Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial
Aim: Evaluation of serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia (AML) using biochip array technology. We searched for links between baseline levels and age, hyperleukocytosis, secondary origin of AML, resistance to induction therapy wi...
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irk-123456789-1453832019-01-22T01:23:36Z Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial Kupsa, T. Vasatova, M. Karesova, I. Zak, P. Horacek, J.M. Original contributions Aim: Evaluation of serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia (AML) using biochip array technology. We searched for links between baseline levels and age, hyperleukocytosis, secondary origin of AML, resistance to induction therapy with cytarabine and daunorubicin and standard risk stratification according to cytogenetics and molecular genetics. Methods: We evaluated the sera of 51 consecutive patients. Serum samples were analyzed by biochip based immunoassays on the Evidence Investigator analyzer. T-tests were used for statistical analysis. Results: We found that higher age is associated with lower levels of interleukin (IL)-12. Patients with secondary disease were older, had higher levels of EGF and IL-7, and lower levels of E-selectin, IL-12 and IL-13. In hyperleukocytosis, the levels of IL-1β, IL-2, TNF-α, VCAM-1, ICAM-1, E-selectin and L-selectin were increased, whereas levels of IFN-γ and MCP-1 were decreased. In patients who failed to achieve complete remission after induction therapy, we found lower E-selectin and P-selectin levels. High risk patients had lower levels of IFN-γ. Conclusion: Some leukemic cell subpopulations have the ability to produce cytokines that modulate the microenvironment by inducing inflammation. This causes endothelial cells to be activated and overexpress adhesion molecules. Hyperleukocytosis and secondary origin of the disease are the major factors influencing the cytokine and adhesion molecule profile in newly diagnosed AML patients. Key Words: cytokines, adhesion molecules, biochip array, acute myeloid leukemia. 2014 Article Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial / T. Kupsa, M. Vasatova, I. Karesova, P. Zak, J.M. Horacek // Experimental Oncology. — 2014. — Т. 36, № 4. — С. 252-257. — Бібліогр.: 21 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145383 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Kupsa, T. Vasatova, M. Karesova, I. Zak, P. Horacek, J.M. Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial Experimental Oncology |
| description |
Aim: Evaluation of serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia (AML) using biochip array technology. We searched for links between baseline levels and age, hyperleukocytosis, secondary origin of AML, resistance to induction therapy with cytarabine and daunorubicin and standard risk stratification according to cytogenetics and molecular genetics. Methods: We evaluated the sera of 51 consecutive patients. Serum samples were analyzed by biochip based immunoassays on the Evidence Investigator analyzer. T-tests were used for statistical analysis. Results: We found that higher age is associated with lower levels of interleukin (IL)-12. Patients with secondary disease were older, had higher levels of EGF and IL-7, and lower levels of E-selectin, IL-12 and IL-13. In hyperleukocytosis, the levels of IL-1β, IL-2, TNF-α, VCAM-1, ICAM-1, E-selectin and L-selectin were increased, whereas levels of IFN-γ and MCP-1 were decreased. In patients who failed to achieve complete remission after induction therapy, we found lower E-selectin and P-selectin levels. High risk patients had lower levels of IFN-γ. Conclusion: Some leukemic cell subpopulations have the ability to produce cytokines that modulate the microenvironment by inducing inflammation. This causes endothelial cells to be activated and overexpress adhesion molecules. Hyperleukocytosis and secondary origin of the disease are the major factors influencing the cytokine and adhesion molecule profile in newly diagnosed AML patients. Key Words: cytokines, adhesion molecules, biochip array, acute myeloid leukemia. |
| format |
Article |
| author |
Kupsa, T. Vasatova, M. Karesova, I. Zak, P. Horacek, J.M. |
| author_facet |
Kupsa, T. Vasatova, M. Karesova, I. Zak, P. Horacek, J.M. |
| author_sort |
Kupsa, T. |
| title |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| title_short |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| title_full |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| title_fullStr |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| title_full_unstemmed |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| title_sort |
baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/145383 |
| citation_txt |
Baseline serum levels of multiple cytokines and adhesion molecules in patients with acute myeloid leukemia: results of a pivotal trial / T. Kupsa, M. Vasatova, I. Karesova, P. Zak, J.M. Horacek // Experimental Oncology. — 2014. — Т. 36, № 4. — С. 252-257. — Бібліогр.: 21 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
252 Experimental Oncology 36, 252–257, 2014 (December)
BASELINE SERUM LEVELS OF MULTIPLE CYTOKINES
AND ADHESION MOLECULES IN PATIENTS WITH ACUTE MYELOID
LEUKEMIA: RESULTS OF A PIVOTAL TRIAL
T. Kupsa1, 2, M. Vasatova3, I. Karesova3, P. Zak2, J.M. Horacek1, 2, *
1Department of Military Internal Medicine and Military Hygiene, University of Defence,
Faculty of Military Health Sciences, Hradec Kralove 50001, Czech Republic
24th Department of Internal Medicine — Hematology, University Hospital and Charles University,
Faculty of Medicine, Hradec Kralove 50005, Czech Republic
3Institute of Clinical Biochemistry and Diagnostics, University Hospital, Hradec Kralove 50005, Czech Republic
Aim: Evaluation of serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia
(AML) using biochip array technology. We searched for links between baseline levels and age, hyperleukocytosis, secondary origin
of AML, resistance to induction therapy with cytarabine and daunorubicin and standard risk stratification according to cytogenetics
and molecular genetics. Methods: We evaluated the sera of 51 consecutive patients. Serum samples were analyzed by biochip based
immunoassays on the Evidence Investigator analyzer. T-tests were used for statistical analysis. Results: We found that higher age
is associated with lower levels of interleukin (IL)-12. Patients with secondary disease were older, had higher levels of EGF and IL-7,
and lower levels of E-selectin, IL-12 and IL-13. In hyperleukocytosis, the levels of IL-1β, IL-2, TNF-α, VCAM-1, ICAM-1,
E-selectin and L-selectin were increased, whereas levels of IFN-γ and MCP-1 were decreased. In patients who failed to achieve
complete remission after induction therapy, we found lower E-selectin and P-selectin levels. High risk patients had lower levels of IFN-γ.
Conclusion: Some leukemic cell subpopulations have the ability to produce cytokines that modulate the microenvironment by inducing
inflammation. This causes endothelial cells to be activated and overexpress adhesion molecules. Hyperleukocytosis and secondary
origin of the disease are the major factors influencing the cytokine and adhesion molecule profile in newly diagnosed AML patients.
Key Words: cytokines, adhesion molecules, biochip array, acute myeloid leukemia.
Acute myeloid leukemia (AML) shows a high degree
of heterogeneity owing to a variety of mutations and
the mechanisms involved in leukemogenesis. Cytokines
and adhesion molecules have been studied as markers
of immune system activation in various diseases inclu-
ding hematologic malignancies and AML [1, 2]. Cyto-
kines are soluble molecules carrying specific informa-
tion for target cells. Acting through a surface receptor,
they provide target cells with specific information about
conditions inside the organism and cause a specific
response. The response may be, e.g. stimulating and
activating in the case of inflammation or in the case
of tissue damage, causing proliferation or apoptosis.
Under abnormal conditions, this physiological role
of cytokines is maladaptive. The effect of inflammation
and altered cytokine signalling on oncogenesis le ading
to tumor progression, has been documented [3, 4].
Blood cells and their marrow-based progenitors are
exquisitely responsive to their environment, and cyto-
kines are an essential part of it. On binding to cytokine
receptor, signal transduction pathways (STP) are
activated and abnormalities in signalling through STP
are common in AML. Cytokines play a role in leuke-
mogenesis, AML cell persistence, treatment outcome
and allogeneic transplantation-related phenomena [5].
Cytokine-related mechanisms of leukemogenesis, AML
cell persistence and resistance to chemotherapy are
complex. Modulation of the cytokine network can disrupt
signalling pathway activation and overcome the high re-
sistance to treatment. It may also increase the selectivity
of AML treatment, reduce the overall treatment-related
toxicity and improve outcomes of AML treatment in all
age groups of patients. Adhesive interactions also trig-
ger signal transduction pathway activation and this
prevents the apoptosis of both normal and malignant
cells. A correlation between expression of defined adhe-
sion molecules and patient outcome has been found for
several malignant diseases inclu ding AML.
