 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN016 38/M M5 46,XY 46,XY 90 <5 70 4.2 279 37 Dead 18 10 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
(7+3)
46,X,-X,add
(11) (p15),- Cytarabine and
UPN018 24/F M4 46,XX 12,del (17) 80 <5 30 8.4 32 79 Dead 26 19 daunomycine Pos Neg Pos Neg
(q11) (7+3)
46XX[25]
Cytarabine and
(7+3)
Cytarabine and
UPN020 18/M M5 46,XY 46,XY 90 0 70 8.2 54 57 Dead 21 5 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN021 48/F M5a 46,XX 46,XX 90 <5 70 7.2 26 67 Dead 10 8 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
(7+3)
Cytarabine and
UPN023 46/M M4 46,XY 46,XY 82 <5 10 10.2 49.8 23 Dead 25 3 daunomycine Pos Neg Pos Neg
(7+3)
46,XX, t(1;6) 46,XX, t(1;6) Cytarabine and

UPN024 67/F M2 (q34;q21) (q34;q21) 70 <5 60 6.9 170 200 Alive 3 1 daunomycine Pos Neg Pos Neg
[20] [20] (7+3)
Cytarabine and
UPN025 37/F M4 46,XX 46,XX 70 <5 60 7.2 89 57 Alive 30 4 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN026 53/M M1 46,XY 46,XY 85 <5 10 10.0 272 76 Alive 13 5 daunomycine Pos Neg Pos Neg
(7+3)
46XX,del(5)
46XX,del (p14)[2]/ Cytarabine and
UPN027 56/F M1 (11)(p13) 46XX,del 80 <5 80 8.8 38 35 Dead 10 6 daunomycine Pos Neg Pos Neg
[10] (11)(p13)[3]/ (7+3)
46,XX[15]
Cytarabine and
(7+3)
Cytarabine and
(7+3)
46,XY,t(6;9) 46,XY,t(6;9) Cytarabine and
UPN030 26/M M5b (p23;q34),der (p23;q34),der 90 <5 20 8.6 77 16 Alive 42 35 daunomycine Pos Neg Pos Neg
(18)r(18) (18)r(18) (7+3)
44,XY,del(1)(
45,XY,del(1)(
p32),-5,del Cytarabine and
p32),- 5,del
UPN031 19/M NA (7)(q11),- 90 <5 20 4.6 23 88 Alive 23 18 daunomycine Pos Neg Pos Neg
(7)(q11.2)[10
17[15]/ (7+3)
]/ 46,XY[15]
46,XY[10]
Cytarabine and
UPN032 70/M M4 46,XY 46,XY 80 <5 75 11.5 48 118 Alive 20 20 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN033 59/F M2 46,XX NA 90 <5 NA 8.0 17 24 Dead 8 NA daunomycine Pos Neg NA Neg
(7+3)
NA
Cytarabine and
UPN034 43/M M4 46,XY NA 58 <5 NA 9.0 89 41 Dead 11 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN035 68/M M1 46,XY NA 20 <5 NA 8.8 10 45 Alive 4 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN036 60/F M5a 46,XX NA 95 <5 NA 9.6 46 99 Alive 30 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN037 70/F M5b 46,XX NA 40 <5 NA 8.6 63 101 Alive 11 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN038 64/F M1 46,XX NA 90 <5 NA 11.7 108 71 Alive 26 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN039 46/F M1 46,XX NA 50 <5 NA 10.5 17.6 65 Alive 38 NA daunomycine Pos Neg Neg
(7+3)
NA
45,X,-
Y,t(1;13)(p22
Cytarabine and
;q12),t(2;19)(
UPN040 23/M M1 NA 90 <5 NA 11.0 6.5 29 Dead 15 NA daunomycine Pos Neg Neg
p11;q13),
(7+3)
inv(11)(q21q
23)[23]
NA
Cytarabine and
UPN041 48/F M4 46,XX NA 90 <5 NA 13.7 9 298 Alive 8.5 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN042 43/F M4 46,XX NA 90 <5 NA 8.3 3.5 152 Alive 30 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN043 48/F M4 46,XX NA 80 <5 NA 11.7 114 24 Dead 6 NA daunomycine Pos Neg Neg
(7+3)
NA
Cytarabine and
UPN044 56/M M5a 46,XY NA 90 <5 NA 10.9 50 16 Alive 23 NA daunomycine Pos Neg Neg
(7+3)
Cytarabine and
UPN045 60/F M2 46XX 46XX 85.7 0 65.8 5.8 30 13.1 Dead 24 9 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN046 38/M M1 47XY,+8 47XY,+8 inc 90.1 <5 59.1 9.2 158 5.5 Dead 8 2 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN047 48/F M1 46XX 46XX 69.6 <5 90 6.5 188 6.2 Dead 35 4 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN048 61/M M1 45,XY 46,XY NA NA NA NA NA NA NA NA 10 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN049 58/M M4 46,XY 46,XY 69 <5 74.8 NA NA NA NA NA 3 daunomycine Pos Neg Pos Neg
(7+3)
46,XX, Cytarabine and

UPN050 60/F M4 46,XX del (11) NA NA NA NA NA NA NA NA 11 daunomycine Pos Neg Pos Neg
(p13p15) (7+3)
46,XX,
Cytarabine and
der (17)
UPN051 65/F M1 46,XX NA NA NA NA NA NA Dead 6.6 3 daunomycine Pos Neg Pos Neg
t(17;17)(p11;
(7+3)
q21)
Cytarabine and
UPN052 69/M M1 46,XY 46,XY NA NA NA NA NA NA Alive 31 7 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN053 67/M M1 46,XY 46,XY NA NA NA NA NA NA Alive 15.1 6 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN054 55/F M4 46,XX NA NA NA NA NA NA NA Alive 7 2 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN055 65/M M4 46,XY NA NA NA NA NA NA NA Alive 15.5 2 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN056 43/F M1 46,XX NA NA NA NA NA NA NA Alive 10.6 6 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN057 47/F M2 NA 46,XX[23] NA <5 90.8 6.7 20.5 219 Dead 12.7 7 daunomycine NA Neg Pos Neg
(7+3)
Cytarabine and
UPN058 49/F M0 No diagnosis 46,XX[20] No DX <5 89.0 13.0 2.7 25 Dead 24.3 13 daunomycine NA Neg Pos Neg
(7+3)
46,XY[18]/46
Cytarabine and
,X, -Y, +8[1]/
UPN059 71/M M0 NA NA 0 53.0 9.0 2 124 Dead 14.7 5 daunomycine NA Neg Pos Neg
45,XY,+8, -
(7+3)
13,-17[1]
Cytarabine and
UPN060 52/M M4 NA 46,XY[26] NA <5 30.9 11.2 5.2 169 Dead 8.4 5 daunomycine NA Neg Pos Neg
(7+3)
Cytarabine and
UPN061 52/F M4 NA 46,XX[30] NA <5 33.5 12.9 3.1 129 Dead 30.1 14 daunomycine NA Neg Pos Neg
(7+3)
46,XY[19] Cytarabine and

UPN062 67/M M2 /random 46,XY[20] 59.5 0 16.9 12.9 3.1 323 Dead 92.0 42 daunomycine Pos Neg Pos Neg
loss[6] (7+3)
Cytarabine and
UPN063 64/M M2 46,XY[30] 46,XY[28] 74.0 <5 75.6 13.6 24 117 Dead 23.9 14 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
46,XX 100%
UPN064 29/F M2 46,XX[30] 90.3 <5 97.3 9.6 92.7 44 Dead 9.6 6 daunomycine Pos Neg Pos Neg
[26/26].
(7+3)
Cytarabine and
UPN065 51/M M2 46,XY[22] 46,XY[20] 83.3 0 54.6 9.8 37 143 Alive 226 23 daunomycine Pos Neg Pos Neg
(7+3)
Cytarabine and
UPN066 32/F M2 46,XX NA 90 <5 NA 9.1 82 52 Alive 16 NA daunomycine Pos Neg NA Neg
(7+3)
Cytarabine and
46,XY,del(7)
UPN067 21/M M0 NA 95 <5 NA 5.8 38 90 Alive 21 NA daunomycine Pos Neg NA Neg
(q11)[20]
(7+3)
Cytarabine and
UPN068 74/M M2 46,XY NA 90 <5 NA 9.1 57 103 Alive 5 NA daunomycine Pos Neg NA Neg
(7+3)
Cytarabine and
UPN069 62/F M5 46,,XX NA 95 <5 NA 8.5 52 70 Dead 7 NA daunomycine Pos Neg NA Neg
(7+3)
46,XY,t(7;11)
(p15;p15),t(1 Cytarabine and
UPN070 28/M M2 5;17)q15~22; NA 95 <5 NA 15.0 52 105 Alive 153 NA daunomycine Pos Neg NA Neg
q21~25)[17]/ (7+3)
46,XY[3]
47,XY, Cytarabine and

UPN071 11/M M2 47,XY 88 <5 69.1 10.3 420 NA Alive 72 NA Pos Neg Pos Neg
+6[17] idarubicin (7+3)
Cytarabine and
UPN072 25/M M2 46,XY[20] 46,XY 66 <5 45 8.6 2.5 NA Dead 96 NA Pos Neg Pos Neg
idarubicin (7+3)
Cytarabine and
UPN073 36/M M2 NA NA NA 0 NA 12.5 NA NA Alive 130 NA Pos Neg NA Neg
idarubicin (7+3)
Cytarabine and
UPN074 36/M M1 46,XY NA 79 <5 NA 9.1 122.4 NA Alive 71 NA Pos Neg NA Neg
idarubicin (7+3)
Cytarabine and
UPN075 37/M M1 46,XY [20] NA 96 0 NA 8.3 169.1 NA Alive 72 NA Pos Neg NA Neg
idarubicin (7+3)
Cytarabine and
UPN076 37/F M1 46,XX[20] NA 70 0 NA 13.8 15.48 19 Dead 2 NA Pos Neg NA Neg
idarubicin (7+3)
47,XY,+8[19] Cytarabine and

UPN077 36/M M5 NA 48 0 NA 10.1 41.85 25 NA 8.6 NA Pos Neg NA Neg
/46,XY[1] idarubicin (7+3)
Cytarabine and
UPN078 40/F M5b 46,XX[19] NA 84 0 NA 9.2 141.4 71 Alive 27.3 NA Pos Neg NA Neg
idarubicin (7+3)
46,XX,[10] - Cytarabine and

UPN079 37/F M2 NA 55 <5 NA 6.5 104.5 37 NA 1.2 NA Pos Neg NA Neg
PB idarubicin (7+3)
47,XX,+8[4]/
Cytarabine and
UPN080 49/F M2 46,XX[15] - NA 60 0 NA 10.0 43 61 Dead 3.9 NA Pos Neg NA Neg
idarubicin (7+3)
PB
FAB : French American and British classification scheme for leukemia
M : Male
F : Female
BM : Bone Marrow
Hb : Hemoglobin
WBC : White blood cell

PLT : Platelets
RFS : Relapse-free survival
OS : Overall survival
DX : Diagnosis
REL : Relapse
Pos : Positive
Neg : Negative
Supplemental Table 7: Coverage analysis of targeted deep sequencing of FLT3-ITD AML
(Frequency Cohort)
Target Coverage Target Coverage Target Coverage

Sample ID Bait Size
>30X >10X >2X
SureSelect Customized beads 2.9 Mb

UPN001 CR 29.40 61.56 87.28

UPN001 DX 88.38 95.90 98.71

UPN001 REL 34.54 74.18 95.75

UPN002 CR 84.54 95.73 98.73

UPN002 DX 52.81 86.59 97.22

UPN002 REL 83.87 96.73 99.10

UPN003 CR 74.60 88.22 96.62

UPN003 DX 88.23 96.52 98.78

UPN003 REL 83.27 95.62 98.65
83.53 95.29 98.54 SureSelect Customized beads 2.9 Mb

UPN004 CR

UPN004 DX

UPN004 REL

UPN005 CR

UPN005 DX

UPN005 REL

UPN006 CR

UPN006 DX

UPN006 REL
93.62 98.21 SureSelect Customized beads 2.9 Mb
UPN007 CR 75.89
UPN007 DX 52.84
UPN007 REL 42.89
UPN008 CR
UPN008 DX

