REGULAR ARTICLE

Interleukin-6 levels predict event-free survival in pediatric AML and

suggest a mechanism of chemotherapy resistance

Alexandra M. Stevens,1 Jennifer M. Miller,2 Jaime O. Munoz,1 Amos S. Gaikwad,1 and Michele S. Redell1
1
Division of Pediatric Hematology/Oncology and 2Department of Pediatrics, Baylor College of Medicine, Houston, TX

The tumor microenvironment can protect cancer cells from conventional anticancer

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Key Points
• IL-6 levels in bone mar-
row predict event-free
survival in pediatric
AML.
• Exogenous IL-6 pro-
tects AML blasts from
chemotherapy-induced
apoptosis.
therapies. Thus, targeting these protective mechanisms could eradicate therapy-resistant
cancer cells and improve outcomes. Interleukin-6 (IL-6) provides extrinsic protection for
several solid tumors and multiple myeloma. In pediatric acute myeloid leukemia (AML),
IL-6–induced STAT3 signaling frequently becomes stronger at relapse, and increases in
IL-6–induced STAT3 activity are associated with inferior survival after relapse. These
findings suggested that the IL-6–induced STAT3 pathway may promote chemotherapy
resistance and disease progression. Thus, we investigated the dysregulation of IL-6 levels in
the bone marrow niche in pediatric patients with AML and the association between IL-6
levels and outcome. We measured levels of over 40 cytokines and growth factors in plasma
from diagnostic bone marrow aspirates of 45 pediatric AML patients and 7 healthy sibling
controls. Of the measured cytokines, only IL-6 levels were associated with event-free
survival. Importantly, the effect of elevated IL-6 was most striking among children classified
as having a low risk of relapse. In these patients, 5-year event-free survival was 82.5% 6 11%
for patients with low IL-6 levels at diagnosis (n 5 14) compared with 17.3% 6 11% for
patients with elevated IL-6 (n 5 13, log-rank P 5 .0003). In vitro, exogenous IL-6 reduced
mitoxantrone-induced apoptosis in cell lines and primary pediatric AML samples. These
results suggest that IL-6 levels at diagnosis could be used to help identify children at high risk
of relapse, particularly those who are otherwise classified as low risk by current algorithms.
Moreover, the IL-6 pathway could represent a target for overcoming environment-mediated
chemotherapy resistance.

Introduction

In the United States, 500 children are diagnosed with acute myeloid leukemia (AML) every year, and the
best available treatments only cure ;60% of these children.1,2 In the last 50 years, incremental
improvements in cure rates for pediatric AML have been achieved by refinements of treatment regimens,
improved supportive care, and optimization of risk-stratification algorithms.2-6 Current algorithms are based
on a few cytogenetic changes and mutations and on an individual’s response to the initial treatment.2,7 For
pediatric patients with AML, the ability of the algorithm to stratify risk at diagnosis is limited. Few prognostic
cytogenetic changes have been identified thus far, and the majority of patients do not have these markers
at diagnosis. Currently, 30% of pediatric patients with AML are classified as high risk (HR) and 70% as low
risk (LR), yet ;30% of LR patients relapse.7 Relapsed AML is frequently resistant to chemotherapy, and it
is difficult to achieve a second remission.8 Thus, one critical step to improve outcomes in pediatric patients
with AML is identifying additional markers that can improve our risk-stratification algorithms.9

Submitted 21 April 2017; accepted 5 July 2017. DOI 10.1182/ © 2017 by The American Society of Hematology
bloodadvances.2017007856.
The full-text version of this article contains a data supplement.

8 AUGUST 2017 x VOLUME 1, NUMBER 18 1387

Table 1. Patient data by unique patient number
UPN Age/sex Cytogenetics FLT3-ITD Risk group Remission analysis Expanded analysis Event Outcome IL-6 level (pg/mL)

1 6.0/F t(9;11) LR Y Y Censored A 6.34

2 0.76/F Normal HR N Y Relapse DD 133.46

3 11.2/F t(8;21) LR N Y Relapse DD 88.62

4 17.0/F 41 LR N N Censored A 3.22

5 0.55/F t(2;11) LR Y N Relapse TD 71.3

6 0.70/F ND LR N Y Relapse DD 41.36

7 11.2/M Normal HR N Y Refractory DD 1.59

8 14.9/M t(6;9) HR N Y Censored A 8369

9 1.5/F Complex LR N Y Relapse DD 12.23
10 10.4/M 13q2 HR N Y Censored A 62.21

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11 11.1/M t(9;11) LR Y Y Censored A 15.51
12 7.7/M t(8;21) LR Y Y Relapse TD 47.97

13 9.7/M t(1;6;11) HR N Y Relapse DD 6.26

14 3.4/F t(8;21) LR Y N Censored A 465.47

15 13.3/M t(10;11) HR N N Refractory DD 19.42

16 14.1/F t(9;11) LR Y Y Censored A 3.31

17 12.5/M 27 HR N N Relapse A ,3
18 4.8/F t(8;21) LR N Y Censored A 11.88

19 1.8/F Tetraploid LR N N Relapse A 13.8

20 3.7/F 11q23 del LR N N Relapse A 6.62
21 0.6/F t(1;11) LR Y N Censored A 11.79

