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Supplemental Methods
Assays to detect presence/absence of internal tandem duplications (ITD) and tyrosine
kinase point mutations (TKD) in FLT3 and mutations in hotspot regions of the NPM1,
CEBPA, WT1, IDH1, IDH2, TET2, DNMT3A, ASXL1, RUNX1 genes and expression
levels of BAALC, ERG and MN1 transcripts were carried out as previously described.1-12
The patients included in this study were treated on Cancer and Leukemia Group B
treatment protocols 1020113, 972014, 942015, 892316 and 852517 and had centrally
reviewed normal karyotypes. All patients provided written informed consent, and all
study protocols were in accordance with the Declaration of Helsinki and approved by
Institutional
Review
Boards
cytarabine/daunorubicin-based
at
each
induction
center.
therapy
All
followed
protocols
by
included
cytarabine-based
consolidation therapy. Briefly, patients on CALGB 10201 (n=68) received induction
chemotherapy consisting of cytarabine and daunorubicin, with or without the BCL2
antisense oblimersen sodium. The consolidation regimen included two cycles of
cytarabine (2 g/m2/d) with or without oblimersen.13 Patients on CALGB 9420 (n=5) and
9720 (n=107) received induction chemotherapy consisting of cytarabine in combination
with daunorubicin and etoposide, with (CALGB 9420) or with/without (CALGB 9720) the
multidrug resistance protein modulator PSC-833.14,15 Patients on CALGB 9420 received
postremission therapy with cytarabine (2 g/m2/d) alone, and patients on CALGB 9720
received a single cytarabine/daunorubicin consolidation course identical to the induction
regimen and were then randomly assigned to low-dose recombinant interleukin-2
maintenance therapy or none.14,15 Patients on CALGB 8923 (n=22) were treated with
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induction chemotherapy consisting of cytarabine in combination with daunorubicin and
were randomly assigned to receive postremission therapy with cytarabine alone or in
combination with mitoxantrone.16 Patients on CALGB 8525 (n=24) were treated with
induction chemotherapy consisting of cytarabine in combination with daunorubicin and
were randomly assigned to consolidation with different doses of cytarabine followed by
maintenance treatment.17
Complete remission, disease-free survival and overall survival times were defined as
previously described.18
To establish a signature of genes differentially expressed between MLL-PTD and MLLWT patients, we evaluated gene-expression profiles obtained using Affymetrix HGU133plus2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). The subset of
patients for whom material was available for gene- and microRNA-expression
microarray analyses (see below) did not differ significantly from the overall cohort, with
respect to baseline clinical and molecular characteristics and outcomes. Summary
measures of gene expression were computed for each probe-set using the robust
multichip average method, which incorporates quantile normalization of arrays.
Expression values were logged (base 2) before analysis. A filtering step was performed
to remove probe-sets that did not display significant variation in expression across
arrays. In this procedure, a χ2 test was used to test whether the observed variance in
expression of a probe-set was significantly larger than the median observed variance in
expression for all probe-sets, using α=.01 as the significance level. A total of 24,649
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probe-sets passed the filtering criterion. Normalized expression values were compared
between MLL-PTD (n=11) and MLL-WT (n=159) patients, and a univariable significance
level of .001 was used to identify differentially expressed probe-sets. A global test of
significance based on a permutation procedure was performed to determine whether or
not the number of differentially expressed probe sets was more than expected by
chance; if not, no signature is reported for the comparison.
For microRNA microarrays (The Ohio State University custom microRNA array version
4.0), signal intensities were calculated for each spot, with an adjustment made for local
background. Spots that were flagged due to low signal-to-noise ratio on more than 75%
of arrays were excluded from analysis. Signal intensities were log-transformed and
quantile normalization was performed on arrays using spots for all human and mouse
microRNA probes represented on the array. Log-signal intensities from replicate spots
(ie, spots representing the same probe) were averaged. For each microRNA probe, an
adjustment was made for batch effects (ie, differences in expression related to the batch
in which arrays were hybridized). Further analysis was limited to 460 unique human
probes that passed the filtering criterion. A comparison of microRNA expression was
made between MLL-PTD (n=11) and MLL-WT (n=163) patients, using a univariable
significance level of .005 to identify differentially expressed microRNA probes.
All microarray analyses were performed using BRB-ArrayTools Version 3.8.1.
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Supplemental References
1.