UPN008 REL

UPN009 CR

UPN009 DX

UPN009 REL
1
UPN010 CR

UPN010 DX

UPN010 REL

UPN011 CR

UPN011 DX

UPN011 REL
93.77 SureSelect Customized beads 2.9 Mb

UPN012 CR 75.21 98.47

UPN012 DX 98.67

UPN012 REL 94.95

UPN013 CR 95.87

UPN013 DX 98.52

UPN013 REL 97.35

UPN014 CR 98.96

UPN014 DX 96.90

UPN014 REL 12.90
UPN015 CR
UPN015 DX
UPN015 REL
UPN016 CR

UPN016 REL

UPN017 CR

UPN017 DX
UPN017 REL
UPN018 CR
UPN018 DX
UPN018 REL
UPN019 CR
UPN019 DX
UPN019 REL
UPN020 CR
UPN020 DX
UPN020 REL
2
UPN021 CR
UPN021 DX
UPN021 REL
UPN022 CR
UPN022 DX
UPN022 REL
UPN023 CR
UPN023 DX
UPN023 REL
UPN024 CR
UPN024 DX
UPN024 REL
UPN025 CR
UPN025 DX
UPN025 REL
UPN026 CR
UPN026 DX
UPN026 REL
UPN027 CR
UPN027 DX
UPN027 REL
UPN028 CR
UPN028 DX
UPN028 REL
UPN029 CR
UPN029 DX
UPN029 REL
UPN030 CR
UPN030 DX
UPN030 REL
UPN031 CR
UPN031 DX
UPN031 REL
UPN032 CR
UPN032 DX
3
UPN032 REL
UPN033 CR
UPN033 DX
UPN034 CR
UPN034 DX
UPN035 CR
UPN035 DX
UPN036 CR
UPN036 DX
UPN037 CR
UPN037 DX
UPN039 CR
UPN039 DX
UPN040 CR
UPN040 DX
UPN041 CR
UPN041 DX
UPN042 CR
UPN042 DX
UPN043 CR
UPN043 DX
UPN044 CR
UPN044 DX
UPN045 CR
UPN045 DX
UPN045 REL
UPN046 CR
UPN046 DX
UPN046 REL
UPN047 CR
UPN047 DX
UPN047 REL
UPN048 CR
UPN048 DX
UPN048 REL
4
UPN049 CR
UPN049 DX
UPN049 REL
UPN050 CR
UPN050 DX
UPN050 REL
UPN051 CR
UPN051 DX
UPN051 REL
UPN052 CR
UPN052 DX
UPN052 REL
UPN053 CR
UPN053 DX
UPN053 REL
UPN054 CR
UPN054 DX
UPN054 REL
UPN055 CR
UPN055 DX
UPN055 REL
UPN056 CR
UPN056 DX
UPN056 REL
UPN057 CR
UPN057 REL
UPN058 CR
UPN058 REL
UPN059 CR
UPN059 REL
UPN060 CR
UPN060 REL
UPN061 CR
UPN061 REL
UPN062 CR
5
UPN062 DX
UPN062 REL
UPN063 CR
UPN063 DX
UPN063 REL
UPN064 CR
UPN064 DX
UPN064 REL
UPN065 CR
UPN065 DX
UPN065 REL
UPN066 CR
UPN066 DX
UPN067 CR
UPN067 DX
UPN068 CR
UPN068 DX
UPN069 CR
UPN069 DX
UPN070 CR
UPN070 DX
UPN071 CR
UPN071 DX
UPN071 REL
UPN072 CR
UPN072 DX
UPN072 REL
UPN073 CR
UPN073 DX
UPN074 CR
UPN074 DX
UPN075 CR
UPN075 DX
UPN076 CR
UPN076 DX
6
UPN077 CR
UPN077 DX
UPN078 CR
UPN078 DX
UPN079 CR
UPN079 DX
UPN080 CR
UPN080 DX
Average 76.82 91.36 97.47
DX : Diagnosis
REL : Relapse
Mb : Megabyte
7
Supplemental Table 8: Summary of somatic mutations in Frequency Cohort
Annotated Amino Acid Allele Status in Sample

Mutation Type Position RefSeq
Gene Change Change Disease ID
APOB Missense chr2:21229521 E3407Q C>G NM_000384 DX UPN014

Frameshift
ARID1A chr1:27105930 G1847fs */+G NM_006015 DX UPN014
insertion
Frameshift
BCOR chrX:39933219 K460fs */+T NM_001123383 DX UPN014
insertion
GATA2 Missense chr3:128200753 N351I T>A NM_001145661 DX UPN014
IDH3B Missense chr20:2640744 G131S C>T NM_174856 DX UPN014
MLL3 Missense chr7:151878824 G2041R C>T NM_170606 DX UPN014
NSD1 Missense chr5:176684085 F1364L C>A NM_172349 DX UPN014

PRDM7 Stopgain chr16:90124792 Q462X G>A NM_001098173 DX UPN014
Frameshift
PYGO2 */+G
insertion chr1:154931543 P311fs NM_138300 DX UPN014
NOTCH4 Missense chr6:32164738 A1722T C>T NM_004557 REL UPN014
USP34 Missense chr2:61575320 D657G T>C NM_014709 REL UPN014

Frameshift
NPM1 chr5:170837543 W288fs */+ CATG NM_199185 DX and REL UPN015
insertion
FAT1 Missense chr4:187557782 S1310L G>A NM_005245 DX and REL UPN015
FAT1 Missense chrX:129147373 G209C G>A NM_021946 REL UPN015
NSD1 Missense chr5:176709485 Y1971C A>G NM_022455 DX and REL UPN015
SF3B1 Missense chr2: 198266834 K700E T>C NM_012433 DX and REL UPN015
SPPL2C Missense chr17:43924130 G620W G>A NM_175882 REL UPN015
POLD1 Missense chr19:176709485 R211H G>A NM_002691 REL UPN015
C5orf60 Missense chr5:179071925 P33S G>A NM_001142306 DX and REL UPN016
CLCA4 Missense chr1:87033185 G345R G>A NM_012128 DX and REL UPN016
DAAM2 Missense chr6:39847218 P604S C>T NM_001201427 REL UPN016
EPPK1 Missense chr8:144940281 D2381N C>T NM_031308 REL UPN016
NFIX Missense chr19:13135971 A55G C>G NM_002501 REL UPN016
WAC Splice site chr:28900702 980-1G>A G>A NM_100486 REL UPN016
MLL3 Missense chr7:151945228 S764F G>A NM_170606 DX and REL UPN016
MLL3 Missense chr7:151845261 R4584Q C>T NM_170606 REL UPN017
EPHB1 Missense chr3:134884855 G544V G>T NM_004441 REL UPN017
FANCI Missense chr15:89858592 R1239L G>T NM_018193 REL UPN017
FLNC Missense chr7:128491604 A1889T G>A NM_001127487 DX UPN017
GJB5 Missense chr1:35223635 T235I C>T NM_005268 REL UPN017
MLL2 Missense chr12:49445272 P732A C>T NM_003482 REL UPN017
SLC34A1 Missense chr5:176825109 R581H G>A NM_003052 REL UPN017
STAG3 Missense chr7:99786085 T148S C>G NM_012447 DX and REL UPN017
STAG3 Missense chr7:99786079 I146T T>C NM_012447 DX and REL UPN017
WT1 Splice site chr11:32417803 G400S C>T NM_000378 DX UPN017
XIRP1 Missense chr3:39229243 R565Q C>T NM_001198621 DX UPN017
1
DNAH9 Missense chr17:11660883 A2290V C>T NM_001372 REL UPN017
APOB Missense chr2:21232158 I2528V T>C NM_000384 DX and REL UPN018
KDM5C Missense chrX53239631 M504V T>C NM_001146702 DX and REL UPN018

Frameshift
insertion
SH2B3 Missense chr12:111884988 S329T G>C NM_005475 REL UPN018
STAG3 Missense chr7:99787151 E267K G>A NM_012447 REL UPN018
TPO Missense chr2:1500457 R769Q G>A NM_175721 DX UPN018

Frameshift
WT1 chr11:32417908 A365fs */-C NM_000378 DX and REL UPN018
deletion
Frameshift
MLL chr11:118343036 E388fs */+ A NM_001197104 DX UPN018
insertion
POM121C Missense chr7:75053499 S294L G>A NM_001099415 DX and REL UPN018
FAT4 Missense chr4:126238838 S424R C>A NM_024582 REL UPN019
FLT3 Missense chr13:28592641 D835V T>A NM_004119 REL UPN019
FLT3 Missense chr13:28592642 D835Y C>A NM_004119 REL UPN019
SGK2 Splice site chr20:42204865 P292L C>T NM_016276 DX and REL UPN019
Framshift */+GTAC
WT1 chr11:32417913 R361fs NM_000378 REL UPN020
insertion AAGA
Frameshift
insertion
Frameshift
ARID1A chr1:27105930 G1847fs */+G NM_006015 DX UPN021
insertion
Frameshift
COL12A1 chr6:75851780 E478fs */+C NM_080645 DX UPN021
insertion
DNMT3A Missense chr2:25457242 R882H C>T NM_175629 DX and REL UPN021
DOT1L Missense chr19:2226655 L1379M C>A NM_032482 DX UPN021
FLT3 Missense chr13:28608266 Y597F T>A NM_004119 REL UPN021
TTN Missense chr2:179472256 I8655T A>G NM_003319 DX UPN021
MLL3 Missense chr7:151874050 N2830H T>G NM_170606 DX UPN021
ZFHX3 Missense chr16:72991961 P695L G>A NM_006885 DX UPN021
RAD21 Stopgain chr8:117866566 L360X A>T NM_006265 REL UPN021
SETD1A Missense chr16:30975505 S244P T>C NM_014712 REL UPN021

Frameshift
USP9X chrX:41075448 S1876fs */+G NM_001039590 DX UPN021
insertion
AHNAK Missense chr11:62284502 T5796M G>A NM_001620 DX UPN022
Frameshift
ASXL1 chr20:31021646 R549fs */-C NM_015338 DX UPN022
deletion
DNMT3A Missense chr2:25463568 I705T A>G NM_175629 DX UPN022
EPHB1 Missense chr3:134670327 R80W C>T NM_004441 DX UPN022
FAT1 Missense chr4:187541867 R1958T C>G NM_005245 DX UPN022
FLT3 Missense chr13:28602340 N676K G>C NM_004119 REL UPN022

Frameshift
RUNX1 chr21:36252864 R139fs */+CCTC NM_001001890 DX UPN022
insertion
SF3B1 Missense chr2:198267359 K666N C>G NM_012433 DX UPN022
POTEE Missense chr2:132021816 A930T G>A NM_001083538 DX UPN022
MUC6 Missense chr11:1017469 T1778S T>A NM_005961 DX UPN022
FAT4 Missense chr4:126411869 S4631L C>T NM_024582 DX UPN022
MLL3 Missense chr7:151902199 R1318I C>A NM_170606 DX and REL UPN022
2
APOB Missense chr2:21230163 L3193F G>A NM_000384 REL UPN023
JHDM1D Missense chr7:139826547 V260I C>T NM_030647 REL UPN023
MLL3 Missense chr7:151945556 V655I C>T NM_170606 REL
PKD1L2 Missense chr16:81249888 S142L G>A NM_001076780 REL UPN023
SRCAP Missense chr16:30724884 L782R T>G NM_006662 DX UPN023
WT1 Stopgain chr11:32417910 S381X G>T NM_000378 DX UPN023
MUC6 Missense chr11:1016854 P1983S G>A NM_005961 REL UPN024

Frameshift
TNRC6C chr17:76063862 W876fs */-G NM_001142640 REL UPN024
deletion
Frameshift
SETD1B chr12:122248078 595fs */+C NM_015048 REL UPN024
deletion
TTN Missense chr2:179398822 V25109I C>T NM_003319 DX and REL UPN025
AHNAK Missense chr11:62293897 K2664N C>A NM_001620 DX UPN025
FLNC Missense chr7:128483313 V861M G>A NM_001127487 DX UPN025
KDM6A Missense chrX:44929364 A822T G>A NM_021140 REL UPN025
MLL3 stopgain chr7:151945256 Q755X G>A NM_170606 DX and REL UPN025
MLL3 Missense chr7:151880065 F1753L A>C NM_170606 DX and REL UPN025

Frameshift
PVRIG */-G UPN025
deletion chr7:99818843 R317fs NM_024070 DX
MUC16 Missense chr19:9067935 S6504Y NM_024690 DX and REL UPN025
POTEE Missense chr2:132021696 M890L A>C NM_001083538 DX and REL UPN025
TTN Missense chr2:179542489 V10140I C>T NM_133378 DX UPN026

Frameshift
FBXO34 chr14:55818226 C373fs */+C NM_017943 DX UPN026
insertion
FLNA Missense chrX:153580617 G2226E C>T NM_001456 REL UPN026
KDM6A Missense chrX:44929377 A826V C>T NM_021140 REL UPN026

Frameshift
MLL3 chr7:152055745 G59fs */-C NM_170606 DX UPN026
deletion
Frameshift
NPM1 chr5:170837544 W288fs */+CCTG NM_199185 DX and REL UPN026
insertion
Frameshift
SRCAP chr16:30718592 R132fs */+C NM_006662 DX UPN026
insertion
TET2 Stopgain chr4:106158157 Q1020X C>T NM_001127208 DX and REL UPN026
MLL4 Missense chr19:36228145 R2511W C>T NM_014727 REL UPN026