22 0.4/F 11q23 del, FLT3-ITD HR N N Relapse DD 13.08

23 12.4/M Normal HR N N Relapse DD 4.55

24 8.3/M 27 HR Y Y DIR TD 10.66

25 14.9/M t(8;21) LR Y Y Relapse DD 34.45

26 4.4/M FLT3-ITD HR N N Relapse DD 38.83

27 9.7/M inv(16) LR Y N Censored A 5.15

28 7.1/M t(8;21) LR N N Censored A 41.81

29 9.7/M Normal HR N N Relapse DD 4.86
30 14.0/M inv(16) LR Y N Relapse A 12059

31 0.4/F t(10;11) HR N N Censored A 4.4

32 8.5/M inv(16), 7q2 LR Y N Relapse A 3.77

33 0.8/M inv(16) LR Y Y Censored A 3.15

34 12.7/F Complex LR Y Y Censored A 10.42

35 3.0/M t(8;21) LR Y Y Censored A 4.78

36 14.3/F inv(16) LR Y N Censored A 4.7

37 3.3/M Normal LR Y N Relapse DD 28.12

38 2.1/F 18, 121 LR Y N Censored A 4.42

39 1.3/F Complex, t(10;11) HR Y Y Censored A 52.27

40 16.6/F t(6;11) HR N N Relapse DD ,3
41 17.3/M Derivative 8 LR Y Y Relapse A 26.96

42 1.3/M t(10;11) HR Y N Censored A 40.12

43 13.6/F 81 HR N N Relapse DD 3.19

44 0.6/F t(11;15) LR Y N Censored A 6.93

45 17.7/M 13q2 HR N N Relapse A 6.86

Age is given in years at the time of initial diagnosis. Risk group is defined as per AAML1031 guidelines with the following additions: mixed lineage leukemia translocations identified as
poor risk factors, t(6;9)(p23;q34), and myelodysplastic syndrome evolving into AML were also considered HR characteristics. Outcome column indicates status at last contact.
A, alive at time of last follow-up; DD, death due to disease; DIR, death in remission; F, female; ITD, internal tandem duplication; M, male; NBM, normal bone marrow; ND, not done; TD,
death in remission due to toxicity; UPN, unique patient number.

1388 STEVENS et al 8 AUGUST 2017 x VOLUME 1, NUMBER 18

 Table 1. (continued)
UPN Age/sex Cytogenetics FLT3-ITD Risk group
NBM1 5.8/F — —
NBM2 12.2/M — —
NBM3 9.2/F — —
NBM4 3.1/F — —
NBM5 13.0/M — —
NBM6 6.7/M — —
NBM7 4.8/M — —
Age is given in years at the time of initial diagnosis. Risk group is defined as per AAML1031 guidelines with the following additions: mixed lineage leukemia translocations identified as
poor risk factors, t(6;9)(p23;q34), and myelodysplastic syndrome evolving into AML were also considered HR characteristics. Outcome column indicates status at last contact.
A, alive at time of last follow-up; DD, death due to disease; DIR, death in remission; F, female; ITD, internal tandem duplication; M, male; NBM, normal bone marrow; ND, not done; TD,
death in remission due to toxicity; UPN, unique patient number.
Previous studies have investigated the relationship between
cytokines and clinical outcome in adult AML, and several cytokines
have been shown to be prognostic. For example, elevated levels of
interleukin-6 (IL-6) in peripheral blood predicted poorer overall
survival (OS), whereas elevated IL-10 correlated with improved
outcomes.10 Additionally, elevated levels of some cytokines in the
adult AML bone marrow (BM) niche have been described, including
IL-6.11 Recently, a study of a small pediatric AML cohort showed
that IL-6 levels in peripheral blood were elevated compared with
healthy age-matched controls.12 However, the cytokine milieu in the
BM niche of pediatric patients with AML has not been investigated
previously. This is partially due to a lack of available banked samples.
In a previous study, we used BM samples from pediatric patients
with AML to identify a novel functional profile in that predicts
prognosis.13 Specifically, we demonstrated that the sensitivity of
AML cells to ligand-induced activation of STAT3 predicts both
event-free survival (EFS) and OS in pediatric patients with AML. IL-6
is one of the ligands that activates STAT3. IL-6 binds a 4-subunit complex
composed of 2 gp130 subunits and 2 IL-6 receptors (IL-6Ra) that
activates JAK2. Activated JAK2 phosphorylates STAT3 at tyrosine 705
(pY-STAT3). Phosphorylated STAT3 proteins dimerize and are trans-
located to the nucleus, where they act as transcription factors regulat-
ing expression of prosurvival genes.14,15 Clinically, activation of STAT3
promotes cell survival in the presence of cytotoxic chemotherapy.13,16-18
Increased STAT3 activity been associated with chemotherapy re-
sistance and inferior survival across many malignancies.19,20
The aim of this study was to investigate the role that IL-6 in the BM
microenvironment plays in determining long-term clinical responses
to cytotoxic chemotherapy. We determined the relationship be-
tween IL-6 levels in the BM at diagnosis and subsequent EFS. We
further determined the effects of IL-6 on chemotherapy-induced
apoptosis and on STAT3 signaling in the critical leukemia stem cell
(LSC) population. Our findings identify IL-6 as a potential marker
with which to improve risk stratification and a potential therapeutic
target in pediatric AML.
Materials and methods
Patient samples
Plasma. In total, 45 pediatric patients with AML who were
treated at Texas Children’s Cancer and Hematology Centers
between 2007 and 2016 were included on this study. Selection of
patient samples was based on both the availability of specimens
8 AUGUST 2017 x VOLUME 1, NUMBER 18
Remission analysis Expanded analysis Event Outcome IL-6 level (pg/mL)
— Y — — 2.62
— — — — ,1.7
— — — — 4.54
— Y — — ,1.53
— Y — — ,1.7
— Y — — 5.67
— Y — — ,1.53
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and on sufficient follow-up time to ascertain EFS. For this reason,
our cohort is enriched for relapses. Plasma samples from the
diagnostic BM aspirate were collected and frozen at 280°C. All
patients initially received treatment according to Children’s
Oncology Group protocols AAML0531 or AAML1031.1,21 The
regimens were very similar on these 2 trials. Risk stratification
differed between the 2 trials, most notably with the inclusion of
minimal residual disease measurement on AAML1031. Thus, for the
purposes of our data analysis, all 45 patients were assigned a risk
group based on the current standard of care at our institution,
following AAML1031 guidelines and also including patients with
t(10;11), t(6;11), t(6;9), and antecedent myelodysplastic syn-
drome as HR.7,22,23 This assignment took into account all established
prognostic markers for pediatric AML. Plasma from healthy pediatric
sibling donors undergoing BM harvest between 2014 and 2016
were used as controls. Cytokine levels were not related to the
sample’s age (supplemental Figure 1).
Viably frozen cells. Samples used for viable cell assays
included both excess pheresis product and BM aspirate samples.
Prior to cryopreservation, samples were enriched for mononuclear
cells using density centrifugation.24 Viably frozen patient samples
were thawed into Iscove modified Dulbecco medium (HyClone,
Pittsburg, PA) with 20% fetal bovine serum (FBS; Invitrogen) with
100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). Cells
were washed once with the medium and then incubated in the same
medium for 2 hours. Cell number and viability were assessed using
the Trypan blue dye exclusion method.25 Samples used for cell-
based assays were confirmed to have at least 80% viability after
thawing. All patients or guardians provided written informed con-
sent for the use of tissue for research purposes in accordance with
the Declaration of Helsinki. These studies were approved by the
institutional review board of Baylor College of Medicine.
Cell lines
The human AML cell lines THP-1 and NB4 were obtained from
ATCC (Manassas, VA). They were grown in RPMI medium (ATCC)
supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL
streptomycin. THP-1 and NB4 cells were grown in a humidified
37°C incubator with 5% CO2.
Quantification of cytokines
We evaluated 41 cytokines using the Milliplex Human Cytokine
Magnetic Bead Premixed 41-plex kit (EMD Millipore). The measured
CYTOKINES IN PEDIATRIC AML 1389
 A *** B ***
15000 500
1000
500 400
400
IL-6 (pg/mL)