Alterations in the interacting functional network
of cytokines and adhesion molecules may have
direct effect on the malignant cells or have indirect
effect on leukemogenesis through altered functions
of bone marrow stromal elements [6, 7]. Further
knowledge gained from multi-analytical determina-
tion of cytokines and adhesion molecules could allow
better diagnosis and management of hematologic
malignancies, since cytokines or their receptors may
also represent a target for specific anticancer therapy
at the molecular level. Recently, some studies reported
the possible diagnostic and prognostic use of cytokine
levels in newly diagnosed AML and myelodysplastic
syndromes [8–10]. The aim of this study was to evalu-
ate baseline serum levels of cytokines and adhesion
molecules in patients treated for AML. We evaluated
changes in cytokine and adhesion molecule levels as-
Submitted: October 7, 2014.
*Correspondce: E-mail: jan.horacek@unob.cz
Abbreviations used: AML — acute myeloid leukemia; CR — complete
remission; EGF — epidermal growth factor; ICAM — intercellular adhe-
sion molecule; IL — interleukin; IFN — interferon; MCP — monocyte
chemotactic protein; MDS — myelodysplastic syndrome; STP — signal
transduction pathways; TNF — tumor necrosis factor; VCAM — vascu-
lar cell adhesion molecule; VEGF — vascular endothelial growth factor.
Exp Oncol 2014
36, 4, 252–257
Experimental Oncology 36, 252–257, 2014 (December) 253
sociated with age, hyperleukocytosis and secon dary
AML origin. Age-related changes were suspected
in several studies and older patients have often been
excluded from analyses. On the other hand, age need
not correlate with biological age or performance sta-
tus. Based on our own experience with good results
using a curative approach in patients aged 65–70 with
ECOG performance status 0–2, we decided to search
for age-related alterations in cytokine and adhesion
molecule levels. A secondary AML usually evolves
from previous myelodysplasia or myelofibrosis and
is more frequent in older patients. Nevertheless, due
to progress in the treatment of malignancies in younger
patients (e.g. Hodgkin’s lymphoma, testicular, early
breast and prostate cancer) and curative treatment,
some younger patients may also suffer from secondary
AML. Hyperleukocytosis may be associated with life
threatening leukostasis, which is based on capillary
obstruction by adherent activated myeloblasts in endo-
thelia. There were two reasons why we evaluated hyper-
leukocytosis separately. First, we expected increased
levels of cytokines directly produced by myeloblasts
in the blood flow. Second, we anticipated high levels
of soluble adhesion molecules as leukostasis and ex-
travascular leukemic cell dissemination is exquisitely
dependent on adhesive interactions [11].
Further, we aimed to determine the prognostic value
of baseline cytokine and adhesion molecule levels. We di-
vided patients according to standard risk stratification
based on cytogenetic and molecular genetic examina-
tion into low-risk, intermediate-1 and -2 and high risk
subgroups to determine whether these subgroups
differed in cytokine and/or adhesion molecule levels.
We then took a detailed look at those patients who failed
to achieve complete remission (CR) after induction
chemotherapy combining cytarabine and daunorubicin.
Failure of induction therapy has direct therapeutic conse-
quences. Irrespective of standard prognostic indicators,
these patients suffered from aggressive disease and
were reinduced by salvage regimen and allotransplanted
if a suitable donor was available. Early identification
of these patients would allow treatment to be more per-
sonalized which is the basic aim of modern medicine.
PATIENTS AND METHODS
Patients. A total of 51 newly diagnosed AML pa-
tients, 19 males and 32 females, mean age 52.5 ± 13.4,
median 56.2 years, were studied. According to cyto-
genetic and molecular genetic evaluation, 11 patients
were classified as low risk, 9 — intermediate-1 risk,
12 — intermediate-2 risk and 19 — high risk disease.
In 24 patients, normal karyotype was present. Of these,
5 had NPM-1 mutated (Nucleophosmin-1), 8 patients
had both NPM-1 and FLT3-ITD (Famosin-like tyrosin
kinase 3 — internal tandem duplications), 3 patients
had solely FLT3-ITD and 8 patients had various muta-
tions including CEBPa (CCAT enhancer binding protein
alpha) and mutations including MLL (Multi-lineage
leukemia) gene. In 27 patients at least 1 cytogenetic
abnormality was present. Of these, 4 patients had CBF
(core binding factor) positive disease, all 4 patients
with acute promyelocytic leukemia had typical trans-
location present and 6 patients had complex changes
including 3 or more translocations. According to FAB
classification, 2 patients had AML M0, 10 patients —
AML M1, 25 patients — AML M2, 4 patients — AML M3,
6 patients — AML M4, 3 patients — AML M5 and 1 pa-
tient — AML M7. Hyperleukocytosis requiring urgent
leukapheresis was present in 12 patients. All patients
were induced with “3+7” induction chemotherapy
consisting of cytarabine 100 mg/m2 per day for 7 con-
secutive days and daunorubicin 90 mg/m2 for the first
3 days of therapy in younger patients. In patients
aged 65 or more, daunorubicin 45–60 mg/m2 was
administered. This was followed by consolidation with
high-dose cytarabine. In those patients who failed
to achieve CR after “3+7” induction regimen, the sal-
vage chemotherapy “FlAG-Ida” was administered.
