Supplementary Information - DNMT3B expression in AML
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1
Participating institutions
2
The following Cancer and Leukemia Group B (CALGB)/Alliance institutions participated
3
in this study and contributed at least 1% of patients. For each of these institutions
4
current principal investigators are listed.
5
6
Wake Forest University School of Medicine, Winston-Salem, NC: David D. Hurd; The
7
Ohio State University Medical Center, Columbus, OH: Clara D. Bloomfield (grant no.
8
U10CA077658); North Shore University Hospital, Manhasset, NY: Daniel R. Budman
9
(grant no. U10CA035279); Duke University Medical Center, Durham, NC: Jeffrey
10
Crawford (grant no. U10CA047577); University of Iowa Hospitals, Iowa City, IA: Daniel
11
A. Vaena; Roswell Park Cancer Institute, Buffalo, NY: Ellis G. Levine (grant no.
12
U10CA059518); University of Chicago Medical Center, Chicago, IL: Hedy L. Kindler
13
(grant no. U10CA041287); Washington University School of Medicine, St. Louis, MO:
14
Nancy L. Bartlett (grant no. U10CA077440); University of Tennessee Cancer Center,
15
Memphis: Harvey B. Niell; Ft. Wayne Medical Oncology/Hematology, Ft. Wayne, IN:
16
Sreenivasa Nattam; Dana Farber Cancer Institute, Boston, MA: Harold J. Burstein
17
(grant no. U10CA032291); Vermont Cancer Center, Burlington, VT: Steven M.
18
Grunberg; University of North Carolina, Chapel Hill, NC: Thomas C. Shea (grant no.
19
U10CA047559); Rhode Island Hospital, Providence, RI: William Sikov; University of
20
Maryland Cancer Center, Baltimore, MD: Maria R. Baer; Eastern Maine Medical Center,
21
Bangor, ME: Thomas H. Openshaw; Mount Sinai School of Medicine, New York, NY:
22
Lewis R. Silverman; Weill Medical College of Cornell University, New York, NY: John
Supplementary Information - DNMT3B expression in AML
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Leonard; SUNY Upstate Medical University, Syracuse, NY: Stephen L. Graziano;
24
Moores University of California San Diego Cancer Center, San Diego, CA: Barbara A.
25
Parker.
26
27
Treatment protocols
28
Patients were treated on one of the following intensive cytarabine/daunorubicin-based
29
CALGB frontline treatment protocols: 8525 (n=23),1 8923 (n=17),2 9420 (n=5),3 9720
30
(n=95),4,5 or 10201 (n=70).6 Among these protocols, CALGB 9420, 9720, and 10201
31
included investigational agents other than chemotherapy. CALGB 9720 4,5 was initiated
32
as a phase III trial in untreated acute myeloid leukemia (AML) patients 60 years and
33
older evaluating multidrug resistance modulation by valspodar (PSC-833) during
34
induction and consolidation therapy with cytarabine, daunorubicin, and etoposide. The
35
valspodar arm was closed after random assignment of only 120 patients because of
36
excessive early deaths. Enrollment on this protocol continued on the chemotherapy-only
37
control arm. CALGB 102016 evaluated the BCL2 antisense, oblimersen sodium
38
(Genasense; G3139) administered with induction and consolidation chemotherapy;
39
preliminary results showed no impact of the antisense on outcome. CALGB 9420 3 and
40
CALGB 97204,5 evaluated a subcutaneous IL-2 regimen in older AML patients as
41
maintenance therapy, which was demonstrated to induce no clear benefit.
42
43
Sample collection and preparation
44
Patients enrolled on the aforementioned treatment protocols gave informed consent for
45
pretreatment bone marrow (BM) and blood collection and their research use in
Supplementary Information - DNMT3B expression in AML
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accordance with the Declaration of Helsinki. Mononuclear cells from pretreatment BM
47
and/or blood were enriched by Ficoll-Hypaque gradient and cryopreserved in liquid
48
nitrogen until they were thawed at 37°C for analysis. DNA and total RNA sample
49
extraction and quality control were performed as reported previously. 7
50
51
Nanostring nCounter assays
52
RNA samples (400 ng) were hybridized in solution to target sequence-specific pairs of
53
biotinylated capture probes and fluorescently labeled reporter probes. The mRNA-probe
54
complexes were washed, immobilized on a streptavidin-coated surface and quantified
55
by fluorescence imaging. The sensitivity, dynamic range, linearity and reproducibility of
56
the nCounter system have been validated previously. 8 The NanoString nCounter assay
57
measures global DNMT3B levels, and does not allow quantification of expression of the
58
individual DNMT3B isoforms.