150 300

IL-10 (pg/mL)
200
100
100
50
0
0
-100
w

w
5) os at

5) os a t

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rro

rro
=4 gn m

=4 gn m
is

is
a

a
(n Dia eru

(n Dia e r u
=7 M

=7 M
L wS

L wS
(n one

(n one
)

)
AM r r o

AM r r o
B

B
al

al
a

a
M

M
m

m
or

or
N

N
C
15000
***

1000
1000
IL-8 (pg/mL)

200
200
150
100
50
0
5) os a t
w
ro

=4 gn m
ar

is
(n Dia e r u
=7 M

L wS
(n one
)

AM r r o
B

a
al

M
m
or
N

Figure 1. Cytokine levels vary in pediatric AML at diagnosis. Cytokine levels from BM plasma samples taken at the time of diagnosis for patients with untreated AML
were compared with those of healthy children. After Bonferroni correction, 3 out of 41 tested cytokines and growth factors had levels at AML diagnosis that were significantly
different when compared with normal controls. Levels of IL-6 (A), IL-10 (B), and IL-8 (C) were significantly elevated in a subset of AML patients at diagnosis compared with normal
children. ***P , .001 by Mann-Whitney U test. The threshold of significance after Bonferroni correction was P 5 .0012.

cytokines included soluble CD40 ligand, epidermal growth factor,
eotaxin/C-C motif chemokine 11, fibroblast growth factor 2,
Fms-related tyrosine kinase 3 ligand, fractalkine, granulocyte-
colony stimulating factor, granulocyte-macrophage colony-stimulating
factor, growth-regulated a, interferon-a2 (IFN-a2), IFN-g, IL-1a,
IL-1b, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12
(p40), IL-12 (p70), IL-13, IL-15, IL-17, IFN-g–inducible protein 10,
monocyte chemotactic protein 1, monocyte chemotactic protein
3, macrophage-derived chemokine/C-C motif chemokine 22,
macrophage inflammatory protein 1a, macrophage inflammatory
protein 1b, platelet-derived growth factor AA, platelet-derived
growth factor AB/BB, RANTES (regulated on activation, normal
T-cell expressed and secreted), transforming growth factor a, tumor
necrosis factor-a (TNF-a), TNF-b, and vascular endothelial growth
factor. Experiments were performed in triplicate and according to the
manufacturer’s instructions with overnight incubation. A MAGPIX
multiplex array reader was used to detect fluorescent signals, and
Milliplex Analyst 5.1 was used to analyze data. Standard curves
were generated using reference concentrations provided in each
kit, and raw relative fluorescence intensity values were converted to
cytokine concentrations in picograms per milliliter. When values for
individual samples were outside the quantification limits of the test,
the result was recorded as the upper or lower quantifiable concen-
tration. Measurement of soluble IL-6Ra (sIL-6Ra), IL-11, oncostatin
M (OSM), and leukemia inhibitory factor (LIF) levels were performed
on a subset of patient samples (Table 1). These receptors and
cytokines were measured using the protocol described above with
beads directed toward these analytes (EMD Millipore). Levels of
these analytes were measured in duplicate.
Assessment of IL-6–induced chemoprotection
AML cell lines were plated in standard growth medium (described
above) and exposed to exogenous IL-6 and sIL-6Ra (50 ng/mL
and 100 ng/mL, respectively) or vehicle for 24 hours. Mitoxantrone,

