The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms”
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irk-123456789-1452062019-01-20T01:23:07Z The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” Letter to editor 2013 Article The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” // Experimental Oncology. — 2013. — Т. 35, № 2. — С. 128-152. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145206 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Letter to editor Letter to editor The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” Experimental Oncology |
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Article |
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The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” |
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The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” |
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The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” |
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second ukrainian-swedish workshop "translational oncology: old and new paradigms” |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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The second Ukrainian-Swedish workshop "Translational oncology: old and new paradigms” // Experimental Oncology. — 2013. — Т. 35, № 2. — С. 128-152. — англ. |
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128 Experimental Oncology 35, 128–152, 2013 (June)
THE SECOND UKRAINIAN-SWEDISH WORKSHOP
“TRANSLATIONAL ONCOLOGY: OLD AND NEW PARADIGMS”
The second Ukrainian-Swedish workshop “Transla-
tional Oncology: Old and New Paradigms” took place
at the R.E. Kavetsky Institute of Experimental Pathol-
ogy, Oncology and Radiobiology National Academy
of Sciences of Ukraine (IEPOR NASU) on May 20–21,
2013. Karolinska Institutet (Stockholm, Sweden) was
co-organizer of the event.
Academician Vasyl F. Chekhun, the director
of R.E. Kavetsky IEPOR NASU, opened the conference
with a welcoming speech. He greeted participants and
stressed the importance of knowledge integration
in field of cancer research. He also pointed out the
necessity of experience exchange and collaboration
between different institutes worldwide.
The goal of the Workshop was to discuss current
views and newly emerging conceptual frameworks
concerning mechanisms of cancer development.
Considering the fact that cancer is major healthcare
problem today, the pursuit of deep understanding
how cancer actually works will lead to new effective
approaches to treatment of proliferative disorders.
Accordingly, all lectures and reports, presented by par-
ticipants, included up-to-date issues and experimen-
tal data dealing with clinical and biological aspects
of cancer research. The Workshop featured excellent
lectures, delivered by invited speakers from Karolinska
Institutet (Sweden) and speakers from IEPOR NASU,
as well as informative reports on proceeding research.
The number of scientists, who took part in the event,
reached more than hundred people this year. Apart
of organizing institutes representatives of other institu-
tions also attended the Workshop. Among them there
were Institute of Cell Therapy, City Clinical Oncological
Center, National Cancer Institute of the Ministry of Pub-
lic Health of Ukraine, National University of Kyiv-Mohyla
Academy, Center of Molecular and Cell Research, Taras
Shevchenko National University of Kyiv, Educational and
Scientific Center “Institute of Biology”, State Institution
“Institute of Pharmaco logy and Toxicology, National
Research Center for Radiation Medicine, Shupik Na-
tional Medical Academy of Post-Graduate Education,
Reference-Centre for Molecular Diagnostic, Ovcha-
renko Institute of Biocolloidal Chemistry, Bogomolets
National Medical University, Institute of Molecular Biol-
ogy and Genetics (Kyiv, Ukraine), Danylo Galytskyj Lviv
National Medical University, Ivano-Frankivsk National
Medical University, Rivne Region Oncology Hospital,
Bila Tserkva National Agrarian University; Bila Tserkva
Regional Oncology Center; Belarusian Research Сenter
for Pediatric Oncology, Hematology and Immunology
(Minsk, Belarus), Cancer Research Institute of SB of the
RAMS (Tomsk, Russia), Institute of Chemical Biology
and Fundamental Medicine of SB of the RAS (Novosi-
birsk, Russia).
The program of the Workshop spanned plenary lec-
tures and oral or poster reports. All talks evoked vivid
interest among listeners and, thus, were thoroughly
discussed. Some of these reports were included into
current issue of Experimental Oncology journal for
your appreciated consideration.
Exp Oncol 2013
35, 2, 128–152
PART I. LECTURES
TUMOR PROGRESSION — TIME FOR A NEW
PARADIGM?
I. Ernberg, MD, PhD, Professor of Tumor Biology
Department of Microbiology, Tumor and Cell
Biology, Karolinska Institutet, Stockholm SE-
17177, Sweden
Ingemar.Ernberg@ki.se
Around the millennium three decades of successful
research into the biology of cancers were concluded
in the most cited review on cancer ever “Hallmarks
of Cancer” (Hanahan and Weinberg, Cell, 2000).
It marked the end of the era when oncogenes and
suppressor genes were discovered en masse, and
when tumor progression at the level of cell and tissue
pathology could be linked with molecular steps like that
of the pioneering description by Bert Vogelstein of colon
cancer progression (“The Vogelgram”). We then under-
stood that cancer was indeed a disease of cells and
depended on mutations of some ten genes. Tumor pro-
gression, when tumors “go from bad to worse”, could
be viewed as an evolutionary, Darwinistic process at the
tumor cell population level, with sequences of mutation,
improved cell survival, selection, another mutation etc.