A total of 31 patients who had intermediate or high risk
disease or failed to achieve CR after “3+7” induction
regimen had suitable donors for allogeneic stem cell
transplantation and were able to undergo the proce-
dure. These patients were allotransplanted. The study
was approved by the local Ethics Committee and all
patients gave written consent.
Serum collection. Peripheral blood was collected
in serum separating tubes, immediately transported
to the laboratory and processed within 2 hours of col-
lection by centrifugation at 1500 rpm × 5 min. All sera
were collected before leukapheresis, administration
of hydroxyurea or induction therapy. If specimens
were not to be analysed immediately, they were stored
frozen in small aliquots at −20 °C as recommended
by the Cytokine Array manufacturer. Repeat freeze/
thaw cycles were avoided.
Methods. All analytes were measured by biochip
array technology using chemiluminescent sandwich
immunoassays applied to the Evidence Investigator
Analyzer (Randox Laboratories Ltd., Crumlin, UK).
The Evidence Investigator Biochip Array technology
is used to perform simultaneous quantitative detec-
tion of multiple analytes from a single patient sample.
The core technology is the Randox Biochip, a solid-
state device containing an array of discrete test re-
gions of immobilised antibodies specific to different
cytokines and growth factors. A sandwich chemilu-
minescent immunoassay is employed for the cytokine
array. Increased levels of cytokine in a specimen will
lead to increased binding of antibody labelled with
horseradish peroxidase (HRP) and thus an increase
in the chemiluminescent signal emitted. The light
signal generated from each of the test regions on the
biochip is detected using digital imaging technology
and compared to that from a stored calibration curve.
The concentration of analyte present in the sample
is calculated from the calibration curve.
We evaluated circulating levels of the following
17 cytokines and 5 soluble adhesion molecules: in-
terleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6,
IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-23), vascular
254 Experimental Oncology 36, 252–257, 2014 (December)
endothelial growth factor (VEGF), tumor necrosis
factor-alpha (TNF-α), interferon-gamma (IFN-γ), epi-
dermal growth factor (EGF), monocyte chemotactic
protein-1 (MCP-1), E-selectin, L-selectin, P-selectin,
intercellular adhesion molecule-1 (ICAM-1) and vascu-
lar cell adhesion mo lecule-1 (VCAM-1). The results are
expressed in nanograms per litre (ng/L) for cytokines
and micrograms per litre (μg/L) for adhesion molecules.
Statistical analysis. Statistical analysis was
performed with the “Statistica” software using two-
tailed t-tests. The values were expressed as means ±
standard deviation. Probability values p < 0.05 were
considered statistically significant.
RESULTS
Age dependent differences. Originally, we ana-
lysed 3 subgroups of patients divided according to age.
The first subgroup consisted of younger patients aged
less than 55 years (n = 25), the second subgroup in-
cluded patients aged 55–65 years (n = 11) and the third
subgroup included patients 65 and older (n = 15). Be-
cause there were no significant differences between
younger and 55–65 year old patients, we analysed both
subgroups together against patients older than 65 years.
In patients aged 65 or more, we found a significant
decrease in IL-12 levels (0.95 ± 1.14 ng/L vs. 4.11 ±
3.76 ng/L, p = 0.025).