1390 STEVENS et al 8 AUGUST 2017 x VOLUME 1, NUMBER 18

Figure 2. Elevated IL-6 levels are associated with inferior
clinical outcome. (A) Using a cut point of 12 pg/mL for the entire A B
log rank p=0.03 log rank p=0.0003
cohort (n 5 45), 5-year EFS was 20.6% 6 10% for those with 100 100
low IL-6 (n=14)

Event free survival

Event free survival

high IL-6 levels at diagnosis vs 43.4% 6 12% for those with low
IL-6 levels (log-rank P 5 .036). (B) For LR patients (n 5 27), low IL-6 (n=24)
a cut point of 12 pg/mL identified patients with inferior EFS 50 50
(17.3% 6 11% vs 82.5% 6 11% for high vs low IL-6 levels,
P 5 .0003). (C) Inferior OS was also noted for the LR patients (cut
high IL-6 (n=21) high IL-6 (n=13)
point of 12 pg/mL, 39% 6 14.8% vs 90.1% 6 8.7% for high vs 0 0
low IL-6 levels, P 5 .007, n 5 27). No other cytokines were 0 2 4 6 8 10 0 2 4 6 8 10
associated with clinical outcome in our cohort. Notably, levels of Time to event (years) Time to event (years)
IL-8 (D) and IL-10 (E) were not associated with EFS. Estimates
of EFS are reported with corresponding Greenwood standard
C D
errors. Groups were compared for significant differences by the

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100 log rank p=0.007 100 log rank p=0.61
log-rank test.

Event free survival

Overall survival
low IL-6 (n=14)

high IL-10 (n=22)

50 50

high IL-6 (n=13)

low IL-10 (n=23)
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time to event (years) Time to event (years)

E
100 log rank p=0.41
Event free survival

high IL-8 (n=23)

50

low IL-8 (n=22)

0
0 2 4 6 8 10
Time to event (years)

etoposide, or cytarabine was added, and cells were incubated for
another 24 hours. Apoptosis was quantified based on Annexin
V/fluorescein isothiocyanate and propidium iodide positivity using
an LSRII flow cytometer (BD Biosciences). Data were analyzed using
FCS Express 5 software (De Novo, Glendale, CA). For primary sample
culture, cells were plated in Iscove modified Dulbecco medium with
20% FBS, exposed to IL-6 and sIL-6Ra (5 ng/mL and 10 ng/mL,
respectively) or vehicle for 6 hours, and treated with mitoxantrone for
18 hours. Mitoxantrone-induced apoptosis was quantified as de-
scribed above. We chose mitoxantrone as the representative
anthracycline, because its fluorescence spectrum overlaps
much less with our analytical fluorochromes than daunorubicin.
IL-6–induced STAT3 activity in LSCs
Diagnostic BM samples from 10 pediatric patients with AML were
thawed, and a viability $80% was confirmed using a Trypan blue
exclusion assay. Primary samples were sorted into LSC-enriched
(LSCe) (CD341/CD382/Aldefluorint) and non–stem AML cell
(CD341/CD382/Aldefluorlow) populations using a MoFlo Cell
Sorter (Beckman Coulter) as described previously.26 Cells from
each subpopulation were divided into 3 aliquots. Two were left
untreated to use as an isotype control and determine constitutive
pY-STAT3 levels. The remaining aliquot was stimulated for 15 min-
utes with 50 ng/mL IL-6 and 100 ng/mL sIL-6Ra. Cells were fixed,
permeabilized, and stained with hCD45-V450, isotype control/
phycoerythrin, and pY-STAT3/phycoerythrin as appropriate (BD
Biosciences). Flow cytometry data were collected on an LSRII flow
cytometer and analyzed using FCS Express 4. For each sub-
population, pY-STAT3 was measured in untreated and stimulated
cells. Data are expressed as the percentage of cells in the positive
region, as defined by the isotype control. A Wilcoxon signed-rank test
was used to search for significant differences in parameters between
subpopulations.
Statistics
For each cytokine, the mean concentration (in picograms per
milliliter) was reported. When cytokine level data passed the
Shapiro-Wilks normality test, healthy donor and AML patient
samples were compared using a Student t test. In cases where
data did not pass the normality test, a Wilcoxon nonparametric test
was used. To account for multiple comparisons within the multiplex
analysis, the Bonferroni method produced an adjusted significance
level of 0.0012 to identify significant differences between cytokines
in patients vs healthy controls. Only differences meeting this

