Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy
Introduction: It is believed that the reason of the leukemic clone cell resistance to treatment with tyrosine kinase inhibitors during chronic myeloid leukemia (CML) is mutations in the genome of an early bone marrow progenitor cells that are CD34-positive. Such cells, regardless of treatment, acqui...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2015
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| Cite this: | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy / I.O. Sviezhentseva, T.P. Perekhrestenko, D.I. Bilko, A.I. Gordienko, M.V. Diachenko, I.S. Dyagil // Experimental Oncology. — 2015. — Т. 37, № 1. — С. 70-72. — Бібліогр.: 17 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859669157778292736 |
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| author | Sviezhentseva, I.O. Perekhrestenko, T.P. Bilko, D.I. Gordienko, A.I. Diachenko, M.V. Dyagil, I.S. |
| author_facet | Sviezhentseva, I.O. Perekhrestenko, T.P. Bilko, D.I. Gordienko, A.I. Diachenko, M.V. Dyagil, I.S. |
| citation_txt | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy / I.O. Sviezhentseva, T.P. Perekhrestenko, D.I. Bilko, A.I. Gordienko, M.V. Diachenko, I.S. Dyagil // Experimental Oncology. — 2015. — Т. 37, № 1. — С. 70-72. — Бібліогр.: 17 назв. — англ. |
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| container_title | Experimental Oncology |
| description | Introduction: It is believed that the reason of the leukemic clone cell resistance to treatment with tyrosine kinase inhibitors during chronic myeloid leukemia (CML) is mutations in the genome of an early bone marrow progenitor cells that are CD34-positive. Such cells, regardless of treatment, acquire ability to proliferation and differentiation. This leads to the re-expansion of the CD34⁺ cells. Aim: to determine the CD34 antigen expression in bone marrow and peripheral blood cells in CML patients with different response to imatinib therapy using the results of hematopoietic cells culturing and the data of flow cytometry. Methods: Bone marrow aspirate from 39 patients who were treated with imatinib was studied with cytogenetic, flow cytometry and culture methods in vitro. Results: In patients with an optimal response to imatinib therapy the number of colonies was 1.8 times lower than the number of those in the group of patients with a suboptimal response to therapy. In turn, in patients with failure of imatinib therapy the number of colonies was the highest and was 2.1 times higher than the patients with optimal response. The results of cytometric studies have shown that the number of CD34⁺ cells in bone marrow was significantly higher compared to the number of CD34⁺ cells in peripheral blood cells and increased with the acquisition of leukemic cells the resistance to imatinib. There was a direct correlation between the number of colonies and clusters in semisolid agar in vitro and the number of CD34⁺ cells in the bone marrow of patients. Conclusions: The correlation between the number of CD34⁺ cells and the number of cell aggregates in semisolid agar in vitro indicates the prognostic value of the method for determining CD34⁺ cells in the patient bone marrow. The parallel increase of their number in the peripheral blood will allow developing express methods for the detection of individual patient response to imatinib therapy. Key Words: chronic myeloid leukemia, imatinib, cell culture in vitro, CD34⁺ cells.
|
| first_indexed | 2025-11-30T12:58:12Z |
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70 Experimental Oncology 37, 70–72, 2015 (March)
FUNCTIONAL ACTIVITY OF CD34-POSITIVE CELLS IN CHRONIC
MYELOID LEUKEMIA PATIENTS WITH DIFFERENT RESPONSE
TO IMATINIB THERAPY
I.O. Sviezhentseva1, T.P. Perekhrestenko2, D.I. Bilko1, A.I. Gordienko2, M.V. Diachenko1, I.S. Dyagil3
1Centre for Molecular and Cell Research of National University of “Kyiv-Mohyla Academy”, Kyiv 04655, Ukraine
2SI “Institute of Hematology and Transfusiology of the NAMS of Ukraine”, Kyiv 04060, Ukraine
3SI “The National Research Center for Radiation Medicine of the NAMS of Ukraine”, Kyiv 04050, Ukraine
Introduction: It is believed that the reason of the leukemic clone cell resistance to treatment with tyrosine kinase inhibitors during
chronic myeloid leukemia (CML) is mutations in the genome of an early bone marrow progenitor cells that are CD34-positive. Such
cells, regardless of treatment, acquire ability to proliferation and differentiation. This leads to the re-expansion of the CD34+ cells.