59
60
Definition of clinical endpoints
61
Complete remission (CR) required a BM aspirate with cellularity >20% with maturation
62
of all cell lines, <5% blasts and undetectable Auer rods; no circulating leukemic blasts,
63
blood neutrophil count ≥1.5 x 109/l and platelets >100 x109/l, and no evidence of
64
extramedullary leukemia, all of which had to persist for four or more weeks. Relapse
65
was defined by more than 5% blasts in marrow aspirates, reappearance of leukemic
66
blasts in the peripheral blood or the development of extramedullary leukemia in patients
67
with previously documented CR.9
68
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Disease-free survival (DFS) was measured from the date of CR until date of relapse or
70
death (regardless of cause), censoring for patients alive and relapse-free at last follow-
71
up. Overall survival (OS) was measured from the date the patient was enrolled onto the
72
study until the date of death, censoring for patients alive at last follow-up.
73
74
Multivariable models
75
To evaluate if DNMT3B expression remains associated with outcome after adjustment
76
for established prognostic factors in cytogenetically normal AML, we constructed
77
multivariable logistic regression models to analyze the probability of CR attainment and
78
multivariable proportional hazards models for DFS and OS using a limited backwards
79
selection procedure. Variables in addition to DNMT3B expression (high vs low) that
80
were considered for univariable analyses for all models were: age (by 10-year
81
increase), sex, race (white vs nonwhite), hemoglobin (continuous), platelet count
82
(continuous), white blood count (WBC; continuous), extramedullary involvement
83
(present vs absent), European LeukemiaNet Genetic Groups (modified Favorable vs
84
Intermediate-I), FLT3-TKD (present vs absent), WT1 (mutated vs wild-type), TET2
85
(mutated vs wild-type), MLL-PTD (present vs absent), IDH1 (mutated vs wild-type),
86
IDH2 (R140 or R172 mutated vs wild-type), ASXL1 (mutated vs wild-type), DNMT3A
87
(R882 mutated vs wild-type; non-R882 mutated vs wild-type), and expression of ERG
88
(high vs low), BAALC (high vs low), miR-155 (high vs low), and miR-3151 (high vs low).
89
Variables significant at α=0.20 from the univariable analyses were considered for
90
multivariable analyses.
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Gene- and microRNA-expression profiling
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For gene-expression microarrays, summary measures of gene expression were
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computed for each gene using the robust multichip average (RMA) method, which
95
incorporates quantile normalization of arrays. Expression values were logged (base 2)
96
before analysis. A filtering step was performed to remove genes that did not display
97
large variation in expression across samples (using the following parameters for each
98
gene: < 1.5-fold change in either direction from the gene's median value in > 20% of
99
patients or data missing in > 50% of the patients). Univariable significance levels of
100
P<0.001 were used to select genes and microRNAs that constituted the signatures. The
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DNMT3B mRNA signatures comprised genes common to the three AML cohorts
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[CALGB/Alliance, German AML Cooperative Group (AMLCG)10 and The Cancer
103
Genome Atlas (TCGA)11] with false discovery rates (FDR) <0.01. For DNMT3B, the
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Affymetrix U133 plus 2.0 arrays measure global DNMT3B expression levels, and do not
105
quantify expression of the individual DNMT3B isoforms.
106
107
For microRNA microarrays, signal intensities were calculated for each spot making an
108
adjustment for local background. Intensities were log-transformed and log-intensities
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from replicate spots were averaged. Quantile normalization was performed on arrays
110
using all human microRNA probes on the array. For each microRNA probe, an
111
adjustment was made for batch effects (i.e., differences in expression related to the
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batch in which the arrays were hybridized). Further analysis was limited to 343 unique
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human microRNAs.