8 AUGUST 2017 x VOLUME 1, NUMBER 18 CYTOKINES IN PEDIATRIC AML 1391


***
15000 # 500
5000
500
IL-10 (pg/mL)
IL-6 (pg/mL)
400
100
50
0 0
Diagnosis Remission
*
15000 #
500
500
IL-8 (pg/mL)
150
150
100
50
0
Diagnosis Remission
stringent threshold were reported. Cytokine levels were analyzed
using cut-point analysis to determine whether levels were asso-
ciated with clinical outcome. Five cut points for each of the 3
differentially expressed cytokines were tested with the selection
interval including the inner 80% of the continuous covariate’s
distribution. The 5 cut points tested were the integer values
closest to the 10th, 25th, 50th, 75th, and 90th percentiles.
The optimal cut point was determined by the minimum P value
approach, and clinical outcomes were compared between groups.
EFS was defined as the time from diagnosis to the identification of
refractory disease, relapse, or toxic death. Estimates of EFS were
reported with corresponding Greenwood standard errors. Groups
were compared for significant differences using the log-rank test.
Adjustment to the significance by the Bonferroni method produced
a significance threshold of 0.01. P values falling between 0.05
and 0.01 are notated within the text and within figure legends. All
analyses were performed using GraphPad Prism version 6.05 for
Windows (GraphPad Software, La Jolla, CA).
Results
Plasma cytokine profiles of pediatric patients with
AML compared with those of healthy donors
We analyzed the cytokine profiles of plasma samples collected
from the diagnostic BM aspirates of pediatric patients with AML
(AML cohort; n 5 45) and from normal BM collected from healthy
sibling donors (NBM cohort; n 5 7). Among the 41 cytokines and
chemokines surveyed, 3 had significantly different levels in the AML
cohort compared with the NBM cohort. For example, IL-6 levels
were significantly elevated in AML patients compared with normal
controls (Figure 1A; P 5 .0001). The median IL-6 level in the AML
cohort was 11.79 pg/mL (interquartile range, 4.62-40.74). In the
NBM cohort, the median IL-6 level was 1.7 pg/mL (interquartile
range, 1.53-4.54). IL-6 levels were below the lower limit of the assay
1392 STEVENS et al
Figure 3. Alterations in cytokine levels return toward
*
normal levels at remission in the majority of patients. For
22 of the AML patients, matched remission BM plasma
400 samples were available. BM plasma IL-6, IL-10, and IL-8 levels
decreased in the majority of patients at remission compared with
300
diagnosis. *P , .05, **P , .01, ***P , .001. #Unique patient
200 number 14.
100 #
Diagnosis Remission
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for 3 of the 45 samples in the AML cohort and 4 of the 7 samples in
the NBM cohort.
In addition to IL-6, levels of IL-10 and IL-8 were significantly different
in the AML cohort compared with the NBM cohort (Figure 1B-C).
Importantly, in our patient group, the concentrations of these
cytokines were elevated in a subset of AML patient samples.
This result suggests that the composition of cytokines within the
BM microenvironment may vary substantially between patients.
This variability in cytokine levels provided the ability to evaluate
for potential relationships between cytokine levels and clinical
outcomes.
Cytokine levels at diagnosis and clinical outcome
We performed cut-point analyses to determine if clinical outcomes
were correlated with cytokine levels. Our previous study showed
that increased IL-6–induced pY-STAT3 at relapse compared with
diagnosis was a predictor of poor outcomes after relapse.20
Therefore, we first examined whether elevated IL-6 levels at
diagnosis were associated with an increased risk of relapse. By
cut-point analysis, 12 pg/mL was identified as the most discrimi-
nating level of IL-6 by which to group patients by clinical outcome.
Using the cut point of 12 pg/mL IL-6 for the entire cohort (n 5 45),
both EFS and OS were evaluated. The 5-year EFS was 20.6% 6
10% for patients with high IL-6 levels at diagnosis compared with
43.4% 6 12% for those with low IL-6 levels (Figure 2A; log-rank
P 5 .036). Differences in 5-year OS were not significant between
groups (supplemental Figure 2A).
Next, we analyzed the subset of patients that would be currently
classified as LR. In this subset (n 5 27), 12 relapses (43%) were
observed, which was a sufficient number of events to evaluate
differences in clinical outcome between groups despite the
relatively small sample size. Among patients with high IL-6
levels (n 5 13), 5-year EFS was 17.3% 6 11% compared with
8 AUGUST 2017 x VOLUME 1, NUMBER 18
 A *
B ns C
600 200 100 log rank p= 0.89

Event free survival

150

OSM (pg/mL)
OSM (pg/mL)

400
low OSM (n=12)
200 100 50

0 50
high OSM (n=8)
-200 0 0
0 2 4 6 8

is

n
) dre of

1) os at

io
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=2 gn a
=5 il a

iss
n

(n Dia lasm
(n Ch sm

gn
is Time to event (years)

m
ia
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n = 10 pairs

Re
D
L P
lth P

w
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at

at
AM rro
H arro

SM

SM
a
M
M

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D ns E ns

80000 80000
sIL-6R (pg/mL)
sIL-6R (pg/mL)

60000 60000

40000 40000

20000 20000

0 0
is

n
) re of

7) os at

io
os
=1 gn a
=6 ild a

iss
(n Dia lasm
(n Ch sm

gn
is

m
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Re

n = 13 pairs
D
L P
th P

w
ea w

at

at
AM rro
H rro

R
6
a
a

6
L-
M
M

L-
sI

sI

Figure 4. OSM and sIL-6Ra do not predict survival in pediatric AML samples. For a subset of patients with available samples, levels of OSM and sIL-6Ra were evaluated. For
OSM, levels between diagnostic AML BM plasma samples and normal controls differed significantly (A), though there was no difference between levels at diagnosis vs remission in a
paired sample analysis (B). No cut point was identified that predicted outcome for OSM level (cut point of 50 pg/mL is shown in panel C). sIL-6Ra BM plasma levels were not significantly
different between normal controls and AML samples at diagnosis (D) or between diagnostic and remission samples in a paired sample analysis (E). *P , .05; ns, not significant.