Frameshift
MUC16 chr19:9012820 P12875fs */+T NM_024690 REL UPN027
insertion
MUC6 Missense chr11:1016836 F1989I A>T NM_005961 REL UPN027
AHNAK Missense chr11:62286693 G5066R C>G NM_001620 DX UPN027
DDX4 Missense chr5:55112340 N567S A>G NM_001166534 DX and REL UPN027
DNMT3A Missense chr2:25457243 R882C G>A NM_175629 DX and REL UPN027
FANCI Missense chr15:89803982 R66C C>T NM_001113378 DX and REL UPN027
FLNA Missense chrX:153595119 P323L G>A NM_001110556 DX and REL UPN027
TTN Missense chr2:179401074 V24402G A>C NM_003319 REL UPN027
FLT3 Missense chr13:28592629 D839G T>C NM_004119 DX UPN027
GATA2 Splice site chr3:128200744 T354M G>A NM_001145661 DX UPN027
NSD1 Missense chr5:176638002 S599P T>C NM_172349 DX UPN027

Frameshift
WT1 chr11:32421560 H327fs */+TGGT NM_000378 DX UPN027
insertion
POM121C Missense chr7:75052322 M405V T>C NM_001099415 DX UPN027
3
XIRP1 Missense chr3:39227495 V1148M C>T NM_194293 DX UPN027
XIRP2 Missense chr2:168104282 Y2127C A>G NM_152381 DX and REL UPN027
ZFHX4 Missense chr8:77616519 V66I G>A NM_024721 DX and REL UPN027
PLEKHH1 Missense chr14:68045912 G971R G>A NM_020715 DX UPN027
FAT1 Missense chr4:187630297 M229V T>C NM_005245 REL UPN027
TTN Missense chr2:179428457 K18403Q T>G NM_003319 DX and REL UPN028
ATRX Missense chrX:76888737 R1660W G>A NM_138270 REL UPN028
BCORL1 Missense chrX:129162639 S1370T T>C NM_021946 REL UPN028

Frameshift
KDM6A chrX:44969406 R1363fs */+A NM_021140 DX UPN028
insertion
chr14:
RTL1 Missense V775M C>T NM_001134888 DX UPN028
101348803
SETD1A Missense chr16:30975502 C243R T>C NM_014712 REL UPN028
MUC6 Missense chr11:1016640 A2054V G>A NM_005961 REL UPN028
ZBTB7A Stopgain chr19:4054087 Q382X G>A NM_015898 REL UPN028
FAT1 Missense chr4:187540041 R2567G G>C NM_005245 REL UPN028
EPPK1 Missense chr8:144946101 G441S C>T NM_031308 DX and REL UPN028
PDXDC1 Missense chr16:15122819 R430L G>T NM_015027 DX and REL UPN028
ARID2 Missense chr12 : 46246471 T1522S C>G NM_152641 DX and REL UPN029
COL12A1 Missense chr6 : 75823017 T1454M G>A NM_080645 DX and REL UPN029
CSMD1 Missense chr8:2857641 G2681D C>T NM_033225 DX and REL UPN029
EFCAB5 Missense chr17:28384816 D774N G>A NM_001145053 DX and REL UPN029
SRSF2 Missense chr17:74733073 F57Y A>T NM_001195427 DX and REL UPN029
MLL3 Missense chr7:151970877 P309S G>A NM_170606 DX and REL UPN029
RP1L1 Missense chr8:10465962 E1882D C>A NM_178857 DX UPN029
MUC16 Missense chr19:9073923 P4508H G>A NM_024690 DX and REL UPN029
NBPF1 Unknown chr1:16918468 Unknown T>C Unknown DX and REL UPN029
C5orf60 Missense chr5:179070147 P136A G>C NM_001142306 DX UPN029
TTN Missense chr2:179588859 P5799S G>A NM_133378 DX and REL UPN030
MUC Missense chr19:9064540 L7636I G>T NM_024690 REL UPN030
RTL1 Missense chr14:101348572 A852T C>T NM_001134888 REL UPN030

Frameshift
WT1 chr11:32417942 R353fs */+TACC NM_000378 REL UPN030
insertion
Frameshift
WT1 chr11:32414224 R426fs */-T NM_000378 REL UPN030
deletion
FAT1 Missense chr4:187539205 M2845I C>T NM_005245 REL UPN030
DCHS2 Missense chr4:155312411 D13E G>A NM_017639 REL UPN030
EZH2 Missense chr7:148508788 V570M C>T NM_001203249 DX and REL UPN031
KDM6A Missense chrX:44936012 S925T T>A NM_021140 DX and REL UPN031
NECAB2 Missense chr16:84035519 P377L C>T NM_019065 REL UPN031
FRG1 Splice site chr4:190874282 317+2T>C T>C NM_004477 DX UPN031
GCGR Missense chr17:79766920 P9L C>T NM_000160 REL UPN032
RYR3 Missense chr15:34030756 L2541I C>A NM_001036 REL UPN032
4
TET2 Stopgain chr4:106196324 Q1553X C>T NM_001127208 DX and REL UPN032
Frameshift
NPM1 chr5:170837545 W288fs */+TCTG NM_199185 DX UPN032
insertion
MUC6 Missense chr11:1016970 S1944N C>T NM_005961 REL UPN032
IGF2BP3 Missense chr7:23509683 S16L G>A NM_006547 DX and REL UPN032
PVRIG Stopgain chr7:99818420 Q212X C>T NM_024070 DX UPN033
DNMT3A Missense chr2:25457242 R882P C>G NM_175629 DX UPN034
MLL3 Stopgain chr7:151962134 C391X G>T NM_170606 DX UPN034
RAD21 Stopgain chr8:117864806 Q435X G>A NM_006265 DX UPN034

Frameshift
TET2 chr4:106155467 R123fs */+T NM_001127208 DX UPN034
insertion
Frameshift
WAC chr10:28897226 p.241_241del */-CT NM_100486 DX UPN034
deletion
LRBA Missense chr4:151186882 R2850C G>A NM_001199282 DX UPN034
EPPK1 Missense chr8: 144944226 R1066C G>A NM_031308 DX UPN034
BAHCC1 Unknown chr17:79428415 Unknown C>T Unknown DX UPN034
OR10G7 Missense chr11:123909309 M134V T>C NM_001004463 DX UPN034
DNMT3A Missense chr2:25463248 R749C G>A NM_175629 DX UPN035
FAT1 Missense chr4:187518126 A4190T C>T NM_005245 DX UPN035
IDH1 Missense chr2:209113112 R132H C>T NM_005896 DX UPN035
TNRC6A Missense chr16:24828190 D1629N G>A NM_014494 DX UPN035
TNRC6A Missense chr16:24828253 D1650N G>A NM_014494 DX UPN035
RP1L1 Missense chr8:10467632 K1326E T>C NM_178857 DX UPN036

Frameshift
insertion
Frameshift
RUNX1 chr21:36253000 L94fs */+G NM_001001890 DX UPN037
insertion
Frameshift
WT1 chr11:32417942 R353fs */+G NM_000378 DX UPN037
insertion
ARID5A Missense chr2: 97217238 L325V C>G NM_212481 DX UPN038
Frameshift
NPM1 chr5:170837544 W288fs */+CCTG NM_199185 DX UPN038
insertion
NBPF1 Unknown chr1:16918351 Unknown T>G Unknown DX UPN039
ACTG2 Missense chr2:74141834 K171R A>G NM_001199893 DX UPN039
DNMT3A Missense chr2:25457242 R882H C>T NM_175629 DX UPN039
RNF213 Missense chr17:78341843 R4019C C>T NM_001256071 DX UPN039
RUNX1 Missense chr21:36252859 G141V C>A NM_001001890 DX UPN039

Non Frameshift 1866_1867de
SRCAP chr16:30736343 */-GTT NM_006662 DX UPN039
deletion l
NOTCH2 Missense chr1:120548030 R113X C>G NM_024408 DX UPN039
NBPF3 Missense chr1:21809667 P494A C>G NM_001256417 DX UPN039
SRGAP2 Unknown chr1:206516335 Unknown C>T Unknown DX UPN039
DNAJB6 Missense chr7:157208773 S321L C>T NM_058246 DX UPN040
EGR1 Missense chr5:137802652 S172A T>G NM_001964 DX UPN040
MLL3 Missense chr7:151921184 P1080L G>A NM_170606 DX UPN040
TP53 Missense chr17:7577108 C145F C>A NM_001126115 DX UPN040
U2AF1 Missense chr21:44524456 S34F G>A NM_001025203 DX UPN040
XIRP2 Missense chr2:168104183 T1872R C>G NM_001199144 DX UPN040
5
BCLAF1 Missense chr6:136597467 G397E C>T NM_001077440 DX UPN040
Frameshift
WT1 chr11:32417941 V354fs */+A NM_000378 DX UPN041
insertion
TTN Missense chr2:179423251 V19914I C>T NM_003319 DX UPN042
TTN Missense chr2:179428921 G18248E C>T NM_003319 DX UPN042
FAT1 Missense chr4:187518053 A4214V G>A NM_005245 DX UPN042
FAT4 Missense chr4:126372029 I3286M A>G NM_024582 DX UPN042
FLNB Missense chr3:58097539 R836G A>G NM_001164317 DX UPN042
LRP2 Missense chr2:170053432 P2896L G>A NM_004525 DX UPN042
TET1 Missense chr10:70405299 R938T G>C NM_030625 DX UPN042
TYRP1 Splicing chr9:12704526 G361A G>C NM_000550 DX UPN042
ZFHX4 Missense chr8:77763592 D1479H G>C NM_024721 DX UPN042

Frameshift
insertion
EPPK1 Missense chr8:144940551 V2291M C>T NM_031308 DX UPN042
NBPF1 Unknown chr1:16918372 Unknown C>A Unknown DX UPN043
ANKRD24 Missense chr19:4207293 L174P T>C NM_133475 DX UPN043
CSMD1 Missense chr8:2887020 S2559I C>A NM_033225 DX UPN043

Frameshift
insertion
TTN Missense chr2:179490113 R5747H C>T NM_003319 DX UPN043
PCDHB16 Missense chr5:140563579 T482S C>G NM_020957 DX UPN043
POLD1 Missense chr19:50905046 G110C C>T NM_002691 DX UPN043
ATRX Missense chrX:76854993 S1910N C>T NM_138270 DX UPN044

Frameshift
MUC17 chr7:100684949 T3418fs */-A NM_001040105 DX UPN044
deletion
CYP4A11 Missense chr1:47402422 H142Y G>A NM_000778 DX UPN044
DNAH9 Missense chr17:11568265 K904T A>C NM_001372 DX UPN044
DNMT3A Missense chr2:25458595 W860R A>G NM_175629 DX UPN044
FBXO34 Missense chr14:55818517 I470S T>G NM_017943 DX UPN044
FLNC Missense chr7:128482859 I801V A>G NM_001127487 DX UPN044
NOTCH2 Missense chr1:120459148 N2066S T>C NM_024408 DX UPN044
PIRT Stopgain chr17:10728713 K84X T>A NM_001101387 DX UPN044
NBPF1 Unknown chr1:16907286 Unknown T>C Unknown DX UPN044
PRDM15 Missense chr21:43248619 K516N C>A NM_001040424 DX UPN044
MLL3 Missense chr7:151945556 V655I C>T NM_170606 DX UPN044
SRGAP3 Missense chr3 : 9094756 S426R G>C NM_001033117 DX and REL UPN045
NBPF1 Unknown chr1:16894519 Unknown C>T Unknown DX and REL UPN045
FAT1 Stopgain chr4:187540153 Y2529X G>T NM_005245 DX and REL UPN046
FAT4 Missense chr4:126370842 V2891I G>A NM_024582 DX and REL UPN046

Frameshift
TET2 chr4:106157628 E843fs */-G NM_001127208 DX and REL UPN046
deletion
6
NBPF1 Unknown chr1:16890680 Unknown G>A Unknown DX and REL UPN046
FAT1 Missense chr4:187540095 F2549L A>G NM_005245 DX and REL UPN046