Secondary AML. The group with secondary AML
consisted of 15 patients with a previous history of MDS
(myelodysplastic syndrome). Two patients progressed
to AML from chronic myelomonocytic leukemia and two
patients from a previous myeloproliferative disorder. They
were much older than those with primary disease (62.9 ±
6.7 vs. 46.6 ± 13.3 years, p = 0.00008). In se condary AML,
we found higher IL-7 (6.13 ± 4.42 ng/L vs. 3.59 ± 1.97 ng/L,
p = 0.047) and EGF levels (26.64 ± 26.58 μg/L vs. 7.49 ±
8.05 μg/L; p = 0.004) (Fig. 1). The levels of IL-12 (1.26 ±
1.46 ng/L vs. 4.32 ± 3.87 ng/L; p = 0.021), IL-13 (2.16 ±
3.02 ng/L vs. 5.09 ± 4.47 ng/L, p = 0.049) and E-selectin
(14.85 ± 10.36 μg/L vs. 28.84 ± 18.16 μg/L; p = 0.018)
were decreased in secondary AML (Fig. 2, 3). The leuko-
cyte counts had a trend to lower counts in the secondary
AML group which were significant after exclusion of pa-
tients with acute promyelocytic leukemia (15.93 ± 15.99 •
109/L vs. 53.35 ± 54.44 • 109/L, p = 0.048). We also found
trends towards higher MCP-1 (246.14 ± 128.35 ng/L vs.
167.56 ± 101.96 ng/L, p = 0.055) and lower IL-10 levels
(1.53 ± 1.16 ng/L vs. 4.99 ± 4.86 ng/L, p = 0.075) in se-
condary AML.
H y p e r l e u k o c y t o s i s . H y p e r l e u k o c y t o -
sis in AML is defined as a blood count containing
at least 50 • 109/L leukocytes. Patients with initial
hyperleukocytosis (n = 12) had higher levels of IL-1β
(4.64 ± 4.44 ng/L vs. 0.84 ± 0.49 ng/L, p = 0.0006),
IL-2 (13.74 ± 15.77 ng/L vs. 3.48 ± 2.85 ng/L, p = 0.009),
TNF-α (4.79 ± 3.39 ng/L vs. 2.14 ± 1.03 ng/L, p = 0.002)
(Fig. 4). We also found lower levels of MCP-1 (102.38 ±
51.29 ng/L vs. 225.89 ± 114.87 ng/L, p = 0.007) and
IFN-γ (0.42 ± 0.49 ng/L vs. 2.03 ± 1.47 ng/L, p = 0.005),
in these patients (Fig. 5, 6).
0
10
20
30
40
50
60
70
80
90
100
EGF sec EGF prim IL-7 sec IL-7 prim
Fig. 1. Serum levels of EGF and IL-7 in primary and secondary
AML. EGF — epidermal growth factor; IL-7 — interleukin 7;
sec — secondary AML; prim — de novo AML
0
5
10
15
20
25
IL-12 sec IL-12 prim IL-13 sec IL-13 prim
Fig. 2. Serum levels of IL-12 and IL-13 in primary and se condary
AML. IL-12 — interleukin 12; IL-13 — interleukin 13; sec — se-
condary AML; prim — de novo AML
0
10
20
30
40
50
60
70
80
E-SEL prim E-SEL sec
Fig. 3. Serum levels of E-selectin primary and secondary AML.
E-SEL — E-selectin; sec — secondary AML; prim — de novo AML
0
5
10
15
20
25
30
IL-2 HLC+ IL-2 HLC- TNF-αHLC+TNF-α HLC- IL-1β HLC+ IL-1β HLC-
Fig. 4. Serum levels of IL-2, TNF-α and IL-1β in patients with and
without hyperleukocytosis. IL-2 — interleukin 2; TNF-α — tumor
necrosis factor-alpha; IL-1β — interleukin 1β; HLC+ — hyper-
leukocytosis; HLC− — without hyperleukocytosis
Experimental Oncology 36, 252–257, 2014 (December) 255
0
1
2
3
4
5
6
7
IFN-γ HLC+ IFN-γ HLC-
Fig. 5. Serum levels of IFN-γ in patients with and without hyper-
leukocytosis. IFN-γ — interferon-gamma; HLC+ — hyperleuko-
cytosis; HLC− — without hyperleukocytosis
0
100
200
300
400
500
600
MCP1 HLC+ MCP1 HLC-
Fig. 6. Serum levels of MCP-1 in patients with and without
hyperleukocytosis. MCP1 — monocyte chemotactic protein-1;
HLC+ — hyperleukocytosis; HLC- — without hyperleukocytosis
Soluble adhesion molecules were greatly increased
in hyperleukocytosis. We found VCAM-1 (1113.65 ±
519.71 μg/L vs. 599.59 ± 180.40 μg/L, p = 0.0002),
ICAM-1 (437.51 ± 177.07 μg/L vs. 296.56 ± 80.99 μg/L,
p = 0.004) and E-selectin (40.65 ± 13.90 μg/L vs.