82.5% 6 11% for those patients with low IL-6 levels (n 5 14; (supplemental Figure 3A-B). Infection at the time of diagnosis was
P 5 .0003; Figure 2B). OS for those with high IL-6 levels was 39.4% 6 documented in 6 patients (13%) and did not correlate with IL-6
15% compared with 90.1% 6 9% for those patients with low IL-6 levels (supplemental Figure 3C).
levels (n 5 14; P 5 .007; Figure 2C). There were no toxic deaths
prior to relapse among these 27 patients. Therefore, disease-free IL-6 levels and clinical characteristics including body
survival is equivalent to EFS in the LR patients. Patients that were mass index
classified as HR had poor OS and EFS regardless of IL-6 levels at Due to the known relationship between obesity and outcome in
diagnosis (supplemental Figure 2B-C). pediatric AML and recent evidence suggesting that IL-6 may have a
The other cytokines that were significantly different in diagnostic role in fatty acid metabolism and obesity,27 we further interrogated
AML samples compared with normal BM, IL-8 and IL-10, were our data for correlations between clinical characteristics and
subjected to cut-point analysis. In contrast to results adult AML,10,11 IL-6 levels. Body mass index did not correlate with IL-6 levels
IL-8 and IL-10 levels were not correlated with EFS (Figure 2D-E). (supplemental Figure 4A; Spearman R 5 0.1244, P 5 .2562,
n 5 30). Furthermore, age, gender, BM blast percentage on initial
IL-6 levels and clinical signs of inflammation biopsy, and white blood cell count at diagnosis did not correlate
with IL-6 levels (supplemental Figure 4B-E). Although our cohort
at diagnosis was too small for a true multivariate analysis, we interrogated our
Because IL-6 is an inflammatory cytokine that plays a key role in the data for relationships between IL-6 levels and cytogenetic and
acute-phase immune response, we performed a chart review to mutation data and found no significant relationships.
determine the likelihood of a concurrent inflammatory process when
the BM plasma sample was collected. Specifically, we looked for Cytokine levels at diagnosis compared with remission
documentation of fever or infection at the time of diagnosis as these In an exploratory analysis, we compared paired diagnostic and
factors could contribute to elevated IL-6 levels. Fever at diagnosis remission plasma samples. In the majority of patients, the levels of
or parental report of fever at presentation was documented in differentially expressed cytokines normalized to that of healthy
29 of 45 patients with AML. Fever correlated weakly with IL-6 levels donors at the time of remission (Figure 3). For example, 20 of the
(Spearman R 5 0.30, P 5 .02) but did not correlate with outcome 22 patients with plasma samples collected at both diagnosis and

8 AUGUST 2017 x VOLUME 1, NUMBER 18 CYTOKINES IN PEDIATRIC AML 1393

 Figure 5. Exposure to IL-6 reduces chemotherapy-induced
vehicle control IL-6 and sIL-6Rα pretreatment
apoptosis. The AML cell lines THP-1 (A) and NB4 (B) had less
mitoxantrone-induced apoptosis when pretreated for 24 hours
A B
NB4 with IL-6 and sIL-6Ra at 50 ng/mL and 100 ng/mL, respectively
THP-1
50 (red), than vehicle-treated cells (blue) (THP-1, n 5 5; NB4, n 5 8;
40 * *
* *P , .05, analysis of variance). (C) Representative primary sample

Annexin V - FITC +
Annexin V - FITC +

40 * demonstrating decreased mitoxantrone-induced apoptosis with

30 *
30 IL-6 pretreatment, and (D) mean apoptosis rates of 7 primary
20 * AML samples with vs without IL-6 pretreatment. Duration of
20
mitoxantrone exposure was 24 hours for cell lines and 18 hours for
10 * 10 primary samples. Values shown are mean 6 standard error of the
mean; P , .05 by Wilcoxon signed-rank test. FITC, fluorescein
0 0
isothiocyanate.
uM

uM
le

nM

nM

nM

nM

le

nM

nM

nM

nM
ic

ic

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h

h
1

1
10

30

10

30

0
ve

ve
10

30

10

30
Mitoxantrone Mitoxantrone

C D
p401 Primary Samples
80 80
p=0.0156
70
60
Annexin V +

Annexin V+

60
40 50

40
20
30
e

nM

nM

nM

nM

nM

nM

nM

nM

nM
cl

cl
hi

hi
01

10

10

0
ve

ve
0.

10

0.

10
0.