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Analyses were performed using BRB-ArrayTools Version 4.3.1 developed by Dr.
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Richard Simon and Amy Peng Lam.
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In patients whose samples were analyzed by both Affymetrix arrays and NanoString
119
nCounter assay, measurements of DNMT3B expression (Spearman Correlation = 0.80,
120
P<0.001) and MIR155 (Spearman Correlation = 0.81, P<0.001) were highly correlated.
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Significant associations of TLR4 and WT1 expression measured by RNA expression
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profiling with high and low DNMT3B expression were confirmed by measurements on
123
the NanoString nCounter assay. Associations of high miR-133b, CDK6, and WT1
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expression with high DNMT3B expression were confirmed by real-time PCR
125
(Supplementary Table S3).
126
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Gene set enrichment analysis (GSEA)
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We used GSEA12 to determine whether genes differentially expressed between high
129
and low DNMT3B expressers were associated with the presence of FLT3 internal
130
tandem duplication (FLT3-ITD) among 177 CALGB/Alliance CN-AML patients analyzed
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for genome-wide gene expression, who comprised 62 patients harboring FLT3-ITD and
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115 without FLT3-ITD. Gene expression data on 6334 genes from these patients was
133
available for analyses. The 195-gene set upregulated in high DNMT3B expressers was
134
analyzed (Supplementary Table S1a). As the ranking metric, we used the standard one,
135
the Signal2Noise ratio. The ranking metric measures each of gene’s correlation with the
136
FLT3-ITD genotype, with a positive value indicating positive correlation with the
137
presence of FLT3-ITD and a negative value indicating correlation with its absence. A
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ranking metric of zero means that there is no correlation of the gene expression with
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FLT3-ITD.
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The final result of GSEA is the enrichment score (ES), which reflects the degree to
142
which the 195-gene set is overrepresented at the top or bottom of the complete ranked
143
list of 6334 genes. A positive ES indicates gene set enrichment at the top of the ranked
144
list; a negative ES indicates gene set enrichment at the bottom of the ranked list. One
145
thousand permutations of the phenotype labels were performed to evaluate the P-
146
values and the FDR.
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DNA methylation
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Genome-wide DNA methylation and levels of DNA methylation across the genome’s
150
functional regions (i.e., genomic features) were measured using the MethylCap-seq
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assay as previously reported.13 This assay does not distinguish between 5-
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methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC).
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REFERENCES
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1.
Mayer RJ, Davis RB, Schiffer CA, Berg DT, Powell BL, Schulman P et al.
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Intensive postremission chemotherapy in adults with acute myeloid leukemia. N
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Engl J Med 1994; 331: 896-903.
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Stone RM, Berg DT, George SL, Dodge RK, Paciucci PA, Schulman P et al.
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Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for
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elderly patients with primary acute myelogenous leukemia. N Engl J Med 1995;
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332: 1671-1677.
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Lee EJ, George SL, Caligiuri M, Szatrowski TP, Powell BL, Lemke S et al.
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Parallel phase I studies of daunorubicin given with cytarabine and etoposide with
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or without the multidrug resistance modulator PSC-833 in previously untreated
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patients 60 years of age or older with acute myeloid leukemia: results of Cancer
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and Leukemia Group B study 9420. J Clin Oncol 1999; 17: 2831-2839.
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4.
Baer MR, George SL, Dodge RK, O'Loughlin KL, Minderman H, Caligiuri MA et
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al. Phase 3 study of the multidrug resistance modulator PSC-833 in previously
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untreated patients 60 years of age and older with acute myeloid leukemia:
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Cancer and Leukemia Group B study 9720. Blood 2002; 100: 1224-1232.
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5.
Baer MR, George SL, Sanford BL, Mrózek K, Kolitz JE, Moore JO et al.
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Escalation of daunorubicin and addition of etoposide in the ADE regimen in acute
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myeloid leukemia patients aged 60 years and older: Cancer and Leukemia Group
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B study 9720. Leukemia 2011; 25: 800-807.
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6.