Whitman SP, Ruppert AS, Radmacher MD, Mrozek K, Paschka P, Langer C, et
al. FLT3 D835/I836 mutations are associated with poor disease-free survival and
a distinct gene-expression signature among younger adults with de novo
cytogenetically normal acute myeloid leukemia lacking FLT3 internal tandem
duplications. Blood 2008; 111: 1552-1559.
2.
Marcucci, G, Maharry K, Radmacher MD, Mrozek K, Vukosavljevic T, Paschka P,
et al. Prognostic significance of, and gene and microRNA expression signatures
associated with, CEPBA mutations in cytogenetically normal acute myeloid
leukemia with high-risk molecular features: a Cancer and Leukemia Group B
Study. J. Clin. Oncol. 2008; 26: 5078-5087.
3.
Becker H, Marcucci G, Maharry K, Radmacher MD, Mrózek K, Margeson D et al.
Favorable prognostic impact of NPM1 mutations in older patients with
cytogenetically normal de novo acute myeloid leukemia and associated geneand microRNA-expression signatures: a Cancer and Leukemia Group B study. J
Clin Oncol 2010; 28: 596-604.
4.
Paschka P, Marcucci G, Ruppert AS, Whitman SP, Mrozek K, Maharry K, et al.
Wilms’ tumor 1 gene mutations independently predict poor outcome in adults with
cytogenetically normal acute myeloid leukemia: a cancer and leukemia group B
study. J. Clin. Oncol. 2008; 26: 4595-4602.
5.
Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D, et al.
IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo
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cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B
study. J. Clin. Oncol. 2010; 23: 2349-2355.
6.
Metzeler KH, Maharry K, Radmacher MD, Mrozek K, Margeson D, Becker H, et
al. TET2 mutations improve the new European LeuekmiaNet risk classification of
acute myeloid leukemia: a Cancer and Leukemia Group B study. J. Clin. Oncol.
2011; 29: 1373-1381.
7.
Metzeler KH, Walker A, Geyer S, Garzon R, Klisovic RB, Bloomfield CD, et al.
DNMT3A mutations and response to the hypomethylating agent Decitabine in
acute myeloid leukemia. Leukemia 2011 Nov 29. [Epub ahead of print]
8.
Metzeler KH, Becker H, Maharry K, Radmacher MD, Kohlschmidt J, Mrózek K,
Nicolet D, Whitman SP, Wu YZ, Schwind S, Powell BL, Carter TH, Wetzler M,
Moore JO, Kolitz JE, Baer MR, Carroll AJ, Larson RA, Caligiuri MA, Marcucci G,
Bloomfield CD. ASXL1 mutations identify a high-risk subgroup of older patients
with primary cytogenetically normal AML within the ELN "favorable" genetic
category. Blood. 2011 Oct 26. [Epub ahead of print]
9.
Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T, et
al. RUNX1 mutations are frequent in de novo AML with noncomplex karyotype
and confer an unfavorable prognosis. Blood 2011; 117: 2348-2357.
10.
Langer C, Radmacher MD, Ruppert AS, Whitman SP, Paschka P, Mrózek K et
al. High BAALC expression associates with other molecular prognostic markers,
poor outcome, and a distinct gene-expression signature in cytogenetically normal
patients younger than 60 years with acute myeloid leukemia: a Cancer and
Leukemia Group B (CALGB) study. Blood 2008; 111: 5371-5379.
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11.
Page 6 of 12
Marcucci G, Maharry K, Whitman SP, Vukosavljevic T, Paschka P, Langer C et
al. High expression levels of the ETS-related gene, ERG, predict adverse
outcome and improve molecular risk-based classification of cytogenetically
normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin
Oncol 2007; 25: 3337-3343.
12.
Langer C, Marcucci G, Holland KB, Radmacher MD, Maharry K, Paschka P et al.
Prognostic importance of MN1 transcript levels, and biologic insights from MN1associated gene and microRNA expression signatures in cytogenetically normal
acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol
2009; 27: 3198-3204.
13.
Marcucci G, Moser B, Blum W, al. e. A phase III randomized trial of intensive
induction and consolidation chemotherapy oblimersen, a pro-apoptotic Bcl-2
antisense oligonucleotide in untreated acute myeloid leukemia patients >60 years
old. J Clin Oncol. 2007;25:360s (suppl; abstr 7012).
14.