Mitoxantrone Mitoxantrone

remission (remission cohort) had significantly decreased IL-6 levels induced by cytarabine, etoposide, and mitoxantrone in AML cell
at remission compared with diagnosis (Figure 3A). The median IL-6 lines and primary AML cell cultures. We stimulated with IL-6 and
level of the remission samples was 2.8 pg/mL, compared with sIL-6Ra, because sIL-6Ra is expressed ubiquitously in the BM
11.2 pg/mL at diagnosis (P 5 .0004). Of note, the one patient with niche, but the membrane-bound form is variably expressed on
a high IL-6 level at remission (Figure 3A) had no documented signs blasts (Figure 4). Exogenous IL-6/sIL-6Ra protected AML blasts
of infection at either diagnosis or remission. from mitoxantrone-induced apoptosis in both AML cell lines tested
(Figure 5A-B). However, exogenous IL-6 did not significantly affect
Evaluation of other IL-6 family cytokines cytarabine- or etoposide-induced apoptosis in these cell lines (data
IL-6 is one of 6 members of the IL-6 family of cytokines. The IL-6 not shown). These results are consistent with our prior finding that
family cytokines signal through receptor complexes containing the environment-induced mitoxantrone resistance is driven primarily by
transmembrane gp130 receptor subunit. Therefore, we expanded soluble factors as opposed to stromal contact interactions, whereas
our cytokine analysis to evaluate plasma levels of other IL-6 cytokine resistance to etoposide and cytarabine requires contact interac-
family members, IL-11, OSM, and LIF. We also quantified the level tions.28 When primary AML samples (n 5 7) were treated with IL-6
of sIL-6Ra in this subset of samples. No differences in the levels of and sIL-6Ra for 6 hours and then exposed to mitoxantrone for
IL-11, LIF, or sIL-6Ra were observed between AML and NBM 18 hours, apoptosis was significantly reduced in cultures. A rep-
samples (Figure 4; supplemental Figure 6). The OSM levels in resentative patient sample (Figure 5C) and composite data from the
diagnostic samples from the AML patients were significantly higher seven tested samples are shown (Figure 5D).
than those from the normal controls (median of 30.3 pg/mL vs
11.7 pg/mL, respectively; P 5 .03). No differences were observed
IL-6 and the LSC population of AML
between diagnosis and remission, and cut-point analysis did not Our previous study showed that IL-6–induced pY-STAT3 is
show an association between OSM level and EFS (Figure 4). frequently increased at relapse, even in patients with minimal
measurable IL-6–induced pY-STAT3 at diagnosis.20 LSCs are
IL-6 and chemotherapy resistance described to be relatively quiescent and thus more likely to survive
To determine how elevated IL-6 levels in the BM niche could upfront chemotherapy. Furthermore, LSCs retain the ability to
contribute to increased relapse risk, we tested the effect generate a clone that emerges at relapse. Therefore, we
of exogenous IL-6 on chemotherapy-induced apoptosis. We investigated the effect of IL-6 on LSCs. Diagnostic AML samples
evaluated the effect of IL-6 pathway stimulation on apoptosis (n 5 8) were sorted into LSCe (CD341 /CD382 /Aldefluorint) and

