SUPPLEMENTAL INFORMATION
PROTOCOLS AND TREATMENT
All patients included in our study were treated on Cancer and Leukemia Group B (CALGB) /
Alliance first-line protocols for patients with acute myeloid leukemia (AML), and received
cytarabine/daunorubicin-based induction therapy. None of the protocols included allogeneic
stem cell transplantation in first complete remission (CR). Patients who enrolled on the
treatment protocols also provided written informed consent to participate in the companion
protocols CALGB 20202 (molecular studies in acute myeloid leukemia), CALGB 8461
(prospective cytogenetic companion), and CALGB 9665 (leukemia tissue bank) which involved
collection of pretreatment bone marrow (BM) aspirates and blood samples.
Patients younger than 60 years were treated on CALGB / Alliance trials 9621 or 19808. Patients
enrolled on CALGB 19808 (n=101) were randomly assigned to receive induction chemotherapy
with cytarabine, daunorubicin, and etoposide with or without PSC-833 (valspodar), a multidrug
resistance protein inhibitor.1 On achievement of CR, patients were assigned to intensification
with high-dose cytarabine and etoposide for stem-cell mobilization followed by myeloablative
treatment with busulfan and etoposide supported by autologous peripheral blood stem-cell
transplantation. Patients enrolled on CALGB 9621 (n=63) were treated similarly to those on
CALGB 19808, as previously reported.2
Older patients (≥60 years) were all treated with cytarabine/daunorubicin-based induction
therapy followed by cytarabine-based consolidation therapy. Patients on CALGB 8525 (n=18)
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.3 For older patients, there were no significant differences in
Metzeler et al.
Supplemental Information
disease-free (DFS) or overall survival (OS) among the different consolidation regimens. Patients
on CALGB 8923 (n=15) received induction chemotherapy consisting of cytarabine and
daunorubicin and were randomly assigned to receive postremission therapy with cytarabine
alone or in combination with mitoxantrone, resulting in no significant difference in DFS or OS
between consolidation arms.4 Patients on CALGB 9420 (n=5) and 9720 (n=97) 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-833.5,6 The PSC-833 arm of CALGB 9720 was closed after random assignment
of 120 patients because of excessive early deaths, and enrollment continued on the
chemotherapy-only control arm. Patients on CALGB 9420 and CALGB 9720 received a single
consolidation course, and patients on CALGB 9720 were then randomly assigned to low-dose
recombinant interleukin-2 maintenance therapy or none.7 Interleukin-2 maintenance was not
associated with differences in DFS or OS. Patients on CALGB 10201 (n=65) 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/m 2/d) with
or without oblimersen. Preliminary results showed no impact of the antisense therapy on
outcome.8
Page 2
Metzeler et al.
Supplemental Information
SUPPLEMENTAL METHODS
microRNA-expression profiling
For microRNA (miR) microarray analyses, we used The Ohio State University Comprehensive
Cancer Center (OSU-CCC) custom miR arrays (version 3.0 for patients younger than 60 years
and version 4.0 for patients aged 60 years or older). Signal intensities were calculated for each
spot, with an adjustment made for local background. Spots that were flagged due to low signalto-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 miR probe, an
adjustment was made for batch effect (ie, differences in expression related to the batch in which
arrays were hybridized). Further analysis was limited to human probes that passed the filtering
criterion. A comparison of miR expression was made between CEhigh and CElow patients, using
the significance analysis of microarrays (SAM) algorithm, using a q value of <.05 as the criterion
for statistical significance.9
Definition of clinical endpoints
Clinical endpoints were defined, in accordance with generally accepted criteria, as follows: CR
required a BM aspirate with cellularity greater than 20% and maturation of all cell lines, less
than 5% blasts and no Auer rods; in the peripheral blood, an absolute neutrophil count of ≥1.5 x
109/L, platelet count of >100 x 109/L, and no leukemic blasts; and no evidence of extramedullary
leukemia, all of which had to persist for at least 1 month.10 Relapse was defined by the
presence of ≥5% BM blasts, or circulating leukemic blasts, or the development of
extramedullary leukemia. DFS was measured from the date of CR until the date of relapse or
Page 3
Metzeler et al.
Supplemental Information
death; patients alive and in CR were censored at last follow-up. OS was measured from the
date of study entry until the date of death, and patients alive at last follow-up were censored.