Frameshift
NPM1 chr5:170837543 W288fs */+TCTG NM_199185 DX and REL UPN047
insertion
GLUD2 Missense chrX:120182436 R300G A>G NM_012084 DX and REL UPN047
MRPL36 Missense chr5:1798950 I34L T>G NM_032479 DX and REL UPN047
SETD1B Missense chr12:122242782 L47M C>A NM_015048 DX UPN047
WAC Stopgain chr10: 28905289 Q479X C>T NM_100486 DX and REL UPN047
ANKMY1 Missense chr2 : 241465261 H162Q G>A NM_017844 REL UPN048
TET1 Missense chr10 : 70405237 E917D G>A NM_030625 REL UPN048
WT1 Stopgain chr11 : 32417910 S364X G>T NM_000378 DX and REL UPN048
Frameshift
NPM1 chr5:170837543 W288fs */+CATG NM_199185 DX and REL UPN048
insertion
Frameshift
PCSK2 chr20:17437050 A352fs */+C NM_001201529 DX UPN048
insertion
ATRX Missense chrX:76854991 D1911Y C>A NM_138270 DX UPN049
TET2 Stopgain chr4: 106196424 S1586X C>G NM_001127208 DX and REL UPN049
Frameshift
insertion
MUC6 Missense chr11:1016854 P1983S G>A NM_005961 DX UPN050
ETV6 Missense chr12 : 12043919 R433H G>A NM_001987 DX and REL UPN050
FBXO38 Missense chr5: 147820079 Y1088C A>G NM_205836 DX and REL UPN050
chr12 :
PTPN11 Missense T42A A>G NM_080601 DX and REL UPN050
112884189
UNC5B Missense chr10:73056469 Q809H G>C NM_001244889 DX UPN050
DNMT3A Missense chr2:25457247 M880I C>A NM_175629 DX and REL UPN050

Frameshift
insertion
Frameshift
insertion
DNMT3A Missense chr2:25457242 R882H C>T NM_175629 REL UPN051
MLL3 Stopgain chr7:151962134 C391X G>T NM_020715 DX UPN051
EP300 Missense chr22:41574351 Q2212H G>T NM_001429 DX UPN051
PLEKHH1 Missense chr14:68044836 P891A C>G NM_020715 DX UPN051
ABR Missense chr17: 912962 S730A G>C NM_001092 REL UPN052
SF3B1 Missense chr2: 198266834 K700E T>C NM_012433 REL UPN052

Frameshift */+GGTC
RUNX1 chr21:36252939 R149fs NM_001754 DX and REL UPN052
insertion CA
AHNAK Missense chr11:62295516 H2125N G>T NM_001620 DX UPN053
DNMT3A Missense chr2:25464555 L653W A>C NM_175629 DX and REL UPN053
WT1 Stopgain chr11 : 32413578 R441X G>A NM_000378 DX and REL UPN053
FRG1 Missense chr4:190876269 I132T T>C NM_004477 DX UPN054
DNMT3A Missense chr2 : 25457243 R882S G>T NM_175629 REL UPN054

Frameshift
insertion
NRAS Missense chr1:115258747 G12V C>A NM_002524 DX UPN054
CYP11B1 Missense chr8:143956536 R412H C>T NM_000497 REL UPN054
RUNX1 Missense chr21: 36252866 R139G G>C NM_001001890 DX and UPN055
7
REL
Frameshift
ASXL1 chr20:31023109 E865fs */-A NM_015338 REL UPN055
deletion
SF3B1 Missense chr2:198267360 K666T T>G NM_012433 DX UPN055
FAT4 Missense chr4:126367465 G2404V G>T NM_024582 REL UPN055
MLL3 Missense chr7: 151902199 R1318I C>A NM_170606 REL UPN056
MLL3 Missense chr7: 151962265 D348N C>T NM_170606 REL UPN056
ASXL1 Missense chr20 : 31023894 D1127N G>A NM_015338 REL UPN056
LRP2 Missense chr2 :170068577 Y2061N A>T NM_004525 REL UPN056
MLL2 Stopgain chr12:49425962 G4176X C>A NM_003482 REL UPN056
NOTCH1 Missense chr9 : 139404319 D945E G>C NM_017617 REL UPN056

Frameshift
NRAS chr1:115251215 L171fs */+T NM_002524 REL UPN056
insertion
NSD1 Missense chr5:176721942 D2525N G>A NM_022455 REL UPN056
Frameshift
insertion
PRAM1 Missense chr19:8564469 E75K C>G NM_032152 REL UPN056
PRDM15 Missense chr21:43248608 G520A C>G NM_001040424 REL UPN056
NFIX Stopgain chr19:13135897 W30X G>A NM_002501 REL UPN057
IDH2 Missense chr15:90631934 R140Q C>T NM_002168 REL UPN057
NBPF1 Unknown chr1:16895623 Unknown G>A Unknown DX UPN058
ASXL1 Stopgain chr20:31022592 R693X C>T NM_015338 REL UPN058

Frameshift
BCOR chrX:39932937 I554fs */-G NM_001123383 REL UPN058
deletion
STAG2 Stopgain chrX:123229240 R1205X C>T NM_006603 REL UPN058
Frameshift
NPM1 chr5:170837543 W288fs */+TCTG NM_199185 REL UPN058
insertion
SPDYE6 Missense chr7:101988944 R310H C>T NM_001146210 REL UPN059
EZH2 Missense chr7:148506461 R628H C>T NM_001203249 REL UPN059
FLT3 Missense chr13:28608275 F594S A>G NM_004119 REL UPN059

Frameshift
RUNX1 chr21:36252950 A122fs NM_001001890 REL UPN059
insertion
ASXL1 Stopgain chr20:31022592 R693X C>T NM_015338 REL UPN059
TP53 Missense 17:7577121 R273C C>T NM_001126115 REL UPN059
IDH2 Missense chr15:90631934 R140Q C>T NM_002168 REL UPN060
POTEH Missense chr22:16287515 D124G T>C NM_001136213 REL UPN060

Frameshift
NPM1 chr5:170837543 W288fs */+TCTG NM_199185 REL UPN060
insertion
DNMT3A Missense chr2:25457242 R882H C>T NM_175629 REL UPN060
RUNX1 Missense chr21:36231783 R174Q G>A NM_001001890 REL UPN061

Frameshift
RUNX1 chr21:36231837 L192fs* */+TGC NM_001001890 REL UPN061
insertion
IDH2 Missense chr15:90631838 R172K C>T NM_002168 DX and REL UPN062
DNAH9 Missense chr17: 11725809 F2969L T>C NM_001372 DX and REL UPN063
IDH2 Missense chr15: 90631934 R140Q C>T NM_002168 DX and REL UPN063
KCNH6 Missense chr17:61601651 S76R C>A NM_030779 REL UPN063
NPM1 Missense chr5:170837543 W288fs */+TCTG NM_199185 DX and REL UPN063
8
B3GAT3 Missense chr11:62383261 W307S C>G NM_012200 DX UPN064
Frameshift
NOTCH4 chr6:32181010 G780fs */+C NM_004557 REL UPN064
insertion
Frameshift
NPM1 chr5:170837543 W288fs */+CATG NM_199185 DX and REL UPN064
insertion
NBPF9 Unknown chr1:144618114 Unknown G>A Unknown DX and REL UPN064
FAT4 Missense chr4:126242341 P1592R C>G NM_024582 DX UPN064
ASXL2 Missense chr2:26101059 R11S C>G NM_018263 DX UPN065

Frameshift
insertion
C12orf51 Missense chr12:112743908 P288L G>A NM_001109662 DX UPN065

Frameshift
THPO chr3:184090395 L323fs */+C NM_000460 REL UPN065
insertion
EPPK1 Missense chr8:144940303 N2373K G>C NM_031308 REL UPN065
KLHL7 Missense chr7:23180541 L199P T>C NM_001031710 DX UPN066
KLHL7 Missense chr7:23180543 T200S A>T NM_001031710 DX UPN066
UNC5B Missense chr10:73048356 W311C G>C NM_001244889 DX UPN066
UNC5B Missense chr10:73048357 S312P T>C NM_001244889 DX UPN066
WT1 Missense chr11:32414232 R423K C>T NM_000378 DX UPN066
EZH2 Missense chr7:148508788 V570M C>T NM_001203249 DX UPN067

Frameshift
TTN chr2:179656902 T520fs */+T NM_003319 DX UPN067
insertion
KDM6A Missense chrX:44936012 S925T T>A NM_021140 DX UPN067
MLL3 Missense chr7:151874398 D2714N C>T NM_170606 DX UPN068
MLL3 Missense chr7:151874401 D2713N C>T NM_170606 DX UPN068
RUNX1 Stopgain chr21:36231783 R174X G>A NM_001001890 DX UPN069
ANKRD24 Missense chr19:4207293 L174P T>C NM_133475 DX UPN070
CNTNAP4 Missense chr16:76486472 Y307F A>T NM_138994 DX UPN070
LRBA Missense chr4:151738288 G1765R C>T NM_001199282 DX UPN070
MSH4 Missense chr1:76356415 S754L C>T NM_002440 DX UPN070
OR10G7 Missense chr11:123908942 F256S A>G NM_001004463 DX UPN070
ZFHX4 Missense chr8:77764484 E1776G A>G NM_024721 DX UPN070
PKD1L2 Unknown chr16:81181843 Unknown C>T Unknown DX UPN70
PVRIG Missense chr7:99817556 P8L C>T NM_024070 DX UPN070
BCLAF1 Missense chr6:136589425 P756S G>A NM_001077440 DX UPN070
BRIP1 Missense chr17 : 59858329 Y556H A>G NM_032043 DX UPN071
CENPF Missense chr1: 214819230 R2106K G>A NM_016343 DX UPN071
EFCAB5 Missense chr17: 28407139 L1026S T>C NM_198529 DX UPN071
JHDM1D Missense chr7: 139796405 S775N C>T NM_030647 DX UPN071
MLL3 Missense chr7: 151882657 R1690G T>G NM_170606 DX UPN071
TET1 Missense chr10: 70333850 K585N G>T NM_030625 DX UPN071

chr11 :
MLL Missense D2677E C>T NM_005933 DX UPN071
118374647
9
Frameshift
NPM1 chr5:170837545 W288fs */+CCTG NM_199185 DX and REL UPN072
insertion
POTEE Missense chr2: 131976077 C34W C>G NM_001083538 DX UPN072
KIT Missense chr4:55593603 W557R T>C NM_000222 DX UPN073
KLHL7 Missense chr7:23163447 H58Y C>T NM_001031710 DX UPN073
RNF213 Missense chr17:78263497 E325K G>A NM_001256071 DX UPN073
RUNX1 Missense chr21:36252950 E111K C>T NM_001001890 DX UPN073
STAG1 Stopgain chr3:136060372 W1156X C>T NM_005862 DX UPN073
XIRP1 Stopgain chr3:39225761 Q1726X G>A NM_194293 DX UPN073
ARID1A Missense chr1:27106024 R1879W C>T NM_006015 DX UPN074
FAT1 Missense chr4:187584755 T1093M G>A NM_005245 DX UPN074
GJB5 Missense chr1:35223154 R75C C>T NM_005268 DX UPN074
IDH2 Missense chr15:90631701 G190S C>T NM_002168 DX UPN074
MLL3 Missense chr7:151932969 L901P A>G NM_170606 DX UPN074
PRDM15 Missense chr21:43248664 M501I C>T NM_001040424 DX UPN074
RNF213 Missense chr17:78269393 V598M G>A NM_001256071 DX UPN074
SETD1B Missense chr12:122255744 E1075Q G>C NM_015048 DX UPN074
U2AF1 Missense chr21:44524456 S34Y G>T NM_001025203 DX UPN074
PHF8 Missense chrX:54028650 R396K C>T NM_001184896 DX UPN074
SRCAP Missense chr16:30721033 P278R C>G NM_006662 DX UPN074
DDX41 Missense chr5:176942967 A133T C>T NM_016222 DX UPN075
GATA2 Missense chr3:128200742 T355A T>C NM_001145661 DX UPN075
GATA2 Splice site chr3:128200744 T354R G>C NM_001145661 DX UPN075
MLL2 Missense chr12:49440509 G1434D C>T NM_003482 DX UPN075
PTPRT Missense chr20:40730913 V1189M C>T NM_007050 DX UPN075
XKR4 Missense chr8:56435873 A347V C>T NM_052898 DX UPN075
MGA Missense chr15:42028627 P1389S C>T NM_001080541 DX UPN075
ODZ2 Missense chr5:167645662 G1580E G>A NM_001122679 DX UPN075
MUC17 Missense chr7:100675956 S420F C>T NM_001040105 DX UPN075
FAT1 Missense chr4:187525647 F3478L A>G NM_005245 DX UPN076
HUWE1 Missense chrX:53589108 E2434D T>A NM_031407 DX UPN077
DNMT3A Missense chr2:25457270 H873N G>T NM_175629 DX UPN078
FLT3 Missense chr13:28608255 L601F G>A NM_004119 DX UPN078

Frameshift
RUNX1 chr21:36252991 V97fs */+CATC NM_001001890 DX UPN078
insertion
FLT3 Missense chr13:28608266 Y597F T>A NM_004119 DX UPN079
Frameshift
NPM1 chr5:170837545 W288fs */+TGCA NM_199185 DX UPN079
insertion
Frameshift
TET2 chr4:106156024 A309fs */+A NM_001127208 DX UPN079
insertion
IDH1 Missense chr2:209113112 R132H C>T NM_005896 DX UPN080
IDH2 Missense chr15:90631934 R140Q C>T NM_002168 DX UPN080
10
Color types Number
Confirmed as a valid mutation by Sanger sequencing 283
Confirmed as a false mutation by Sanger sequencing 24
Germline SNP 7
Total Sanger events 314
True positive rate 91.03
Difficulty in designing primers; PCR failure; poor signal of Sanger sequencing 38
DX : Diagnosis
REL : Relapse
RefSeq : The Reference Sequence
11
Supplemental Table 9: List of primers used for the validation of somatic mutations detected in Frequency
Cohort
Gene Name Forward Primer (5' to 3') Reverse Primer (5' to 3')
ACTG2 CTGCACTTCAGTCTGGGTGA GAAGGCTGGAAGAGGGTCTC