18.39 ± 14.11 μg/L, p = 0.0006) (Fig. 7, 8). The levels
of L-selectin were at the upper limit of the Array sen-
sitivity in the vast majority of patients with hyperleuko-
cytosis, similar to several patients without hyperleu-
kocytosis. Hence, the results are probably affected.
However, we measured increased levels of L-selectin
(3262.18 ± 265.10 μg/L vs. 2307.96 ± 1010.81 μg/L,
p = 0.006) in hyperleukocytosis (data not shown).
0
500
1000
1500
2000
2500
3000
VCAM1 HLC+ VCAM1 HLC- ICAM1 HLC+ ICAM1 HLC-
Fig. 7. Serum levels of VCAM-1 and ICAM-1 in patients with
and without hyperleukocytosis. VCAM1 — vascular cell adhe-
sion molecule-1; ICAM1 — intercellular adhesion molecule-1;
HLC+ — hyperleukocytosis; HLC− — without hyperleukocytosis
0
10
20
30
40
50
60
70
80
E-SEL HLC+ E-SEL HLC-
Fig. 8. Serum levels of E-selectin in patients with and without
hyperleukocytosis. E-SEL — E-selectin; HLC+ — hyperleukocy-
tosis; HLC− — without hyperleukocytosis
Resistance to induction therapy. Those who had
not achieved CR after induction chemotherapy and were
reinduced with the salvage regimen (n = 11), had lower
E-selectin (11.63 ± 7.34 μg/L vs. 26.93 ± 17.81 μg/L,
p = 0.028) and P-selectin levels (76.19 ± 23.29 μg/L vs.
136.04 ± 64.74 μg/L, p = 0.022). On the other hand,
those who failed to achieve CR had significantly higher
CRP (C-reactive protein) levels (63.4 ± 54.8 mg/L vs.
27.4 ± 26.0 mg/L, p = 0.017) (Fig. 9, 10).
0
50
100
150
200
250
300
350
E-SEL no CR E-SEL CR P-SEL no CR P-SEL CR
Fig. 9. Serum E-selectin and P-selectin levels in patients who
failed to reach CR after induction therapy. E-SEL — E-selectin;
P-SEL — P-selectin; no CR — failed to reach CR after induction
therapy; CR — attained CR after induction therapy
0
20
40
60
80
100
120
140
160
180
200
CRP no CR CRP CR
Fig. 10. Serum CRP levels in patients who failed to reach CR after
induction therapy. CRP — C-reactive protein; no CR — failed
to reach CR after induction therapy; CR — attained CR after
induction therapy
Risk stratification and cytokine levels. Fi-
nally, we analysed subgroups based on cytogenetic
and molecular genetic risk stratification. There was
no significant difference between the groups but when
256 Experimental Oncology 36, 252–257, 2014 (December)
we ana lysed the high risk subgroup (n = 19) against
the 3 other prognostic subgroups (n = 32) we found
decreased IFN-γ levels (1.02 ± 1.11 ng/L vs. 2.03 ±
1.49 ng/L, p = 0.049) in high risk patients (Fig. 11).
No other significant differences were found in the levels
of other evaluated cytokines or adhesion molecules
in any analysis.
0
1
2
3
4
5
6
7
IFN-γ HR+ IFN-γ HR-
Fig. 11. Serum levels of IFN-γ in high risk patients. IFN-γ —
interferon-gamma; HR+ — high risk; HR− — other risk
DISCUSSION
Altered levels of cytokines and adhesion molecules
have been found in many pathological states and have
been linked to autoimmune diseases, allergies and
cancer, including AML [3–5, 12–14]. Cytokines and ad-
hesion molecules form an unique interacting functional
network. This led us to study both systems together.
Our results are in agreement with the general
finding that patients with secondary AML are usually
older than those with primary disease. The decrease
in IL-12 levels found in elderly patients is probably attri-
buted to se condary AML. We venture to suggest that
the origin of AML is a more important factor for cytokine
and adhesion molecule levels than age per se. The finding
of lower IL-13 levels in secondary AML is in agreement with
previous results, where MDS patients had lower IL-13 le-
vels than AML [9]. Further, the trend to lower IL-10 levels
in secondary disease may explain the recently published
negative prognostic impact of low IL-10 levels [10].