Multivariable analyses
Multivariable logistic regression models were generated for attainment of CR, and multivariable
proportional hazards models were constructed for DFS and OS using a limited backwards
elimination procedure. Variables considered for model inclusion and evaluated in univariable
models were: the dichotomized CE gene expression score (using a median cut), mutations in
NPM1, CEBPA, ASXL1, TET2, DNMT3A, WT1, RUNX1, IDH1 and IDH2, and FLT3-tyrosine
kinase domain (FLT3-TKD) mutations, FLT3-internal tandem duplications (FLT3-ITD), and MLLpartial tandem duplications (MLL-PTD), expression levels of ERG, BAALC, and miR-155, age
group (<60 years vs ≥60 years), sex, race (white vs. non-white), white blood count, hemoglobin,
platelet count, peripheral blood blast count, and presence/absence of extramedullary
involvement. Variables significant at α=.20 from the univariable analyses were considered for
multivariable analyses. For the time-to-event endpoints, the proportional hazards assumption
was checked for each variable individually.
Page 4
Metzeler et al.
Supplemental Information
SUPPLEMENTAL RESULTS
Survival analyses according to quartiles of the numeric CE score
Patients were divided into four groups with increasing CE scores (Quartile 1 through Quartile 4),
using the 25th, 50th, and 75th percentiles of all patients as thresholds. The resulting KaplanMeier plots for DFS and OS (Supplemental Figure 1a and 1b, respectively) supported the use of
a median cut to divide our patient cohort into CEhigh and CElow subgroups.
miR expression signatures associated with the CE gene expression score in younger and
older patients
Two different versions of the OSU-CCC custom miR microarray platform were used for younger
(<60 years) and older (≥60 years) patients. A total of 535 miR probes were common to both
platforms. Separate miR signatures were generated for each age group. Within the younger
patients, expression levels of 43 miRs significantly correlated with the CE score (Supplemental
Figure 3a, Supplemental Table 2). Thirty-nine miRs showed higher expression in CEhigh
patients, while 4 miRs were expressed at higher levels in CElow patients. In the older age group,
expression of 21 miRs correlated with the CE score, and all of them showed higher expression
in patients with a high CE score (Supplemental Figure 3b, Supplemental Table 3). A set of 15
‘core’ miRs was associated with CE scores both among younger and among older patients.
These 15 miRs, representing the overlap between the two signatures described above, all
showed higher expression in patients with high CE scores. The degree of overlap between the
signatures in younger and older patients was statistically highly significant (P=1.1x10-13 by
Fisher’s exact test).
Page 5
Metzeler et al.
Supplemental Information
SUPPLEMENTAL TABLES
Supplemental Table 1. Multivariable models including the European LeukemiaNet (ELN)
Genetic Groups instead of FLT3 internal tandem duplications, NPM1 and CEBPA mutations
separately
Complete remission
Variable
OR (95% CI)
CE score (high vs low)
P
0.33
ELN Genetic Group (Favorable vs Intermediate-I)
1.86 (1.00 - 3.44)
.05
Age group (≥ 60 years vs < 60 years)
0.38 (0.22 - 0.66)
<.001
BAALC expression (high vs low)
0.25 (0.14, 0.46)
<.001
WBC (per 50-unit increase)
0.65 (0.51, 0.82)
<.001
HR (95% CI)
P
CE Score (high vs low)
1.86 (1.36 - 2.55)
<.001
ELN Genetic Group (Favorable vs Intermediate-I)
0.53 (0.39 - 0.73)
<.001
Age group (≥ 60 years vs < 60 years)
2.13 (1.58 - 2.86)
<.001
WT1 (mutated vs wild-type)
2.40 (1.39 - 4.13)
.002
WBC (per 50-unit increase)
1.26 (1.10 - 1.45)
.001
IDH1 (mutated vs wild-type)
1.55 (1.03 - 2.34)
.04
HR (95% CI)
P
1.92 (1.46 - 2.52)
<.001
Disease-free survival
Variable
Overall survival
Variable
CE Score (high vs low)
ELN Genetic Group (Favorable vs Intermediate-I)
.10
Age group (≥ 60 years vs < 60 years)
2.74 (2.08 - 3.62)
<.001
WT1 (mutated vs wild-type)
3.15 (2.00 - 4.97)
<.001
miR-155 expression (high vs low)
1.66 (1.27 - 2.17)
<.001
WBC (per 50-unit increase)
1.16 (1.06 - 1.26)
<.001
BAALC expression (high vs low)
1.51 (1.14 - 1.98)
.004
ASXL1 (mutated vs wild-type)
1.68 (1.12 - 2.52)
.01
DNMT3A (codon R882 mutation present vs absent)
1.48 (1.09 - 2.01)
.01
Abbreviations: CE, core enriched; WBC, white blood count; OR, odds ratio; HR, hazard ratio; CI,
confidence interval.