AHNAK CCAAGATCAAAATGCCCAAG TCCCCTGTAACTTGCCTGTC
AHNAK AAAAGCCCCAAAGCTGACAT TGGAGTTCACCCTCCAGTTT
AHNAK ATTGAAGGTCCAGACGCAAA GGCCTTTTAGTTTCCCCTCT
AHNAK AAGGCCCTAAGTTCAAGATGC GAGATCTTGGGGGCTTTGAT
AHNAK GCCAGAGATGAACATCAAAGC ATCAGGCATGGAGATCTTGG
ANKMY1 CCATTTCCCTGGCATTTG AAAACAAGCCCACTGGCTAA
ANKMY1 AACTTGTTGTGTTGCCAGGA CAGCTGAGTGGGTTTCTGCT
ANKMY1 CGTTCAGCAGAAACCCACTC AGACAGTTGGTGGTGCAGTG
APOB TCAGCTTACCCTGGATAGCAA TTCCCTTCTCCAAACAGTGC
APOB TGGTGAAATTCAGGCTCTGG ATGGTCCCAAGGATGGTCTT
APOB AAATGGAGAAGCAAATCTGGA TGGTGAATGGAGACACTTCAA
APOB CATCACACTGAATACCAATGCTG GCTTGTGGTCAACTGCTCCT
APOB TTGCATTAGATGATGCCAAAA GCACTCTAACATCAATAGCCTCAA
ARHGAP39 AGGTGTACAACAGAGGGTTCG CCCTCTTCAGGAAGGGTGAC

ARID1A AAGGACAAGCCAGCTTCAGA AATGCCAAATGGAAACTTGC
ARID2A TTCAGATGTGCCTCAGCAAC TAACTTACCTGCGGGACCAC
ASH1L ACCATATTTCTATTACTTCTTGTCAGG TCCAAATGTGGAAGAAAAAGG

ASH1L AATGGGAGGTGCTTCTGATG TGGATTCAGAAGTCAAACTTGG
ASXL1 CTGCCAGGCACATCCTCT ACTTCAGGGCCCCAGAGTTA
ASXL1 TCCTGGGAAAGTGATGATGA TTTCTCCCACTCCTCTCCAA
ASXL1 GGACCCTCGCAGACATTAAA AGGCATCTCCTAGCCCATCT
ASXL1 GAGCACCCCTGGAAAGTGTA AGAATGGGACCATTGTCTGC

ASXL1 CAAGGTGAATGGTGACATGC CAAGACCAGTGCCTGTTTCA
ASXL2 AACCTTGCAAAGTAAGCAGCTC TCCCATTTTCAAGGCTTCAG
ASXL2 TTCCCTTTTCTCCCACCTTT ATCGCAACCCCGACACTA
ATRX CTTCAAAAGACAACAGAGGAGGT CACTGTCAGAAGAATTACGC
ATRX TTAACCAAATACGGGAGCAGA TCACAGCAGACTAAGATGAACCT
ATRX TGATGCATATTTCAGTGGGAAT AAAAGGCAAAAACCTGAAGGA

BCORL1 AGCTTCCCGCATCTGACA AGAGATGAGCGTGGGCACT
BCORL1 CTGGGAGCTGTCTCCTGCT CATGTGCTTGGTCTTGCACT
CLCA4 TTGCTTCCTTTAACAACATCAA TTTGCAGTACGATTTTCACCTG

CNTNAP4 AGCAGAGCAAAGGGTTGAAG AAGAGGAGGATACCCCCTGA
CYP4A11 TAAGCCTAACCCACCTGCAT AAAGCGTGGCTGTGTTGAG
CYP4A11 ACCTCCTTTTCCTGCATTTG TGACCTGAGAGTGTTGTTCCA
DAAM2 CCCACTCACCTTGTCTTCCT CATCCACATGACTGCCCTTA
DDX41 GCAAAGAGTCTGCCAAGGAG CAGAACATAACGGGGTGGAG
DNMT3A AGGTCACAGAAACCAGCACA GGTATTTCCGCCTCTGTGG
DOT1L CAGCTTCAGTGATGGTGCTT CCGCAGAGATGAAGAGGTTG
DOT1L CAGCTACTTGGGAGGCTGAG TCAGATCAGCTTGCACCATC
EPHB1 AAAAATTGCTGTTTGTTTTGGTT AGGCTGCTCTTGGATTGAGA

EPPK1 GATCCAGGAGAAGCTGCTGT AGAAAACTGCACGGAGGAAG
EPPK1 GCAGCTACCGGCTTCATC GCTGCAGGTACGTGAGGTTC
EPPK1 CCTTCAGTGGCTCCCAAAT CAGCTCCCAGAGGGACAC
EPPK1 CGTCAAGCGCTACCTGGAG CGGACGATGAGGTCCTTCT
EPPK1 GCAGCTACCGGCTTCATC GCTGCAGGTACGTGAGGTTC
EPPK1 GGCTACACCGACCCCTACAC CAACCCAGACACACAAGTATGC
EZH2 TCAGCAGCTTTCACGTTGAC CTCCCAAAGTGCTGGGATT
EZH2 GGTGATTAATTTTAACCTGGAACAA CACACTGGTGTCAGTGAGCAT
FANCI CTCCCAAAGTGCTGGGATTA TGCCTGATCACCTTTTAGCC
FANCI GGAGGAGGAAGAGGCATTCT CAGCGCTCTCTAGTGGAAAGA
FANCI TCAAAGCCCTTAACCATTGC CGGCCTGCTTCTTTGTTTT
FAT1 CCAAATCAACCATTGCAAGA CTCAAACGTTATCCGGCATT
FAT1 AGAAACGGGGAAGTGCATTA AAGGAGTCAGTTGCGCGTAT
FAT1 GCCTATATTATGTGCCAAGTGTTC TCTGGATCAAATGCCTCGAT
FAT1 TGCAGCATGAGCACTCCTTA TTGTCTTTGGCCCTCACAGT

FAT1 CCCACCTCATACAGCGTTTC CCCTGTCCAGTTCTGATGGT
FAT1 TACGGATGGGGATTCTGGTA CATAGGTGATGTCGGCATTG
FAT1 CCTGTCAGTGTCTGATGGAGTT TCGAAATTGTGGTGCATTGT
FAT1 ACACATGCGGAGTAGCATGA CTCCAGCTGCTGAAAACCTC
FAT1 CCAGCAGTTGGACTTTGAGA ATGGAAAAGGGCATCAAATG
FAT1 ACCATGAGATGGTGGCTTCT ATGGCTGTGGAGGATAGCTG
FAT1 TCTTCCCTTTTCGCTACACC CGCCAAGCAGCTCTTCTCTA
FAT4 GCAACAGATGCTGACTCTGG GATCTGGAAACCCTTCTTTCG

FAT4 TTTTCCCTCCAACAGGTGTC AACATGATTTGCACCCCAAT
FAT4 CCAGCACAGGGGATATTGTC TGAAGCCAGTGACATTTTGG
FAT4 GAAGGCACAAATGGACAGGT GAGGAGAGAACAGGGGAAGC
FAT4 GCAATAGATGACAAAGATTTTGGA TTGATTGCAAAAATGCCTTC
FAT4 AGATCCTTCTCCGGCAGAGT TGGAAAATTCTGGCAGATGG
FAT4 ATGGGGATGACATGACTGTG TGTGTCGAGACAGAGGGTTG
FAT4 TGCTGGACGTGAATGACAAT AGACCAGAGTCGCCATCAGT
FAT4 GGTGGCCTTGCACAACTTA TGGGTGATGCTGAAATCTTG
FAT4 TGCTTTGAAGAACGTGACCA AGAGGCGTCCAACAGTTTTT
FAT4 GTGTCCCACAGCTCTCCTCT AAATCTGGCTGCACCATTTC
FLNC TGTCACCAAAGATGCTGGAG ATGACCCCTCACACCTTGAA

FLNC GGAACAGGGAGGGATGATTC ACATCCAGCTTGGCCTTG
FLNC CCAGGTAAGGGGCAGGTG ACATGTTTCTGAGGGTGTGG
FLNC AGCGGGTAAAGGTGTACGG CATCTCCAGCTGCCTTCTG
FLT3 TCCATCACCGGTACCTCCTA CCCTGAAGCTGCAGAAAAAC
FLT3 CATTGTCGTTTTAACCCTGCT TTGCGTTCATCACTTTTCCA

FLT3 TACAGGCATGACCCACCAT CCACTTGGGTTTGAGAGTTCA
FLT3 TGCTAACCTTCTCAATCAACCA CAGCGATGGGGACTAACTTC
FRG1 AAGAAGGTAAAATAGATCTTGATTCCA GATAGGAAATCCATTAAAAGTAGATCA

FRG1 ACACAGCCACACACATACGC TTGTGGGCAAAAGGACTAATG
FRG1 GAGTCACCACTCAGCCACAT TTTTGAGATCTCTTTAAGGCTCTATG
FRG1 TTGCAGAAGAAAATAGCCACA GGAAATCCATTAAAAGTAGATCACAAA
GATA2 TTCCATGGTTCTGAGATGCTT AGCCAAGCTGGATATTGTGG
GATA2 TTCCATGGTTCTGAGATGCTT CCTTCTGGCGCTCACTCAG
HUWE1 TCAAGGAACCAGGTAAATGTTG CAGCCACTCAACAAAGGACA
HUWE1 TGGTTGCCCCTTAACCACTA TTCTCTCAAGCAAAAGAGCAAA
HUWE1 TATCCCATCACCTTCCCACT CATCCTCCTTAAGGGAAAATAGG
HYAL1 TCTATGGCTTCCCTGACTGC AAAGTACCCCAAGGCTGGAC
IDH1 AACGACCAAGTCACCAAGGA TGTGTTGAGATGGACGCCTA
IDH2 GCCCACACATTTGCACTCTA TGTGGCCTTGTACTGCAGAG
IDH3B 5'-TCATCAAGTCTGTCATCGGTCA-3' 5'-AATGCAAATGGACATTCTCTGG-3'
IGF2BP3 GACAGAAGGAAGCTAAGGCTCA
GCTGTCTTCCTCCAATTTTCA
IGF2BP4 TTCACCAAGCATTCAGTTGC AAAATTCGGGGACTTGGAAC
IGF2BP5 CCATGGAACATCTGCTTTGA AACCCCGCTATCCAAAAACT
JAK1 CCAGCAGCACTGACCTTTTA AAAACACTGAGAATGCCTTGG
JHDM1D GCTACACGAACTCTGAAACCTG GACTTCAGAGGCCTGAAAACC
JHDM1D CAAGACTCTGTCTCAAAAACAAACA CCACTGAAAAACTAGGGCTATATG
JHDM1D TTTTACCAAAAATGTCATCAGAAAA CGTGATATGTCAGCATTGTGC
KDM5C GAGCCAGGGAATCAAGTCAG CCCCTTAGCTGTTACCGTCA
KDM5C AACCAGAGATCACTGTCCACAA GGTCTCACCCAGGAAAACTG
KDM6A TCACCCCATAAAATGCGTTA TGATACACACACTCTCTCTCATTCTT

KDM6A GGATACAGTGCCGTAAAATGC TGTGTCCTTTCAAAACTCCAAA
KDM6A TCAGCTTTGTTTTGATTCTCACA CCTTAAAGCTGGGCACCTG
KDM6A GGATCCACATCCCACATCTC CGCCCCCAAACTATTTAATC
KDM6A TGAGGTTTTCAGAATTGATGTTG TTTGCACCATTTTGCTTTGA
KDM6A AGGCACACTGGAGAGACACC ACTTCTGCTGAGCTGGGGTA
KDM6A TGTCGAGGGACTTCCTAATCA ACCTGCATGCCTTCAGAACT
KLHL7 TGAAACAGAAACGGATCCAGA TTTCCCTTGTCATGTGTCCA