Aim: to determine the CD34 antigen expression in bone marrow and peripheral blood cells in CML patients with different response
to imatinib therapy using the results of hematopoietic cells culturing and the data of flow cytometry. Methods: Bone marrow aspirate
from 39 patients who were treated with imatinib was studied with cytogenetic, flow cytometry and culture methods in vitro. Results: In pa-
tients with an optimal response to imatinib therapy the number of colonies was 1.8 times lower than the number of those in the group
of patients with a suboptimal response to therapy. In turn, in patients with failure of imatinib therapy the number of colonies was
the highest and was 2.1 times higher than the patients with optimal response. The results of cytometric studies have shown that
the number of CD34+ cells in bone marrow was significantly higher compared to the number of CD34+ cells in peripheral blood cells
and increased with the acquisition of leukemic cells the resistance to imatinib. There was a direct correlation between the number
of colonies and clusters in semisolid agar in vitro and the number of CD34+ cells in the bone marrow of patients. Conclusions: The cor-
relation between the number of CD34+ cells and the number of cell aggregates in semisolid agar in vitro indicates the prognostic value
of the method for determining CD34+ cells in the patient bone marrow. The parallel increase of their number in the peripheral blood
will allow developing express methods for the detection of individual patient response to imatinib therapy.
Key Words: chronic myeloid leukemia, imatinib, cell culture in vitro, CD34+ cells.
Chronic myeloid leukemia (CML) is a myeloproliferative
neoplasm that characterized the expansion of the early he-
matopoietic progenitor cells pool which express CD34 an-
tigen [1, 2]. It is known that the diffe rentiation of bone
marrow cells expression of CD34 antigen on the cell
surface of myeloid hematopoiesis decreases or disap-
pears [3]. In general, the function of CD34 antigen is still
not clear, but it is believed that it plays an important role
in the interaction between hematopoietic cells and bone
marrow microenvironment [4, 5].
The cause of CML is tyrosine kinase activity of BCR-
ABL oncoprotein, which is expressed by derivative
chromosome 22. It is called the Philadelphia chromo-
some (Ph-chromosome) [6]. Due to the aforemen-
tioned activity of this oncoprotein the leukemic clone
cell is not only characterized by altered potential for
self-renewal and differentiation, but also by the re-
duced the levels of adhesion to bone marrow stroma.
As a result a large number of early CD34-positive
hematopoietic cells get to the peripheral blood [7, 8].
The BCR-ABL tyrosine kinase is a therapeutic target
of the imatinib which is the first-generation of tyrosine
kinase inhibitors (TKI) [9]. Early clinical studies have
revealed that 65–85% of patients had a complete cyto-
genetic remission received after 12 months of imatinib
therapy which was much more effective compared
to other therapeutic agents, known at that time. However,
a part of the patients (approximately 30%) did not achieve
a complete cytogenetic response within 1 year of treat-
ment [10]. Some researchers consider that the resis-
tance is caused by mutations in the genome of bone
marrow progenitor cells that are CD34-positive. Such
cells, regardless of treatment, get on ability to active
proliferation and differentiation. This leads to the re-
expansion of the pool of CD34+ cells [3]. That is why
immunophenotypic monitoring of CD34+ cells in periphe-
ral blood and bone marrow should be conducted for
the prognosis of the disease and evaluation of the ef-
fectiveness of therapy in CML patients [11, 12].
The aim of this work was to determine the features
of CD34 antigen expression in bone marrow and pe-
ripheral blood in CML patients with different response
to imatinib therapy using the results of hematopoietic
cells culturing and the data of flow cytometry.
MATERIALS AND METHODS
We studied the bone marrow samples of 39 CML
patients during the chronic phase of disease treated with
imatinib for at least 12 months. The average age of patients
was 54.0 ± 7.41 years. All patients before the study signed
a voluntary informed consent. All patient samples were ob-
tained under Research Ethics Board. According to the cri-
teria for assessing the effectiveness of tyrosine kinases
therapy of The European LeukemiaNet the patients were
divided into three groups: with optimal (n = 15), subopti-
mal response (n = 10) and failure (n = 14) of the imatinib
therapy. The optimal response means a complete cyto-
Submitted: November 06, 2014.
*Correspondence: Email: iilona@ukr.net; tat2007@bigmir.net
Abbreviations used: CFU-GM — colony-forming unit-granulocyte/
macrophage; ClFU — cluster-forming unit; CML — chronic myeloid
leukemia; TKI — tyrosine kinase inhibitors.
Exp Oncol 2015
37, 1, 70–72
Experimental Oncology 37, 70–72, 2015 (March) 71
genetic response after 12 months of therapy with imatinib
(0% Ph-positive cells). Suboptimal response was defined
when the bone marrow research showed from 1% to 35%
of Ph-positive cells after 12 months of imatinib therapy.
The lack of response to the drug indicated the presence
of a large number of cells with Ph-chromosome, which
is greater than 35% in the bone marrow [13].