An odds ratio greater than (less than) 1.0 means a higher (lower) complete remission rate for the higher
values of the continuous variables and the first category listed for the categorical variables. A hazard ratio
greater than 1 (less than 1) corresponds to a higher (lower) risk of an event for higher values of
continuous variables and the first category listed of a dichotomous variable. Variables were considered
for inclusion in the multivariable models if they had a univariable P-value of <.2. See the Supplemental
Information for a full list of variables evaluated in univariable analyses.
Page 6
Metzeler et al.
Supplemental Information
Supplemental Tables 2 and 3: see separate Excel file
Page 7
Metzeler et al.
Supplemental Information
1
SUPPLEMENTAL FIGURES
2
Supplemental Figure 1: Age-adjusted survival curves according to quartiles of the CE stem
3
cell gene expression score. (a) Disease-free survival, (b) overall survival.
4
Page 8
Metzeler et al.
Supplemental Information
5
6
Supplemental Figure 2: Survival of patients with cytogenetically normal AML according to their
7
CE stem cell gene expression score and age group. (a) Disease-free survival of patients <60
8
years, (b) overall survival of patients <60 years, (c) disease-free survival of patients ≥60 years,
9
(d) overall survival of patients ≥60 years.
10
Page 9
Metzeler et al.
Supplemental Information
11
12
Supplemental Figure 3: microRNA (miR) expression signatures associated with the CE stem
13
cell gene expression score. (a) patients <60 years, (b) patients ≥60years.
14
15
Page 10
Metzeler et al.
Supplemental Information
16
17
SUPPLEMENTAL REFERENCES
18
1.
19
inhibition using valspodar (PSC-833) does not improve outcomes for patients younger than age
20
60 years with newly diagnosed acute myeloid leukemia: Cancer and Leukemia Group B study
21
19808. Blood 2010; 116: 1413-1421.
22
2.
23
studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance
24
modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60
25
years: final induction results of Cancer and Leukemia Group B study 9621. J Clin Oncol 2004;
26
22 :4290-4301.
27
3.
28
postremission chemotherapy in adults with acute myeloid leukemia. N Engl J Med 1994; 331:
29
896-903.
30
4.
31
therapy in older patients with de novo acute myeloid leukemia: a randomized trial comparing
32
mitoxantrone and intermediate-dose cytarabine with standard-dose cytarabine. Blood 2001; 98:
33
548-553.
34
5.
35
phase I studies of daunorubicin given with cytarabine and etoposide with or without the
36
multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age or
37
older with acute myeloid leukemia: results of Cancer and Leukemia Group B study 9420. J Clin
38
Oncol 1999; 17: 2831-2839.
39
6.
40
Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients
Kolitz JE, George SL, Marcucci G, Vij R, Powell BL, Allen SL et al. P-glycoprotein
Kolitz JE, George SL, Dodge RK, Hurd DD, Powell BL, Allen SL et al. Dose escalation
Mayer RJ, Davis RB, Schiffer CA, Berg DT, Powell BL, Schulman P et al. Intensive
Stone R, Berg D, George S, Dodge RK, Paciucci PA, Schulman PP et al. Postremission
Lee EJ, George SL, Caligiuri M, Szatrowski TP, Powell BL, Lemke S et al. Parallel
Baer MR, George SL, Dodge RK, O'Loughlin KL, Minderman H, Caligiuri MA et al.
Page 11
Metzeler et al.
Supplemental Information
41
60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B study
42
9720. Blood 2002; 100: 1224-1232.
43
7.
44
Interleukin-2 immunotherapy does not improve outcome of patients age 60 years and older with
45
acute myeloid leukemia in first complete remission: Cancer and Leukemia Group B study 9720.
46
J Clin Oncol 2008; 26: 4934-4939.
47
8.
48
randomized trial of intensive induction and consolidation chemotherapy ± oblimersen, a pro-
49
apoptotic Bcl-2 antisense oligonucleotide in untreated acute myeloid leukemia patients >60
50
years old. J Clin Oncol 2007; 25 (abstract 7012).
51
9.
52
Sci U S A 2003; 100: 9440-9445.
53
10.
54
Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and
55
response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813-819.
Baer MR, George SL, Caligiuri MA, Sanford BL, Bothun SM, Mrózek K et al. Low-dose
Marcucci G, Moser B, Blum W, Stock W, Wetzler M, Kolitz JE et al. A phase III
Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad
Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD et al.
Page 12