LRBA TGAAGATATTTCATGTTGGGTGA GTTTCTAATGTGCCCCCAAA
LRP2 TTTGATAGTGTGGCTGATTCAAA TGCCATCCCTTCTCTAGCTC
LRP2 CCAATCGAACAGTGCTTGTG GAGATGAGAGCACCCACCAT
LRP2 TGCCTTCCAAGCTGATAACC AGCATCCCATCAGCTGAAAA
LRP2 AACAACAGTGAGGAGGGAGGT TCATATCCAGGCCCAGACTC
LRP2 GGTAGGAAACTGGGTGGCTA GATGATTTGAAGAAGCCATGC
LRP2 TCCGATGTTGTTTTTAGATTGC GTGAGCCCTCCAGGTGATT
LRP2 TCCATTTTCCAGGCTCAGTC CCCACACAGTGCTCTCCAC
MLL TCTGAGCTCTCACCTCTCACC CTTTGGAAACCCTACCCACA
MLl GCCTCCATCAACAGAAAGGA GACGGGGCACTGAATCTACT

MLL CTCGTCTTCGTCTTCGTCATC AAGGGTCAGGGGGAGGAG
MLL2 CACCTGTAAAGGGTCCTGCT AGATATGCAGCCCCCTCACT
MLL2 CCTGAGGAGCCACACCTATC GCTCTCCAAGCAAGGGAGAT
MLL2 GTGTCACGCCTGTCTCCAC GGACAGGTGCAATTCCTCAG
MLL2 TCTGAGGCTTCCAGGACAAC CGTACTGCCTGACTCTGCTG
MLL3 TGCAGTCTTCAGGAGGGTCT TGAGACAAACATGGAAAATTGA
MLL3 TTCATGTGGGTCTGTCCTCA AAAACAAGGCAGATGGGAAA
MLL3 TGAAAGGTTCATGATGCAAATC TGATGAAGTCACTGAATGAATATAGC
MLL3 TTTTATCTCAGTGGCATTTGGA GAGGAAGGTAAGAAAAGAGAAAGGA
MLL4 GTCCTCAACATCTCCCCTCA GGTTTTGCCATGTTGCCTAC
MLL4 GTTTGTTTCAAGGGCCAAAA GGATTCCTCCTGCTCCTCTT
MLL4 CTCGAATCAAAGTGCCCAAC AGTCCTGACACCACCTCCAT
MSH4 TGCTCAGCAGGATGTCTCTAAG AAAGCTATTTGTGGGTGTCCA

NFIX GCTGCTTTTCTCCTTCTCT TCCCCTTAAACAAAATCACCA
NPM1 TTTTTATGACTGATTAAAGTGTTTGGA CAGGCATTTTGGACAACACA
NPM1 TCTCGAACTCCTGACCTCGT ACAGGAGTGAGCCACTGAGC
NPM1 TCACTGGAGTTCGATGGTCA TGTCCTCTCCAATTTCTAGACTCA
NPM1 TCTGCTTTGCCCTCTGGTAG CAGTGTGACTGGAATTTACATGG
NSD1 CACTGGAGAAAACCTCAGTTCC TGCTGCTTTCCCAGATGTC
NSD1 GGTTTAGCATTTGGTAGATTCTTGA AGATCACGCCACTGCACTC
NSD1 TATGGCAGAACCCCCAGTTA GACCAGGACTCCGGTAAGAG
OR10G7 GACACCTCAGCCAACGAGAT CACCCTGAGCAAATACTGACC

PDXDC1 AGCTCACAGAGTTGTTGGAAGA ACATGCAGGGCGATTAGTCT
PDXDC1 CTGGGGCTAATATTGGGATG AGCAATGCCAGAAGGACTTG
POM121C GGAAAATCCGAAGTAGAGTTCC GCTGCTCAGAAGGCTGGAT
POM121C CTTCCAGCCTGTCTTTAGCA CAGGGACTGGCTGAAGGTG
POM121C GGGAGGACGCTGAAATACTG GAAAATGGGCTTGAACATGG
POM121C TTTGGCCAGATTGTTAGCAG TCCAAAGTGCTGGGATTACA
POTEE CCCAGATCATGTTTGAGACCT GGCAGCTCGTAGCTCTTCTC
POTEE AGCAAGATGGGCAAGTGGT AGGGTATGTCCCCATCATCC
POTEE GAAACCCGGAGTTACCTGCT CACCACTTCCCCATCTTGTT
POTEE CTGGACAGACGATGTCAACTT TCGATATCAGCACCATATAAGAGC
POTEE GGGAACTGCCTGACTACCTC CATGCCAGGGTACATGGTG
POTEE GGTTTGAAGTTTTGTCCAATTTTT ACATGCCAGAGCCGTTGT
PRDM15 TCCCCTCTGTAAACCTCAACA CCTGAGCTCCAGAACATTCC
PRDM15 CCGCGACTCAGTTTACCAAT CCAAACATCCCTCTGCAGTT
PRDM15 TGCTTGGTGATGAAAGGTGA CCCTGCTGACTGCTCCAT
PTPN11 CGTTCCTTGGGTTTCTTTCA ACCGTCATGCATTTCTGACA

PTPN11 TTGCTTTTCACAGTGTTTTCTGA TTCCTAAACATGGCCAATCTG
PTPN11 CCTGGCTCTGCAGTTTCTCT AAGCTCCTCTGCAGTGAAAAA
PTPN11 CCTCTGTCCGTGCCTTTATG TTTCGAAGAAGCCCTCTCAG
PTPN11 TGAAAGAACAACATGAACCCATA GGTGAAACCCCATCTGTAGG
RAD21 GCAGGGGAATATAGTCAGTCCA TGTGCCATACAGTTGCAAAA
RAD21 TTGGAAGATAGAAATCAGTGGTGA TGGAAAGACAGGAGGCTTCA
RAD21 GACTTCAGTAAGCAAATGTGACATAA GCTGTTCAAGACTCAGAATTAGCA
RUNX1 AAATTCCGGGAGTGTTGTCA GCAACTTTTTGGCTTTACGG
RUNX1 CATTGCTATTCCTCTGCAACC ACCGAGTTTCTAGGGATTCCA
SETD1A GAGCCCATTCCTCTCTCCTT CTATGCTGCCCTCCAAAAGA
SETD1A CTGGGCAATAGAGCGAGACT CCAACTCCCTCTTGGAGACA
SETD1A CTGGGCAATAGAGCGAGACT TGGGCATCTTCTTCCCAGT
SETD1B CTTCCCTTCTGGTGATCCAA GAACCTCCCGAAGACTGCTC
SETD1B GATTGGATTCTTTCGCGTGT CCTTTCTCGAGGCACTTGTC
SETD1B AAATGTCTCGTGTTCGCTCA AGAAGGTGGGTGTGAAGCTG
SETD1B AGAGGAGGTGGACATCGAGA ATAGCAGTCGTTCCCCTTTG
SETD1B TCCTCCTCAACCTCATCCAC CAGCCCTAAGGACCACAGAC
SETD1B GACGGTGACCCTGGACTTC GTCGTTAAGCCAGTCCATGC
SETD1B CGAGATGGTCATCGAGTACG TATAACCCTTGGCCAACCTG
SF3B1 TTTGCTGTTGTAGCCTCTGC GATCACTTGAGCCCAAAGGT
SF3B1 TGCTGACAGGCTATGGTTCA GGCGGATACCCTTCCATAAA
SGK2 TGCACAAGACAGGGGTCATA AAGTGAGCAGTGCCTGGTTT

SH2B3 TGAATTCATGGATGGCTGAG GGGGTAGCAGGACAGGAAAT
SMC1A CCATTATTGTGGACTCGGAGA TCCAGTACTGAGCCTGTCCA
SMC1A TTTTGGGCAAGAACATGGAT CAAGGGAATCCACACCTTGT
SPPL2C TCAGCTGGCTCTAGGCAGAA TCAAACCCTTCCTCTTGTGG
SRCAP CCAGTGTGACTCCACCATTG AGGAGGAGCTGGAGAAGGAG
SRCAP CCAGGATTTGGTAGCTGGTG GAGGCTAGAGGCACAGGACA
SRCAP ACAGGTGGAGATGGAGATGG CAGGTGCTCAGGAATGGTTC
SRCAP CGTGGTGCTTCAGAAAGGTT GTTGCCGTGGTGGTAGAAGT
SRCAP CTTGAGGAAAAGCGCAAAAA CTCTGCATCATTGCCTTCCT
STAG3 ATTTGGCCATGTCTACACCA TCTTTTACCCCCAAGAGACTGA
STAG3 TTTAGGGTCATCCTGCTCTGA ACCCCTTACAGGATGGGTCT

SUV39H2 GATGAATGCTTCTTTGGTTCAG TTGGTAGGCACCTCAATGTTT
SUZ12 AATGTTTACCTTTGCAGGATGT TTGAAAATTTGCACAACAAAATG
SUZ12 GGAGGGGGTGGCAGTTAC GTAATCTCCGAAAGGGGGACA
TET1 CAGTTACTGGACAGCAGCAAA TCGATGCTATCCAAATGTCG
TET1 CAATGAAGGTGATCAACCAAAA GTGACTTTGTGGATGCTTGG
TET1 AACACTCAGGGGTTCCCATT GCCCCTTGACTGTTTGCTTA
TET2 TGCTGCTCTAAGGTGGCATC GCTGTTCTTTTGGTTGGTGTC
TET2 TCCACAGAACTTTTGCGACTT CAGATCCATCGGCTGAGACT
TP53 GGCTTTGGGACCTCTTAACC TGCTAGGAAAGAGGCAAGGA

U2AF1 GGTGCTTAATACCACGGAAAA AGTCGATCACCTGCCTCACT
UNC5B GGCATGGGGTTATGTGGATA GACCCTTACATGGCTCCAGA

USP9X CACAGAGAAACTTACTTGGGCTTT TCTTGGGATGCACAAAAACA
USP9X TGGTGACAATTTATGAAGTGGTG AGTCAACTCCGGAAGATCATTT
USP9X CAAGTTGATGGGGGATGAAC AAAGCCCACTGACAGGTGAC
WAC AGCATCTTCTGTGTGCTCCA CCATTCCTAAACTGGGGATTT
WAC AGCCAGAGAAGTCCATCACC AAGGCATTAAGCACTGAGCTAAC
WAC AGGTTCAGGCCTAGCTTCCT CTTTTCCGAATGTCCCTCAA
WT1 ATGGGGATCTGGAGTGTGAA AACCTGGGTCCTTAGCAGTG
WT1 GGGGACTGGGGAAATCTAAG GCCACGCACTATTCCTTCTC
WT1 CCTGAGCTCCCATTCATTTG GGGGGAAAATACTGGAAAAA
XRIP1 TAGTCGGAGGTGATGTGCAG GAACATCCAGGTGCAGGACT

ZRSR2 CATCAAAGAGTCGGGAGAGG AACCCATCTGCGTTCATAGC
ZRSR2 AAGTCTTGATTGCCTGTTCCA CTCTCCCAAAAGGGGAACTC

Supplemental Table 10: Variant allele frequency (VAF) of DNMT3A mutations in FLT3-ITD AML
Position Amino
Sample Allele % VAF %VAF %VAF
Gene Chr RefSeq (DX and Acid
ID Change (CR) (DX) (REL)
REL) Change
UPN002 DNMT3A chr2 NM_175629 25457242 R882H C>T 1 32 43
UPN005 DNMT3A chr2 NM_175629 25457243 R882C G>A 40 52 54

UPN006 DNMT3A chr2 NM_175629 25457192 R899fs */- G 0 45 30

UPN010 DNMT3A chr2 NM_175629 25470993 P257fs */-G 0 47 34

UPN022 DNMT3A chr2 NM_175629 25463568 I705T A>G 0 47 2


UPN050 DNMT3A chr2 NM_175629 25457247 M880I C>A 28 45 52
UPN054 DNMT3A chr2 NM_175629 25457243 R882S G>T 0 44 1

UPN060 DNMT3A chr2 NM_175629 25457242 R882H C>T 19 NA 46
UPN034 DNMT3A chr2 NM_175629 25457242 R882P C>G 0 53 NA

UPN035 DNMT3A chr2 NM_175629 25463248 R749C G>A 5 40 NA
UPN039 DNMT3A chr2 NM_175629 25457242 R882H C>T 0 40 NA
UPN042 DNMT3A chr2 NM_175629 25457242 R882H C>T 0 42 NA
UPN044 DNMT3A chr2 NM_175629 25458595 W860R A>G 0 42 NA

UPN078 DNMT3A chr2 NM_175629 25458595 W860R A>G 0 42 NA
VAF : Variant allele frequency
DX : Diagnosis
REL : Relapse
Supplemental Table 11: Analysis of FLT3-ITD insertion at FLT3 locus using Pindel and ITDetector
Sample Position Position Length Length ITD sequence VAF VAF