We found evidence that hyperleukocytosis has
direct impact on levels of several cytokines and
soluble adhesion molecules. We think that increased
levels of IL-1 beta and IL-2 reflect production of these
cytokines by myeloblasts in the blood circulation.
The IL-1, IL-2 and b-chemokine of the CC subgroup
CCL-3 (CC-ligand-3) were shown to stimulate leu-
kemic cell proliferation. IL-1β is the predominant
secreted form of IL-1 [15]. Possibly, the myeloblasts
trigger autocrine inflammatory loops enhancing their
own proliferation which causes hyperleukocytosis
and TNF-α inflammatory overproduction. E-selectin
is expressed by endothelia. Its expression is regulated
on the transcriptional level and is induced by TNF-α,
IL-1 and oncostatin M. In response to myeloid growth
factors, endothelia increase expression of E-selectin,
VCAM-1 and ICAM-1 and promote augmented leu-
kocyte adhesion in a p38 MAPK dependent manner.
Soluble adhesion molecules originate from proteolytic
cleavage of surface-expressed adhesion molecules.
In lymphoma-bearing mice, the lymphoma cells are
the major source of soluble L-selectins [16–18].
We speculate that in hyperleukocytosis the soluble
L-selectin mo lecules originate from myeloblasts,
whereas increased levels of VCAM-1, ICAM-1 and E-se-
lectin originate from inflammatory activated endothelia.
In those patients who failed to reach CR after “3+7”
induction therapy, the levels of E- and P- selectins were
found to be decreased. Further, the higher CRP level
in these patients indicates an activated inflammatory
response. It is unclear whether these high CRP levels
were caused by systemic infection or reflect the activity
of highly aggressive disease. From previous studies,
we know that cytokine levels do not correlate with in-
fection [9], but we lacked data for adhesion molecules.
Decreased levels of selectin adhesion molecules seem
to be in contradiction with the findings in hyperleukocy-
tosis, as one would expect inferior outcomes in patients
with hyperleukocytosis which was found to increase
E-selectin levels. What we have to take into account
is the ability of leukemic blasts to bind to soluble ad-
hesion molecules. In KG-1, AML cell line interactions
with both P- and E- selectins under flow have been
documented. Upon adhesion of myeloblasts to se-
lectin adhesion molecules, the adherent myeloblasts
may sustain toxic concentrations of chemotherapeutic
agents and thus be rescued from death [19, 20]. This
mechanism suggests how adhesive interactions pro-
tect AML cells during chemotherapy. For this reason,
possibly, the levels of E- and P-selectins in patients
resistant to induction chemotherapy were low be-
cause the soluble adhesion molecules were attached
to leukemic blasts and thus not measured. The origin
of the MCP-1 and IFN-γ decrease remains unclear and
is worth further investigation.
The risk stratification of AML is exactly defined
by cytogenetics and molecular genetics. We attemp ted
to match standard risk stratification with specific
alterations in cytokine or adhesion molecule levels.
We found decreased IFN-γ in the high risk subgroup
which was not described previously [9]. On critical
reappraisal however, the high risk subgroup in our
analysis included both patients with normal karyotype
and those with high risk molecular genetics, the same
as patients with high risk cytogenetics and complex
karyotype. Thus, the high risk group was not homo-
genous and this may have been reflected in the results.
Based on analysis of patient sera and healthy
blood donors as a non-leukemic control, we previ-
ously reported that changes in cytokine and adhesion
molecule levels are related to disease activity [21].
Our results indicate that serum levels of specific
cytokines and adhesion molecules are significantly
altered in AML patients and this enables us to further
understand the mechanisms of disease progression
and resistance to treatment. We highlight the impor-
tance of E- and P-selectin levels in AML and to the best
of our knowledge this is the first published evidence
in vivo. Further studies in a larger number of patients
Experimental Oncology 36, 252–257, 2014 (December) 257
will be needed to confirm our data and define the po-
tential role of these and additional markers in the risk
stratification and therapy of AML patients.
ACKNOWLEDGEMENTS
The work was supported by a long-term organiza-
tion development plan 1011 (Faculty of Military Health
Sciences, Hradec Kralove) and by a specific research
project “Analysis of defined prognostic factors in acute
myeloid leukemia” (Faculty of Military Health Sciences,
Hradec Kralove).
CONFLICT OF INTEREST STATEMENT
Author’s conflict of interest disclosure: None declared.
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