Baer MR, George SL, Dodge RK, et al. Phase 3 study of the multidrug resistance
modulator PSC-833 in previously untreated patients 60 years of age and older
with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood.
2002;100:1224-1232.
15.
Lee EJ, George SL, Caligiuri M, et al. Parallel phase I studies of daunorubicin
given with cytarabine and etoposide with or without the multidrug resistance
modulator PSC-833 in previously untreated patients 60 years of age or older with
acute myeloid leukemia: results of cancer and leukemia group B study 9420. J
Clin Oncol. 1999;17:2831-2839.
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16.
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Stone RM, Berg DT, George SL, et al. Postremission therapy in older patients
with de novo acute myeloid leukemia: a randomized trial comparing mitoxantrone
and intermediate-dose cytarabine with standard-dose cytarabine. Blood.
2001;98:548-553.
17.
Mayer RJ, Davis RB, Schiffer CA, et al. Intensive postremission chemotherapy in
adults with acute myeloid leukemia. Cancer and Leukemia Group B. N Engl J
Med. 1994;331:896-903.
18.
Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD et
al. Report of the National Cancer Institute-sponsored workshop on definitions of
diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813819.
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Supplemental Data
Figure S1. A heatmap depicting distribution of additional molecular markers in 13 CNAML patients with MLL-PTD. Each column represents one patient. Rows represent the
presence (red) or absence (grey) of 12 types of mutations in 10 different genes, and
high (dark blue) or low (grey) expression levels of three additional genes. White color
denotes missing data. The bottom row shows to which ELN Genetic Group each patient
belonged. The Favorable and Intermediate-I Genetic Groups are indicated by,
respectively, yellow and green.
MLL -PTD
NPM1
FLT3 -ITD
FLT3- TKD
RUNX1
CEBPA
WT1
Mutation positive
Mutation negative
not determined
DNMT3A
ASXL1
TET2
IDH1
IDH2 R140
IDH2 R172
ERG
BAALC
MN1
ELN
Low expression
High expression
not determined
ELN Favorable
ELN Intermediate I
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Table S1. Relative fold-change in expression of Affymetrix probe sets
that target MLL (Accession # NM_005933) and the MLL mRNA region
detected by each probe set
Probe set
Fold-change:
MLL-PTD/
MLL-WT
P-value
MLL mRNA regions detected (NM_005933)
1565436_s_at
212079_s_at
212078_s_at
216624_s_at
212080_at
1.61
0.0002994
Exon 3-exon 4
1.74
0.0003348
Exon 4
1.41
0.0037313
Exon 4-exon 7
1.19
0.0993096
Exon 33-exon 36
0.96
0.7578895
3’-UTR (note the 3’-end of the probe set target
sequence is not included in NM_059333)
1559856_s_at
226981_at
212076_at
1.03
0.7733507
Exon 10 splice variant
1.03
1.02
0.873657
0.8766589
3’-UTR; but also recognizes non-MLL sequence
3’-UTR
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Participating Institutions
The following Cancer and Leukemia Group B (CALGB) institutions, principal
investigators, and cytogeneticists participated in this study:
Wake Forest University School of Medicine, Winston-Salem, NC: David D. Hurd, P.
Nagesh Rao, Wendy L. Flejter and Mark J. Pettenati (grant no. CA03927); The Ohio
State University Medical Center, Columbus, OH: Clara D. Bloomfield, Karl S. Theil,
Diane Minka and Nyla A. Heerema (grant no. CA77658); University of Iowa Hospitals,
Iowa City, IA: Daniel A. Vaena and Shivanand R. Patil (grant no. CA47642); North
Shore–Long Island Jewish Health System, Manhasset, NY: Daniel R. Budman and
Prasad R. K. Koduru (grant no. CA35279); Roswell Park Cancer Institute, Buffalo, NY:
Ellis G. Levine and AnneMarie W. Block (grant no. CA02599); Duke University Medical
Center, Durham, NC: Jeffrey Crawford, Mazin B. Qumsiyeh, John Eyre and Barbara K.
Goodman (grant no. CA47577); University of Chicago Medical Center, Chicago, IL:
Hedy L. Kindler, Diane Roulston, Yanming Zhang and Michelle M. Le Beau (grant no.
CA41287); Washington University School of Medicine, St. Louis, MO: Nancy L. Bartlett,
Michael S. Watson, Eric C. Crawford, Peining Li, and Jaime Garcia-Heras (grant no.