1394 STEVENS et al 8 AUGUST 2017 x VOLUME 1, NUMBER 18

Figure 6. IL-6–induced pY-STAT3 is increased in the leukemia
stem cell enriched (LSCe) subpopulation. (A) Histograms of
A
sorted subpopulations of a representative diagnostic AML BM sample.
Constitutive (yellow) and IL-6–induced (blue) pY-STAT3 are shown for
the non–stem AML population (left) and the LSCe population (right).
(B) A significant difference in the mean percentage of cells with
IL-6–induced pY-STAT3 in the LSCe subpopulation compared with
Count
the non–stem AML subpopulation was found. No differences were
seen in constitutive pY-STAT3 between sorted populations (n 5 8).
Bar graphs show mean 6 standard error of the mean. *P , .05. PE,
phycoerythrin.
B
60%
50%
% pY-STAT3-PE +
40%
30%
20%
10%
non–stem AML populations (CD341 /CD382 /Aldefluorlow),26
and the levels of constitutive and IL-6–induced STAT3 phos-
phorylation were measured by flow cytometry (see supplemental
Figure 6 for the gating strategy used). The LSCe population
comprised 1.2% to 12% of sorted cells (mean, 5.6%). The LSCe
populations had a higher percentage of cells with IL-6–induced
pY-STAT3 compared with the paired non–stem AML populations
(44% 6 7.3% vs 28% 6 8.3%, respectively; P 5 .039, n 5 8).
There were no differences in levels of constitutive pY-STAT3
between LSCe and non–stem AML populations (8.2% 6 2.1% vs
7.7% 6 2.1%; Figure 6). These data show that IL-6–induced
STAT3 signaling pathways are frequently more robust in the LSCe
population than the non–stem AML population in pediatric
patients with AML. Together, our results suggest that IL-6 in the
microenvironment promotes relapse by supporting LSCs and
augmenting chemotherapy resistance.
Discussion
Accurate risk stratification is critical for determining the intensity of
therapy. Clinicians need to select a treatment that is aggressive
enough to cure the disease while limiting treatment-related toxicity.
Additionally, they need to know when it is appropriate to incorporate
targeted agents. To address this need, we investigated various
cytokines in plasma from BM aspirates to determine their potential as
prognostic markers and biological factors contributing to treatment
failure. In this study, IL-6, IL-10, and IL-8 levels were significantly
different in the AML cohort compared with the NBM cohort. These
data are similar to several previous studies that showed elevated
levels in peripheral blood and BM of IL-6, IL-10, IL-8, IL-4, TNF-a, and
8 AUGUST 2017 x VOLUME 1, NUMBER 18
40 80
30 60
Non stem AML LSC enriched
(LSCe)
20 40
10 20
0 0
101 102 103 101 102 103
pY-STAT3
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Unstimulated IL-6 (50 ng/mL) + sIL-6R (100ng/mL)
* Non-Stem AML
LSC enriched
0%
IL-6 50 ng/ml Constitutive
IL-17A in adult patients with AML.10,11 Among the various cytokines
tested, only elevated IL-6 levels predicted lower survival in our cohort.
The significance of this relationship was emphasized when data were
limited to patients classified as LR by current criteria. Importantly,
based on extensive clinical records, the elevated IL-6 levels were not
strongly correlated with clinical signs of inflammation or infection.
Thus, IL-6 may be valuable as a marker of HR disease and as a target
for new therapies for pediatric patients with AML.
Our data generate the question of the source of the elevated
IL-6 within the BM niche. One potential source is AML blasts
autosecreting IL-6. IL-6 autosecretion has been demonstrated
previously in solid tumors and in adults with AML.29-32 An alternative
source of IL-6 production is normal stromal cells or monocytes
within the BM. It is possible that through paracrine signals, AML
blasts are driving IL-6 production by these normal cells.33 Our
observation of decreased IL-6 levels in plasma collected during
remission would be compatible with either explanation but the lack
of correlation between IL-6 levels, and BM blast percentage at
diagnosis supports a paracrine process. Additional studies are
planned to better delineate the source of elevated IL-6 within the
BM niche at diagnosis.
Next, we investigated potential biological mechanisms by which
elevated IL-6 levels could reduce survival in pediatric patients with
AML. Since STAT3 signaling is known to regulate antiapoptosis
gene expression, one possible mechanism could be by promoting
chemoresistance. In this study, we showed that exogenous IL-6
reduced mitoxantrone-induced apoptosis in AML cell lines and primary
cultures. Importantly, this effect was seen only with mitoxantrone. This
is consistent with our prior work demonstrating that soluble factors are
CYTOKINES IN PEDIATRIC AML 1395
most important in mediating chemoresistance to mitoxantrone while
adhesion mediated factors are important for resistance to etoposide
and cytarabine.28 Since mitoxantrone is used in current upfront
pediatric AML treatment protocols in North America, this finding is
potentially clinically significant.
Another possible mechanism by which IL-6 promotes relapse could
be by supporting the LSCs. To this point, we showed that a higher
proportion of cells in the LSCe subpopulation had increased
pY-STAT3 levels following treatment with exogenous IL-6 compared
with non–stem AML cells. Based on these results, we hypothesize that
IL-6–induced pY-STAT3 activity could potentially help cancer cells,
particularly the critical LSC subpopulation, survive initial rounds of
chemotherapy and allow them to subsequently expand, leading to
investigate agents that target the IL-6–induced STAT3 pathway
to determine which patients are most likely to respond to the
intervention and the clinical feasibility of targeting this pathway.
Acknowledgments
The authors thank the Cytometry and Cell Sorting Core at Baylor
College of Medicine and acknowledge the expert assistance of
Tatiana Gotslova.
The Cytometry and Cell Sorting Core at Baylor College of
Medicine is funded by the National Institutes of Health (NIH), National
Institute of Allergy and Infectious Diseases (grant P30 AI036211),
NIH, National Cancer Institute (grant P30 CA125123), and NIH,
National Center for Research Resources (grant S10 RR024574).
Collection and storage of human specimens by the Children’s

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relapsed disease. This hypothesis is supported by our previous study
showing increased levels of IL-6–induced pY-STAT3 at relapse
compared with those at diagnosis.20 The small sample size and lack
of inclusion of patients from different institutions are limitations of our
study. Additionally, our cohort is enriched for relapses with 43% of LR
patients experiencing a relapse, a higher rate than is seen within
children currently classified as LR.7 A large validation study utilizing
peripheral blood samples from multiple sites and a high sensitivity
assay is currently underway. Determining whether this potential
predictive marker will have sufficient positive predictive value when
tested in a cohort with fewer events will be important in the further
evaluation of the clinical utility of IL-6. Despite these limitations of our
study, we have identified a potential mechanism of chemoresistance in
pediatric AML. Importantly, the IL-6–induced STAT3 pathway can be
targeted with the US Food and Drug Administration–approved sIL-6
receptor monoclonal antibody tocilizumab. Studies are underway to
evaluate the feasibility of incorporating this agent into existing treatment
regimens as a chemosensitizing agent.
Oncology Group is supported through funding from the NIH,
National Cancer Institute (grant U24CA114766). This work was
supported by grants from the Thrasher Research Fund Early Career
Award (A.M.S.), Children’s Leukemia Research Association (A.M.S.),
a gift from the Turn it Gold Fund (A.M.S.), and NIH, National Cancer
Institute grants R01CA17026 (M.S.R.) and K12CA090433 (A.M.S.).
Authorship
Contribution: A.M.S. designed and performed experiments, analyzed
and interpreted data, collected clinical data, and wrote the manu-
script; J.M.M. collected clinical data; J.O.M. conducted experiments;
A.S.G. assisted with performing experiments; and M.S.R. designed
experiments, analyzed and interpreted data, and wrote the
manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.

In summary, our data support the prospective validation of IL-6
levels in plasma from BM as a factor in risk stratification algorithms.
In addition, treatments that target the IL-6 signaling pathway could
potentially eradicate chemoresistant LSCs and prevent relapse.
However, additional studies are needed. Among these, we need to
ORCID profiles: A.M.S., 0000-0002-7292-2464; M.S.R., 0000-
0002-2515-3703.
Correspondence: Alexandra M. Stevens, Texas Children’s Can-
cer and Hematology Centers, 1102 Bates St, Suite 750, Houston,
TX 77030; e-mail: amsteven@txch.org.

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