To determine the functional activity of hematopoietic
cells of patients in vitro culturing of bone marrow mono-
nuclear cells in semisolid agar (“Difco”, USA) (concen-
tration 0.33%) with a use of the medium for culturing
RPMI-1640 (“Sigma”, USA), 20% fetal calf serum and
antibiotics (50 IU/ml penicillin, 50 mg/ml streptomycin)
was carried out. Cultivation was performed for 13 days
under conditions of absolute humidity, 5% CO2 and
at 37 °C. A cluster-forming units included no more than
40 cells, at the same time the colony consists of from
40 to several hundred cells (Fig. 1). A proliferative
potential was determined as the ratio of the number
of colonies and the number of clusters.
a
b
Fig. 1. Colony (a) and cluster (b) formed by culturing bone mar-
row cells of patients with CML in vitro
To assess the phenotypic characteristics of he-
matopoietic cells of patients the method of direct flow
cytometry was used. For this purpose, bone mar-
row mononuclear cells were stained with mono-
clonal antibodies (“Becton Dickinson”, USA) using
the method provided by the manufacturer. Flow
cytometry study was performed using a flow cytome-
ter laser FACScan (“Becton Dickinson”, USA) with
an argon laser which has a wavelength of 488 nm.
Data collection was performed by flow cytometry using
the LYSYS-II Ver. 1.1 (“Becton Dickinson”, USA) soft-
ware. The program WinMDI 2.8 (Joseph Trotter, Scripps
Institute, La Jolla, CA) was used to analyze the results.
The statistical analysis of the results was carried
out using nonparametric Mann — Whi tney tests for
comparing two populations. The conclusion of the sta-
tistical significance of the results made at p < 0.05. For
estimation of interrelations between obtained indexes
the correlational analysis with determination of Pear-
son’s correlation coefficient has been conducted.
RESULTS AND DISCUSSION
In general, the determination of the number of colo-
nies in semisolid agar in vitro is used as a test system
to check the impact of chemotherapeutic agents
on the leukemic clone cells [14]. However, such stu dies
are usually implemented under conditions of the pre-
sence of chemotherapeutic agents in the culture medi-
um. Culture of bone marrow subjected to therapy with
TKI and with the absence of imatinib in the cultivation
medium may allow reproducing the features of bone
marrow cells proliferation without the inhibitory effect
of the drug. The previous studies have demonstrated
that the determination of functional activity of bone
marrow cells of CML patients in semisolid agar in vitro
with different response to imatinib therapy may have
prognostic value for assessing the further develop-
ment of the disease [15, 16]. However, a problem
of finding the predictive test systems that would be ef-
ficient and quick remains open. Therefore, it appeared
reasonable to compare the results of cultivation with
the number of cells expressing CD34 antigen.
The results of cultivation suggest that in patients with
an optimal response to imatinib therapy the number
of colonies was 1.8 times lower than the number of those
in the group of patients with suboptimal response
to thera py (Table 1). In turn, in patients with fa ilure of ima-
tinib therapy the number of colonies was the highest
and was 2.1 times higher than the patients with optimal
response. In the case of clusters was recorded tendency
to increase this indicator in patients with suboptimal
response and treatment failure. In addition, the patients
with inefficiency and suboptimal response to imatinib
therapy had the significantly higher proliferative potential
than in patients with optimal response. This may indicate
that early progenitor cells dominated the more diffe-
rentiated cells which form clusters in the culture. In turn,
in the bone marrow of patients who have increasing
numbers of leukemic clone cells proliferation processes
dominate over the processes of differentiation that may
be the cause of the increase of the number of colonies
in semisolid agar in vitro [17].
Table 1. Characteristics of the functional activity of bone marrow cells
in patients with different response to imatinib therapy
The response
to imatinib therapy
The number
of colonies
The number
of clusters
Proliferative
potential
Optimal response 34.42 ± 1.46 46.01 ± 2.27 0.94 ± 0.04
Suboptimal response 63.75 ± 2.57* 54.25 ± 4.68 1.19 ± 0.02*
Failure of the therapy 73.81 ± 3.2* 54.53 ± 3.7 1.41 ± 0.05*
*Statistically significant differences with group of optimal response (p < 0.05).
72 Experimental Oncology 37, 70–72, 2015 (March)
The results of cytometry analysis showed that
in patients with the acquisition of resistance to imatinib
the number of CD34+ cells increases in the bone mar-
row and peripheral blood (Table 2). It should be noted,
in patients with different response to therapy the num-
ber of CD34 antigen in bone marrow and peripheral
blood was significantly different. This may indicate that
during the progression of CML and during the acquisi-
tion of resistance to the drug by leukemic clone cells
the pool of early progenitor cells not only expands,
but also, due to the weakening of contacts between
the cells of the microenvironment and leukemic clone
cells, the early progenitor cells fall in blood flow in large
numbers, promoting a poorly differentiated hemato-
poietic cells in peripheral blood of patients [5].