CA77440); Dana Farber Cancer Institute, Boston, MA: Harold J. Burstein, Ramana
Tantravahi, Leonard L. Atkins, Paola Dal Cin and Cynthia C. Morton (grant no.
CA32291); Eastern Maine Medical Center, Bangor, ME: Harvey M. Segal and Laurent J.
Beauregard (grant no. CA35406); Long Island Jewish Medical Center CCOP, Lake
Success, NY: Kanti R. Rai and Prasad R. K. Koduru (grant no. CA11028); University of
North Carolina, Chapel Hill, NC: Thomas C. Shea and Kathleen W. Rao (grant no.
CA47559); Ft. Wayne Medical Oncology/Hematology, Ft. Wayne, IN: Sreenivasa
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Nattam and Patricia I. Bader; SUNY Upstate Medical University, Syracuse, NY: Stephen
L. Graziano, Larry Gordon and Constance K. Stein (grant no. CA21060); Minneapolis
VA Medical Center, Minneapolis, MN: Vicki A. Morrison and Sugandhi A. Tharapel
(grant no. CA47555); University of Maryland Cancer Center, Baltimore, MD: Martin J.
Edelman, Joseph R. Testa, Maimon M. Cohen and Yi Ning (grant no. CA31983);
Dartmouth Medical School, Lebanon, NH: Konstantin Dragnev, Doris H. Wurster-Hill
and Thuluvancheri K. Mohandas (grant no. CA04326); Vermont Cancer Center,
Burlington, VT: Steven M. Grunberg, Elizabeth F. Allen and Mary Tang (grant no.
CA77406); Mount Sinai School of Medicine, New York, NY: Lewis R. Silverman and
Vesna Najfeld (grant no. CA04457); Weill Medical College of Cornell University, New
York, NY: John Leonard and Ram S. Verma (grant no. CA07968); Rhode Island
Hospital, Providence, RI: William Sikov, Teresita Padre-Mendoza, Hon Fong L. Mark
and Shelly L. Kerman (grant no. CA08025); University of Massachusetts Medical
Center, Worcester, MA: William V. Walsh and Vikram Jaswaney (grant no. CA37135);
McGill Department of Oncology, Montreal, Quebec: J. L. Hutchison and Jacqueline
Emond (grant no. CA31809); University of Minnesota, Minneapolis, MN: Bruce A.
Peterson, Diane C. Arthur and Betsy A. Hirsch (grant no. CA16450); University of Illinois
at Chicago: David J. Peace, Maureen M. McCorquodale and Kathleen E. Richkind
(grant no. CA74811); University of Nebraska Medical Center, Omaha, NE: Anne
Kessinger and Warren G. Sanger (grant no. CA77298); Western Pennsylvania Hospital,
Pittsburgh, PA: John Lister and Gerard R. Diggans; Walter Reed Army Medical Center,
Washington, DC: Brendan M. Weiss and Rawatmal B. Surana (grant no. CA26806);
University of Cincinnati Medical Center, Cincinnati, OH: Orlando J. Martelo and Ashok
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K. Srivastava (grant no. CA47515); Columbia-Presbyterian Medical Center, New York,
NY: Rose R. Ellison and Dorothy Warburton (grant no. CA12011); Georgetown
University Medical Center, Washington, DC: Minnetta C. Liu and Jeanne M. Meck (grant
no. CA77597); Massachusetts General Hospital, Boston, MA: Jeffrey W. Clark, Leonard
L. Atkins, and Cynthia C. Morton (grant no. CA 12449); SUNY Maimonides Medical
Center, Brooklyn, NY: Sameer Rafla and Ram S. Verma (grant no. CA25119);
University of Missouri/Ellis Fischel Cancer Center, Columbia, MO: Michael C. Perry and
Tim H. Huang (grant no. CA12046); Medical University of South Carolina, Charleston,
SC: Mark R. Green and Daynna J. Wolff (grant no. CA03927); University of Puerto Rico
School of Medicine, San Juan, PR: Eileen I. Pacheco, Paola Dal Cin, Leonard L. Atkins
and Cynthia C. Morton; University of California at San Diego: Barbara A. Parker and
Renée Bernstein (grant no. CA11789); Christiana Care Health Services, Inc., Newark,
DE: Stephen S. Grubbs and Digamber S. Borgaonkar (grant no. CA45418).