Gene ITD sequence (DX)
ID (DX) (REL) (DX) (REL) (REL) (DX) (REL)
ATTTTCTCTTGG ATTTTCTCTTGG
AAACTCCCATTT AAACTCCCATTT
UPN001 FLT3 28608273 28608273 45 45 40 45
GAGATCATATTC GAGATCATATTC
ATATTCTCT ATATTCTCT
TAAATTTTCTCTT
ATTCTCTGAAAT
GGAAACTCCCAT
UPN002 FLT3 28608296 28608294 30 64 CAACGTAGAAGT 26 17
TTGAGATCATAT
ACTCAT
TCATATTCTCTG
AAATCAACGTAG
AAG
TCTTGGAAACTC TCTTGGAAACTC
CCATTTGAGATC CCATTTGAGATC
UPN003 FLT3 28608294 28608294 58 58 25 14
ATATTCATATTC ATATTCATATTC
TCTGAAATCAAC TCTGAAATCAAC
GTAGAAGTAC GTAGAAGTAC
GGGCGATCATAT GGGCGATCATAT
TCATATTCTCTG TCATATTCTCTG
AAATCAACGTAG AAATCAACGTAG
UPN004 FLT3 28608322 28608322 57 57 25 29
AAGTACTCATTA AAGTACTCATTA
TCTGAGGAGCCG TCTGAGGAGCCG
G G
TAAATTTTCTCTT TAAATTTTCTCTT
UPN005 FLT3 28608225 28608225 48 48 GGAAACTCCCAT GGAAACTCCCAT 12 23
TTGAGATCATAT TTGAGATCATAT
TCATATTCTCT TCATATTCTCT
TTCTCTTGGAAA TTCTCTTGGAAA
CTCCCATTTGAG CTCCCATTTGAG
UPN006 FLT3 28608279 28608279 48 48 18 9
ATCATATTCATA ATCATATTCATA
TTCTCTGAAATC TTCTCTGAAATC
GTATGAAATTTT GTATGAAATTTT
CTCTTGGAAACT CTCTTGGAAACT
CCCATTTGAGAT CCCATTTGAGAT
UPN007 FLT3 28608302 28608302 57 57 12 15
CATATTCATATT CATATTCATATT
CTCTGAAATCAA CTCTGAAATCAA
CG CG
AAGATCAACGTA AAGATCAACGTA
UPN008 FLT3 28608300 28608300 24 24 GAAGTACTCATT GAAGTACTCATT 18 0
ATC ATC
TTTGAGATCATA TTTGAGATCATA
TTCATATTCTCT TTCATATTCTCT
GAAATCAACGTA GAAATCAACGTA
UPN009 FLT3 28608309 28608309 67 64 21 17
GAAGTACTCATT GAAGTACTCATT
ATCTGAGGAGCC ATCTGAGGAGCC
GGTCACC GGTC
GTCAAACTCTAA GTCAAACTCTAA
UPN010 FLT3 28608279 28608279 61 61 21 25
ATATTCTCTGAA ATATTCTCTGAA
ATC ATC
AAACTCTAAATT AAACTCTAAATT
TTCTCTTGGAAA TTCTCTTGGAAA
CTCCCATTTGAG CTCCCATTTGAG
UPN011 FLT3 28608312 28608312 65 65 15 10
ATCATATTCATA ATCATATTCATA
TTCTCTGAAATC TTCTCTGAAATC
AACGT AACGT
CATATTCTCTGA CATATTCTCTGA
UPN012 FLT3 28608284 28608284 21 21 35 44
AATCAACGT AATCAACGT
AACTCCCATTTG AACTCCCATTTG
AGATCATATTCA AGATCATATTCA
TATTCTCTGAAA TATTCTCTGAAA
UPN013 FLT3 28608308 28608308 67 67 15 10
TCAACGTAGAAG TCAACGTAGAAG
TACTCATTATCT TACTCATTATCT
GAGGAGCTAC GAGGAGCTAC
ACCAAACTCTAA ACCAAACTCTAA
UPN014 FLT3 28608216 28608216 64 64 14 20
ATCT ATCT
TATTCATATTCT TATTCATATTCT
UPN015 FLT3 28608280 28608280 21 21 12 39
CTGAAATCA CTGAAATCA
AATCAACGTAGA AATCAACGTAGA
AGTACTCATTAT AGTACTCATTAT
UPN016 FLT3 28608314 28608314 39 39 28 20
CTGAGGAGCCGG CTGAGGAGCCGG
TCA TCA
CCATCACCTGAT CCATCACCTGAT
CCTAGTACCTTC CCTAGTACCTTC
CCTGCAAAGACA CCTGCAAAGACA
AATGGTGAGTAC AATGGTGAGTAC
GTGCATTTTAAA GTGCATTTTAAA
UPN017 FLT3 28608298 28608298 110 110
GATTTTCCAATG GATTTTCCAATG
GAAAAGAAATG GAAAAGAAATG
CTGCAGAAACAT CTGCAGAAACAT
TTGGCACATTCC TTGGCACATTCC
ATTCTTA ATTCTTA
TATTCATATTCT TATTCATATTCT
UPN018 FLT3 28608280 28608280 21 21 12 18
CTGAAATCA CTGAAATCA
ATCCTAACGAGC ATCCTAACGAGC
GGNATTTTCTCT GGNATTTTCTCT
UPN019 FLT3 28608277 28608277 51 51 TGGAAACTCCCA TGGAAACTCCCA 24 30
TTTGAGATCATA TTTGAGATCATA
TT TT
AATTGAGTTACC
AAACTCTAAATT AATTGAGTTACC
TTCTCTTGGAAA AAACTCTAAATT
CTCCCATTTGAG TTCTCTTGGAAA
UPN020 FLT3 28608303 28608303 95 63 34 20
ATCATATTCATA CTCCCATTTGAG
TTCTCTGAAATC ATCATATTCATA
AACGTAGAAGTA TTCTCTGAAA
CTCATTATCTGA
GGAGCC
AACTCTAAATTT AACTCTAAATTT
TCTCTTGGAAAC TCTCTTGGAAAC
UPN021 FLT3 28608265 28608265 45 45 16 23
TCCCATTTGAGA TCCCATTTGAGA
TCATATTCA TCATATTCA
AAGTACTCATTA AAGTACTCATTA
TCTGAGGAGCCG TCTGAGGAGCCG
UPN022 FLT3 28608286 28608286 42 42 12 0
GTCACCTGTACC GTCACCTGTACC
ATCTGT ATCTGT
CTCCTTGATACC CTCCTTGATACC
CNAATTTTCTCT CNAATTTTCTCT
UPN023 FLT3 28608298 28608298 47 47 25 2
TGGAAACTCCCA TGGAAACTCCCA
TTTGAGATCA TTTGAGATCA
TGGCACATTCCA TCCCATTTGAGA
TTCTTACCAAAC TCATATTCATAT
TCTAAATTTTCT TCTCTGAAATCA
UPN024 FLT3 28608313 28608313 76 69 15 11
CTTGGAAACTCC ACGTAGAAGTAC
CATTTGAGATCA TCATTATCTGAG
TATTCATATTCT GAGCCGGTC
CTGA
TACCAAACTCTA TACCAAACTCTA
AATTTTCTCTTG AATTTTCTCTTG
GAAACTCCCATT GAAACTCCCATT
UPN025 FLT3 28608215 28608215 67 67 7 4
TGAGATCATATT TGAGATCATATT
AATCAACAC AATCAACAC
ATTTGGCACATT ATTTGGCACATT
CCATTCTTACCA CCATTCTTACCA
AACTCTAAATTT AACTCTAAATTT
UPN026 FLT3 28608196 28608196 78 78 TCTCTTGGAAAC TCTCTTGGAAAC 5 5
TCCCATTTGAGA TCCCATTTGAGA
TCATATTCATAT TCATATTCATAT
TCTCTG TCTCTG
UPN027 FLT3 28608321 28608321 105 105 ATATTCTCTGAA ATATTCTCTGAA 16 0
ATCAACGTAGAA ATCAACGTAGAA
GTACTCATTATC GTACTCATTATC
TGAGGAGCCGGT TGAGGAGCCGGT
CACCTGTAC CACCTGTAC
ACTCTAAATTTT ACTCTAAATTTT
UPN028 FLT3 28608314 28608314 93 93 20 0
CGTAGAAGTACT CGTAGAAGTACT
CATTATCTGAGG CATTATCTGAGG
AGCCGGTCA AGCCGGTCA
TCAACGTAGAAG TCAACGTAGAAG
TACTCATTATCT TACTCATTATCT
UPN029 FLT3 28608277 28608277 60 60 GAGGAGCCGGTC GAGGAGCCGGTC 10 13
ACCTGTACCATC ACCTGTACCATC
TGTAGCTGGCTT TGTAGCTGGCTT
CTCTAAATTTTC CTCTAAATTTTC
TCTTGGAAACTC TCTTGGAAACTC
UPN030 FLT3 28608306 28608306 61 61 26 5
TCTGAAATCAAC TCTGAAATCAAC
G G
GTAACTCCCATT GTAACTCCCATT
UPN031 FLT3 28608275 28608275 36 36 TGAGATCATATT TGAGATCATATT 19 7
GGAAACTCCCAT GGAAACTCCCAT
UPN032 FLT3 28608262 28608262 24 24 12 4
CTCTGAAATCAA
CGTAGAAGTACT
UPN045 FLT3 28608239 NA 43 NA CATTATCTGAGG NA 4 1
AGCCGGTTCTAG
GGGTG
UPN046 FLT3 28608264 28608264 18 18 19 22
ATATTC ATATTC
UPN047 FLT3 28608284 28608284 21 21 38 45
AATCAACGT AATCAACGT
ACTCTAAATTTT ACTCTAAATTTT
UPN048 FLT3 28608308 28608308 90 90 27 28
CGTAGAAGTACT CGTAGAAGTACT
CATTATCTGAGG CATTATCTGAGG
AGCACT AGCACT
UPN049 FLT3 28608279 28608279 33 33 ATATTCATATTC ATATTCATATTC 6 27
TCTGAAATC TCTGAAATC
GTTCAAACTCTA TTCCATTCTTAC
AATTTTCTCTTG CAAACTCTAAAT
GAAACTCCCATT TTTCTCTTGGAA
UPN050 FLT3 28608311 28608206 64 66 18 5
TGAGATCATATT ACTCCCATTTGA
CATATTCTCTGA GATCATATTCAT
AATCAAC ATTCTC
CTTGGAAACTCC CTTGGAAACTCC
CATTTGAGATCA CATTTGAGATCA
UPN051 FLT3 28608301 28608301 59 59 TATTCATATTCT TATTCATATTCT 25 29
CTGAAATCAACG CTGAAATCAACG
TAGAAGTACTC TAGAAGTACTC
CTAGATCAACGT CTAGATCAACGT
UPN052 FLT3 28608296 28608296 20 20 9 16
AGAAGTACTCAT AGAAGTACTCAT
UPN053 FLT3 28608286 28608286 24 24 18 17
AAATCAACGTAG AAATCAACGTAG
CTTACCAAACTC CTTACCAAACTC
TAAATTTTCTCTT TAAATTTTCTCTT
GGAAACTCCCAT GGAAACTCCCAT
UPN054 FLT3 28608102 28608102 65 65 19 10
AAAT AAAT
GGTACCTTCCCT GGTACCTTCCCT
GCAAAGACAAA GCAAAGACAAA
TGGTGAGTACGT TGGTGAGTACGT
GCATTTTAAAGA GCATTTTAAAGA
UPN055 FLT3 28608325 28608325 88 88 13 10
TTTTCCAATGGA TTTTCCAATGGA
AAAGAAATGCTG AAAGAAATGCTG
CAGAAACATTTG CAGAAACATTTG
GCACAT GCACAT
UPN056 FLT3 28608270 28608270 24 24 23 20
UPN062 FLT3 NA NA NA NA NA NA 19 13
CATTCCATTCTT CATTCCATTCTT
UPN063 FLT3 28608284 28608284 80 80 AAACTCCCATTT AAACTCCCATTT 19 18
ATCAACGTC ATCAACGTC
GAATCCCATTTG
CCCATCATATTC
AGATCATATTCA
UPN064 FLT3 28608289 28608279 45 24 ATATTCTCTGAA 28 10
TATTCTCTGAAA
ATC
TCAACGTAGAAG
UPN065 FLT3 28608302 28608302 39 39 NA NA 30 35
GAAATCAACGTA GAAATCAACGTA
GAAGTACTCATT GAAGTACTCATT
UPN071 FLT3 28608312 28608312 39 39 36 30
ATCTGAGGAGCC ATCTGAGGAGCC
GGT GGT
UPN072 FLT3 NA NA 18 18 NA NA NA NA
ITD : Internal tandem duplication
VAF : Variant allele frequency
DX : Diagnosis
REL : Relapse
Supplemental Table 12: MuSiC (Mutational Significance in Cancer) analysis identified significantly mutated genes
1 2 3 4 5 6 7 8 9 10 11 12
#Gene Indels SNVs Tot Muts Covd Bps Muts Pmbp P-value FCPT P-value LRT P-value CT FDR FCPT FDR LRT FDR CT
DNMT3A 02 25 27 1064270 27.25 0 0 0 0 0 0