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Marcucci G, Moser B, Blum W, Stock W, Wetzler M, Kolitz JE et al. A phase III
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randomized trial of intensive induction and consolidation chemotherapy ±
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oblimersen, a pro-apoptotic Bcl-2 antisense oligonucleotide in untreated acute
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myeloid leukemia patients >60 years old. J Clin Oncol 2007; 25(suppl): 360s
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(abstract 7012).
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7.
Baldus CD, Tanner SM, Ruppert AS, Whitman SP, Archer KJ, Marcucci G et al.
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BAALC expression predicts clinical outcome of de novo acute myeloid leukemia
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patients with normal cytogenetics: a Cancer and Leukemia Group B study. Blood
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2003, 102: 1613-1618.
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8.
Payton JE, Grieselhuber NR, Chang LW, Murakami M, Geiss GK, Link DC et al.
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High throughput digital quantification of mRNA abundance in primary human
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acute myeloid leukemia samples. J Clin Invest 2009; 119: 1714-1726.
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9.
Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD et
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al. Report of the National Cancer Institute-sponsored workshop on definitions of
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diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813-
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819.
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10.
Metzeler KH, Hummel M, Bloomfield CD, Spiekermann K, Braess J, Sauerland
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MC et al. An 86-probe-set gene-expression signature predicts survival in
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cytogenetically normal acute myeloid leukemia. Blood 2008; 112: 4193-4201.
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of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059-2074.
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Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes
12.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et
al. Gene set enrichment analysis: a knowledge-based approach for interpreting
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genome-wide expression profiles. Proc Natl Acad Sci U S A 2005; 102: 15545-
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15550.
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13.
Yan P, Frankhouser D, Murphy M, Tam HH, Rodriguez B, Curfman J et al.
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Genome-wide methylation profiling in decitabine-treated patients with acute
202
myeloid leukemia. Blood 2012; 120: 2466-2474.
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Supplementary Table S1a. One hundred ninety-five upregulated genes in high
DNMT3B expressers (each test is at P<0.001 and FDR<0.01). This Table is provided in
a separate Excel file.
Supplementary Table S1b. One hundred sixty-eight downregulated genes in high
DNMT3B expressers (each test is at P<0.001 and FDR<0.01). This Table is provided in
a separate Excel file.
Supplementary Information - DNMT3B expression in AML
Supplementary Table S2. DNMT3A mutations detected in CN-AML patients analyzed
for DNMT3B expression
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211
212
Mutations
n
Patients with any DNMT3A mutations
66
Total number of DNMT3A mutations
71
Mutations at codon R882
39
-
-
p.(R882H)
22a
-
-
p.(R882C)
15b
-
-
p.(R882S)
1
-
p.(R882G)
1c
Frame shift mutations
4
-
p.(A574Lfs)
1
-
p.(H739Sfs)
1
-
p.(R899Pfs)
1
-
p.(D845Tfs)
1c
Mutations affecting splice sitesd
4
-
c.1936G>A
1
-
c.2596A>G
1e
-
c.2598-20_c.2598-5del
1
-
c.2598-2A>G
1a
Nonsense mutations
3
-
p.(E561*)
1
-
p.(E814*)
1
-
p.(R887*)
1
Short in-frame deletions
2
-
p.(572_574del)
1
-
p.(F732del)
1
Missense mutations not affecting codon R882
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-
p.(S714C)
-
Other mutations
19
3b
16e,f,g
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217
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219
220
221
222
223
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a
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One patient had both an p.(R882H) mutation and a splice site mutation (c.2598-2A>G).