Table 2. Expression of CD34 antigen on hematopoietic progenitor cells
in the bone marrow and peripheral blood of CML patients with different
response to imatinib therapy (% ± m%)
The response
to imatinib therapy
The number of CD34+
bone marrow cells
The number of CD34+
peripheral blood cells
Optimal response 9.23 ± 2.01 4.43 ±1.07
Suboptimal response 31.65 ± 5.85* 12.08 ± 5.35*
Failure of the therapy 44.37 ± 6.72* 17.95 ± 4.42*
*Statistically significant differences with group of optimal response (p < 0.05).
A correlation analysis was conducted to identify
the correspondence between the number of CD34+ cells
in the bone marrow and in peripheral blood of patients tre-
ated with imatinib. These results indicate a direct correlation
between the number of colonies and clusters in semisolid
agar in vitro and the number of CD34+ cells in the patient
bone marrow (r = 0.83 and r = 0.58, p < 0.05) (Fig. 2).
0
20
40
60
80
100
120
140
0 20 40 60 80
The number of CD34+ cells in the patients bone marrow
Th
e
nu
m
be
r o
f c
ol
on
ie
s
pe
r 1
00
0
00
e
xp
la
nt
ed
c
el
ls
0
20
40
60
80
100
0 20 40 60 80
The number of CD34+ cells in the patients bone marrow
Th
e
nu
m
be
r o
f c
lu
st
er
s
pe
r 1
00
0
00
e
xp
la
nt
ed
c
el
ls
a
b
Fig. 2. Correlation between number of CD34+ cells in the pa-
tients bone marrow, the number of colonies (a) and the number
of clusters (b)
Thus, the number of CD34+ cells in the bone marrow
of patients increases with the acquisition of leukemic
clone cells resistance to imatinib. The correlation between
the number of CD34+ cells and the number of cell aggre-
gates in semisolid agar in vitro, which may be predictive
method for determining the future course of CML, shows
the prognostic value of method for determining the CD34+
cells in the bone marrow of patients; and a parallel increase
of their number in the peripheral blood of patients will allow
to develop express methods for the detection of individual
patient response to imatinib therapy.
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Copyright © Experimental Oncology, 2015
|
| id | nasplib_isofts_kiev_ua-123456789-145458 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-11-30T12:58:12Z |
| publishDate | 2015 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Sviezhentseva, I.O. Perekhrestenko, T.P. Bilko, D.I. Gordienko, A.I. Diachenko, M.V. Dyagil, I.S. 2019-01-22T10:46:07Z 2019-01-22T10:46:07Z 2015 Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy / I.O. Sviezhentseva, T.P. Perekhrestenko, D.I. Bilko, A.I. Gordienko, M.V. Diachenko, I.S. Dyagil // Experimental Oncology. — 2015. — Т. 37, № 1. — С. 70-72. — Бібліогр.: 17 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145458 Introduction: It is believed that the reason of the leukemic clone cell resistance to treatment with tyrosine kinase inhibitors during chronic myeloid leukemia (CML) is mutations in the genome of an early bone marrow progenitor cells that are CD34-positive. Such cells, regardless of treatment, acquire ability to proliferation and differentiation. This leads to the re-expansion of the CD34⁺ cells. Aim: to determine the CD34 antigen expression in bone marrow and peripheral blood cells in CML patients with different response to imatinib therapy using the results of hematopoietic cells culturing and the data of flow cytometry. Methods: Bone marrow aspirate from 39 patients who were treated with imatinib was studied with cytogenetic, flow cytometry and culture methods in vitro. Results: In patients with an optimal response to imatinib therapy the number of colonies was 1.8 times lower than the number of those in the group of patients with a suboptimal response to therapy. In turn, in patients with failure of imatinib therapy the number of colonies was the highest and was 2.1 times higher than the patients with optimal response. The results of cytometric studies have shown that the number of CD34⁺ cells in bone marrow was significantly higher compared to the number of CD34⁺ cells in peripheral blood cells and increased with the acquisition of leukemic cells the resistance to imatinib. There was a direct correlation between the number of colonies and clusters in semisolid agar in vitro and the number of CD34⁺ cells in the bone marrow of patients. Conclusions: The correlation between the number of CD34⁺ cells and the number of cell aggregates in semisolid agar in vitro indicates the prognostic value of the method for determining CD34⁺ cells in the patient bone marrow. The parallel increase of their number in the peripheral blood will allow developing express methods for the detection of individual patient response to imatinib therapy. Key Words: chronic myeloid leukemia, imatinib, cell culture in vitro, CD34⁺ cells. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy Article published earlier |
| spellingShingle | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy Sviezhentseva, I.O. Perekhrestenko, T.P. Bilko, D.I. Gordienko, A.I. Diachenko, M.V. Dyagil, I.S. Original contributions |
| title | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| title_full | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| title_fullStr | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| title_full_unstemmed | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| title_short | Functional activity of CD34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| title_sort | functional activity of cd34-positive cells in chronic myeloid leukemia patients with different response to imatinib therapy |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145458 |
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