NPM1 29 00 29 277106 90.22 0 0 0 0 0 0
WT1 10 05 15 514846 29.13 2.70E-13 1.11E-16 6.65E-18 2.84E-11 1.17E-14 6.98E-16
FLT3 00 14 14 709398 19.74 7.90E-09 2.13E-11 2.58E-12 6.22E-07 1.68E-09 2.03E-10
IDH2 00 11 11 428596 25.67 1.70E07 4.66E-11 6.94E-11 1.07E-05 3.67E-09 4.37E-09
MLL3 01 22 23 2486781 09.25 5.28E-08 1.18E-08 3.50E11 3.32E-06 7.41E-07 2.21E-09
RUNX1 05 08 13 1840859 07.06 0.001397492 9.34E-05 1.78E-05 0.073368306 0.002102609 0.000802845
KDM6A 01 07 08 736442 10.86 0.006782571 0.001099848 9.43E-05 0.267063724 0.011946619 0.003300324
GATA2 00 05 05 202953 24.64 0.019798563 0.003180528 0.000145658 0.519712271 0.022263696 0.004588224
TET2 04 06 10 1559839 06.41 0.009918937 2.77E-05 0.000210666 0.34716278 0.000821588 0.006032702
BCORL1 04 04 08 1146126 06.98 0.015838414 2.87E-05 0.000290323 0.498910055 0.000821588 0.007620989
FAT1 02 10 12 1959192 06.64 0.024115162 0.004542027 0.001097057 0.584328918 0.028053699 0.019198504
NSD1 00 08 08 1191901 06.71 0.041807060 0.000239977 0.001433373 0.857638509 0.004446626 0.023763819
ARID2 00 06 06 1521115 03.94 0.062792610 9.08E-07 0.001571853 1 4.09E-05 0.024756686
PTPN11 00 06 06 981238 06.11 0.070848892 4.49E-05 0.002067217 1 0.001087419 0.029598796
SRCAP 02 08 10 1859744 05.38 0.085180779 0.012700894 0.004613368 1 0.054064617 0.060550461
EZH2 00 04 04 570802 07.01 0.190513743 3.53E-05 0.006315166 1 0.000926901 0.079571086
TET1 00 10 10 464774 30.12 0.024398578 0.000581505 0.000362190 0.004813 0.031140975 0.166659476
ARID1A 03 01 04 931649 04.29 0.394384565 0.001776699 0.028812300 1 0.031403037 0.240815269
FAT4 00 09 09 1917682 04.69 0.187642387 0.055184241 0.019841974 1 0.163990904 0.185296976
Columns 7, 8 and 9 provides p-values for Fisher’s Combined P-value Test (FCPT), a Likelihood Ratio Test (LRT) and a Convolution Test (CT).
Columns 10,11 and 12 provides false discovery rate (FDR) values for FCPT, LRT and CT, after multiple-testing correction.
Supplemental Table 13: KEGG pathways by MuSic PathScan
Genes affected in Patients KEGG Pathway KEGG ID P-values FDR
RUNX1 (12), FLT3TKD (9), WT1 hsa05202 5.0106E-28 2.9485E-27

(13), KDM6A (5), HOXA9 (1), TP53 Transcriptional misregulation in cancer
(2), DOTIL (2), CEBPA(2)
DNMT3A (27) EZH2 (3) Cysteine and methionine metabolism hsa00270 1.7939E-18 1.9329E-18
ASHIL (2), NSD1 (4), MLL2 (3), hsa00310 6.9527E-16 7.5444E-16

MLL3 (12), SETD1A (2), DOTIL Lysine degradation
(2), SUV39H2 (1), SETD1B (3)
FLT3-TKD (9), RUNX1(12), NRAS Acute myeloid leukemia hsa05221 1.9239E-09 2.1084E-08
(2), CEBPA (2)
IDH1(3), IDH2 (8), IDH3B (2) Citrate cycle (TCA cycle) hsa00020 2.6999E-08 2.7177E-08
Glutathione metabolism hsa00480 3.6564E-07 5.5389E-07
SF3B1 (4), SF3A1 (1), U2AF1 (2), Spliceosome hsa03040 0.028204 0.03537
PRPF40B (1)
RAD21 (2), TP53 (2), SMC1A (1), Cell cycle hsa04110 0.175100 0.2480
STAG1 (1), STAG2 (2)
Supplemental Table 14A. shRNA targeting sequences
shRNA Sequence
MLL3 shRNA1 5'-GAAGAGGATGCTGAAGCAGAAGTGGATAA-3'
MLL3 shRNA2 5'-GGTTCAAGTCCACACTGAAGAGCAACAGA-3’
MLL3 shRNA3 5'-CACTACTTAGTTCATCCTCGGCTCCAACA-3'
MLL3 shRNA4 5'-TCTCCACAGCTAAGAAGATCAGTATCTGT-3'
Scramble shRNA 5’-GCACTACCAGAGCTAACTCAGATAGTACT-3'

Supplemental Table 14B. siRNA targeting sequences
siRNA Sequence
MLL3 siRNA1 5'-CAAGGCUACUCAACCUUGAUU-3'
MLL3 siRNA2 5'-GAACGCACCUUAUAGUAAAUU-3'
MLL3 siRNA3 5'-CCAUUCGUGUGCACCUAAUUU-3
FAT1 siRNA 5'-GAAAUCACCUACUCAAUAA-3'
FAT1 siRNA 5'-GAACUAACAUGGCUGGUUU-3'
FAT1 siRNA 5'-GGAAGGAAUUGGAAUCGUU-3'
FAT1 siRNA 5'-GAACUGAUGCCUCGGGUUU-3'

Supplemental Table 14C. siRNA targeting sequences
siRNA (Murine) Sequence
MLL3 siRNA1 5‘-GGAAGAGGAUGCUGAAACAtt-3'
MLL3 siRNA2 5'- GAGAUGGGUUCACUUAGAAtt-3'
MLL3 siRNA3 5'- GAACCAUUGUUGACAGAAAtt-3’

Supplemental Table 15 : Selected gene primers for real time quantitative PCR
qPCR primer Sequence
MLL3 Forward 5’-TAGGATGTCGTCGGAGGAGG-3’
MLL3 Reverse 5’-TCCCCCTACTTCGAGGTTTCT-3’
MLL3 Forward 5’- GAAAATGACACAATGTCGAATGC-3’
MLL3 Reverse 5’- TTCACCCAGAGCCTCCTCTT-3’
TP53 Forward 5’-CCCCTCCTGGCCCCTGTCATCTTC-3’
TP53 Reverse 5’-GCAGCGCCTCACAACCTCCGTCAT-3
P21 Forward 5’-CCTCATCCCGTGTTCTCCTTT-3’
P21 Reverse 5’-GTACCACCCAGCGGACAAGT-3’
HOXA7 Forward 5’-CGCCAGACCTACACGCG-3’
HOXA7 Reverse 5’-CAGGTAGCGGTTGAAGTGGAA-3’
HOXA9 Forward 5’-GCTTGTGGTTCTCCTCCAGT-3’
HOXA9 Reverse 5’-CCAGGGTCTGGTGTTTTGTA-3’
MEIS1 Forward 5’-GCATGCAGCCAGGTCCAT-3’
MEIS1 Reverse 5’-TAAAGCGTCATTGACCGAG-3’

Supplemental Table 16: Mutational frequencies between our study and AML (TCGA) from FLT3-ITD subtype
Gene Frequency (%) Frequency (%) Gene Frequency (%) Frequency (%)
FLT3ITD AML (TCGA ) FLT3ITD AML (TCGA )
NPM1 37 49 MLL3 11 0
DNMT3A 34 31 FLT3 11 0
WT1 18 13 FAT1 10 0
TET2 10 10 SRCAP 08 0
IDH2 09 05 NSD1 05 0
RUNX1 15 03 IDH1 04 0
FAT4 06 03 ASXL1 06 0
APOB 05 03 KDM6A 06 0
GATA2 04 03 LRP2 06 0
STAG2 01 05 SF3B1 05 0
SMC3 0 05 EZH2 04 0
Mutated genes found only in our FLT3-ITD cohort are marked in red; those only in AML TCGA for FLT3-ITD subtype
are marked in blue; mutation present in both the cohort are shown in black

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SUPPLEMENTAL INFORMATION

Joanna Schiller10, Karl-Anton Kreuzer10, Steven M. Kornblau11,12, Hagop M. Kantarjian11,12, Torsten

Yang1, Seishi Ogawa2 and H. Phillip Koeffler1,13,16

Supplemental Methods (pages 3 – 8)

Supplemental Figures (Pages 9 – 30)

Clustering and clonal analysis

Frequency analysis using targeted deep sequencing (TDS)

Significantly mutated gene (SMG) analysis and pathway analysis

identify co-occurring and exclusively occurring genes across the cohort.

Single nucleotide polymorphism array (SNP-array) analysis

analysis tool for genechip (CNAG) software, as previously described25,26.

Cell culture and antibodies

are listed in Supplemental Table 14A, B and C.

RT-PCR analysis and quantitative real-time PCR (qRT-PCR)

Supplemental Table 15.

Gel electrophoresis and Immunoblotting

hr. Blots were developed using ECL reagent (Thermo Scientific).

Supplemental Figure 4. Hotspot mutations identified in FLT3-ITD AML. Schematic representation of

acid in different cases are depicted.

same mutation at diagnosis.

Supplemental Figure 7. Recurrent mutations in DNMT3A were validated by Sanger sequencing in

chromatograms shows a clear heterozygous mutation.

the genes coding for spliceosome and cohesin complex.

formation assay (D). Data in A-D represent mean ± s.d.; n = 3. ** P ≤ 0.01.

WBC : White blood cell

RFS : Relapse-free survival

UPN001 DX 135.352824 94.48 86.97 81.05 Whole exome SureSelect

UPN001 CR 111.308110 92.85 82.77 73.98 Whole exome SureSelect

UPN001 REL 130.495242 94.24 86.04 78.69 Whole exome SureSelect

UPN002 DX 125.169604 93.21 83.69 75.19 Whole exome SureSelect

UPN002 CR 134.479008 93.86 85.64 78.45 Whole exome SureSelect

UPN002 REL 116.546046 93.02 83.13 74.03 Whole exome SureSelect

UPN003 DX 126.410161 94.03 85.40 77.71 Whole exome SureSelect

UPN003 CR 118.594015 93.60 83.92 75.42 Whole exome SureSelect

UPN003 REL 115.175664 93.97 84.90 76.66 Whole exome SureSelect

UPN004 DX 84.156426 92.92 84.71 73.60 Whole exome SureSelect

UPN004 CR 107.242603 93.42 87.14 78.62 Whole exome SureSelect

UPN004 REL 96.875570 93.05 85.47 75.35 Whole exome SureSelect

UPN005 DX 112.571034 96.30 91.68 86.78 Whole exome SureSelect

UPN005 CR 120.131054 96.57 91.88 83.18 Whole exome SureSelect

UPN005 REL 99.040552 96.53 91.60 82.53 Whole exome SureSelect

UPN006 DX 220.967296 97.05 95.05 92.83 Whole exome SureSelect

UPN006 CR 82.382913 96.09 90.05 78.65 Whole exome SureSelect

UPN006 REL 61.154439 92.66 71.55 46.74 Whole exome SureSelect

UPN007 DX 177.042505 96.91 94.56 91.46 Whole exome SureSelect

UPN007 CR 112.040334 96.35 91.98 84.29 Whole exome SureSelect

UPN007 REL 99.266013 96.63 92.13 83.87 Whole exome SureSelect

UPN008 DX 124.983011 96.64 93.50 89.85 Whole exome SureSelect

UPN008 CR 141.391579 96.64 92.91 85.57 Whole exome SureSelect

UPN008 REL 129.957987 96.61 93.33 88.23 Whole exome SureSelect

UPN009 DX 72.724605 92.84 80.85 66.72 Whole exome SureSelect

UPN009 CR 79.195375 92.29 83.83 71.59 Whole exome SureSelect

UPN009 REL 92.052943 92.57 83.79 72.30 Whole exome SureSelect

UPN010 CR 103.073496 96.21 91.44 82.60 Whole exome SureSelect

UPN010 REL 178.527554 96.70 94.36 91.77 Whole exome SureSelect

UPN011 DX 84.713373 92.98 84.50 72.96 Whole exome SureSelect

UPN011 CR 106.600816 91.87 77.50 61.87 Whole exome SureSelect

UPN011 REL 88.980051 90.99 74.93 59.10 Whole exome SureSelect

UPN012 DX 145.973217 99.67 97.29 91.50 Whole exome SureSelect

UPN012 CR 147.468752 99.33 93.19 82.14 Whole exome SureSelect

UPN012 REL 146.913329 99.21 93.58 83.76 Whole exome SureSelect

UPN013 DX 154.885369 99.62 95.92 88.10 Whole exome SureSelect

UPN013 CR 135.028303 99.59 95.08 86.00 Whole exome SureSelect