One patient had both an p.(R882C) mutation and a non-R882 missense mutation p.(S714C).
c One patient had both an p.(R882G) mutation and a frame shift mutation p.(D845Tfs).
d Effect on the RNA / protein level not tested.
e One patient had both a splice site mutation c.2596A>G and a non-R882 missense mutation
p.(L723I).
f The following missense mutations not affecting codon R882 were each identified in single patients:
p.(A570P), p.(R635L), p.(R635P), p.(S638P), p.(G706R), p.(V716I), p.(L723I), p.(R736H), p.(L754P),
p.(V758M), p.(M761V), p.(D768E), p.(K841E), p.(P849L), p.(K855E), p.(Y908C).
g One patient had two different missense mutations not affecting codon R882, namely p.(R635P) and
p.(K855E).
b
Supplementary Information - DNMT3B expression in AML
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226
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Supplementary Table S3. Associations of TLR4 and WT1 expression assessed by
NanoString and miR-133b, CDK6 and WT1 expression assessed by real-time PCR with
high and low DNMT3B expression
Characteristic
TLR4 nanostring expression
Median
Range
WT1 nanostring expression
Median
Range
miR-133b PCR expression
Median
Range
CDK6 PCR expression
Median
Range
WT1 PCR expression
Median
Range
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High DNMT3B
(n=24)
Low DNMT3B
(n=23)
0.18
-2.14 to 3.23
3.18
-2.36 to 5.33
0.27
-4.09 to 1.76
-3.49
-6.49 to -0.092
0.002
0.000053 to 0.0064
0.0002
0.000018 to 0.0054
2.29
0.76 to 5.55
0.69
0.28 to 2.44
0.093
0.0018 to 0.34
0.003
0.00027 to 0.052
P
<0.001
<0.001
<0.001
<0.001
<0.001
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Supplementary Figure S1. Heat map of distribution of mutations known to be
231
prognostic in cytogenetically normal AML patients according to DNMT3B expression
232
(105 high expressers and 105 low expressers). Each row represents a mutation, and
233
each column a patient. For CEBPA, it is indicated whether a single (1) or double (2)
234
mutation was present.
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236
237
238
Gene
NPM1
FLT3- ITD
DNMT3A
TET2
IDH1
IDH2- R140
IDH2- R172
CEBPA
RUNX1
FLT3- TKD
WT1
ASXL1
MLL- PTD
Gene
NPM1
FLT3- ITD
DNMT3A
TET2
IDH1
IDH2- R140
IDH2- R172
CEBPA
RUNX1
FLT3- TKD
WT1
ASXL1
MLL- PTD
Patients with high DNMT3B expression
1
1
1
1
1 1
2
1 2 1 1 2
Patients with low DNMT3B expression
1
1
1
1
1 2
2
2 2 2 1
2 2
Mutated for NPM 1, DNM T3A, TET2, IDH1, IDH2- R140, IDH2- R172, CEBPA, RUNX1, WT1, or ASXL1
Positive for FLT3- ITD, FLT3- TKD, or M LL- PTD
Wild-type for NPM 1, DNM T3A, TET2, IDH1 , IDH2- R140, IDH2- R172, CEBPA, RUNX1, WT1, or ASXL1
Negative for FLT3- ITD, FLT3- TKD, or M LL- PTD
Not Determined
239
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Supplementary Figure S2. GSEA enrichment profile of a set of 195 genes upregulated
241
in high DNMT3B expressers in CN-AML patients with or without FLT3-ITD showing that
242
the high DNMT3B expression and FLT3-ITD signatures are significantly correlated
243
[P=0.006; FDR=0.006; familywise-error rate (FWER)=0.003]. The figure depicts the
244
distribution of genes upregulated in high DNMT3B expressers according to their rank
245
position among 6334 genes sorted according to expression association with FLT3-ITD
246
positive versus negative. These expression data were derived from 62 FLT3-ITD-
247
positive and 115 FLT3-ITD-negative CN-AML patients. In the top portion of the plot, the
248
green curve shows the running enrichment score for the 195-gene set upregulated in
249
high DNMT3B expressers, as the analysis walks down the gene list. The score at the
250
peak of the green curve is the enrichment score (ES) for the 195-gene set, i.e.,
251
ES=0.695. The middle portion of the plot, with black thin bars, shows the ranking score
252
for each member of the 195-gene set. Almost all of the genes upregulated in high
253
DNMT3B expressers are also associated with FLT3-ITD, since they have a positive
254
Signal2Noise score. The bottom portion of the plot shows the value of the ranking
255
metric as one moves down the complete 6334-gene dataset. The ranking metric
256
measures a gene’s correlation with the FLT3-ITD genotype.
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