This seemed so transparent and logically sound that the
scientific community at large believed it had understood
the biology of cancer. Now we only needed to trace
these mutated genes and eliminate their erroneous
behaviour one way or another. It also gave a strong im-
petus to “personalized cancer medicine”, now we only
needed the genome of individual tumors for designing
the cure. One impressive major extrapolation was that
all the 200 different forms of cancer develop through
a similar mechanism. With this modern paradigm of can-
cer biology (or the Hanahan — Weinberg — Vogelstein/
HWV-paradigm) as a basis, ten years later it was clear
that the success rate in exploiting its implications for
cancer treatment was quite low.
During the same time the understanding of cell and
tissue biology took quantum leaps in new directions,
which clearly exposes the HWV-paradigm as totally
insufficient and oversimplified, although not directly
wrong. The conclusions are two: we do not really un-
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 129
derstand cancer biology, and cancer is a very complex
disease reflected by the enormous complexity of its
“host” — the cell. Major findings have been: epigenetic
deregulation is as important as mutations, cancer tis-
sues are highly heterogenous with cell hierarchies (in-
cluding stem-cell like cells) and a participating stroma,
microRNAs represent a large new set of regulatory
genes, low level chronic inflammation is a key compo-
nent in early carcinogenesis, there are not only some ten
mutations in cancer cells but rather several thousands,
and cancer cells usually switch metabolism from oxida-
tive glycolysis to fermentation (the Warburg effect).
How can we comprehend all these significant find-
ings in our understanding of cancer biology? Firstly
we should be humble. Secondly we should explore new
tools and technologies in interdisciplinary initiatives
to adopt an approach that accommodates complex
or complicated features of biological systems like can-
cer. This will be a long and uncertain way. Meanwhile
it must be perfectly motivated to continue to develop
treatments based on trial-and-error without sufficient
information about the enemy.
EXPLOITING THE P53 PATHWAY TO ACHIEVE
CURE IN CHRONIC MYELOGENOUS
LEUKEMIA
S. Lain, PhD, Associated Professor
Department of Microbiology, Tumor and Cell
Biology, Karolinska Institutet, Stockholm, Sweden
sonia.lain@ki.se
In 2008 we reported the discovery and antitumor
activity of a small molecule named tenovin-6. In addition,
we showed that this compound activates the tumour sup-
pressor p53 as well as inhibits the protein deacetylase
activity of sirtuin SirT1. In 2012, a series of remarkable
studies were published showing that tenovin-6 increases
apoptosis in Chronic Myelogenous Leukemia (CML) stem
cells and reduces their growth in vitro and in vivo in combi-
nation with imatinib. These exciting preclinical results have
encouraged us to synthesize new tenovin-6 analogues
and investigate their activity in cells and biochemical as-
says. Here I will present new findings obtained with our
current collection of tenovin analogues and suggest which
of these features may influence therapeutic activity. I will
also discuss our progress in the identification and mecha-
nism of new compounds for testing in leukemia preclinical
models and patient samples.
INDIVIDUALIZATION
OF CANCER TREATMENT: SUCCESSES AND
CHALLENGES
S. Souchelnytskyi, PhD, Associated Professor
Department of Oncology-Pathology, Karolinska
Institutet, Stockholm, Sweden
serhiy.souchelnytskyi@ki.se
Biomedical research delivered a new look on the
medicine. New developments of biochemistry, mo-
lecular biology, engineering, biomedical research
deliver new insights in the cancer mechanisms, new
treatment methods and new drugs.
Medicine success needs new techniques.
New techniques are coming from biochemistry,
chemistry, physics, molecular and cell biology.
More than 100 anti-cancer drugs are used in the
clinic today, and new drugs are under development
for use tomorrow. The challenge is to match a patient
with the right drugs. The clinicians face this challenge
with practically every patient, because of the limita-
tions of diagnostics. Information obtained with current
diagnostics is still far from predicting securely how
the tumor and patient would respond to a treatment.
The recent developments of cancer research have
opened for the qualitative improvement of diagnostics.
We have developed Functional Molecular Diagnostics
(FMDx) which is for the clinical use. FMDx tests re-
sponsiveness of individual patient’s tumors to different
drugs by testing responsiveness of the living tumor
samples in organ culture (Organ Culture FMDx), test-
ing targets and modulators of the drugs’ action (Func-
tional Biochemical Assays), and by unbiased testing
of the tumor’s proteome profile (Proteomics FMDx).
FMDx is a set of assays performed on living and
processed tumor cells following removal from patients
upon surgery or biopsy. These assays measure in a real
time how the patients’ tumor may respond to differ-
ent drugs before the patient is offered treatment, and
whether the tumor is of an aggressive type. The suc-
cessfulness of FMDx is based on novel proprietary
technologies.
FMDx assays are of 3 types — organ culture-based,
functional biochemical assays and proteomics-based.
The assays may be used all together or separately,
depending on the clinical requirements. All 3 types
of assays analyze a tumor with different methods, but
for the common goal of finding the best treatment.
This increases the confidence of the recommendations
for treatment. FMDx has been used for diagnostics
of cancer patients, and in my talk, Functional Molecular
Diagnostics applications will be discussed.
HIGH-RISK HPV L1 CAPSID PROTEIN
AS A MARKER OF CERVICAL
INTRAEPITHELIAL NEOPLASIA IN HIGH-
RISK HPV-POSITIVE WOMEN WITH MINOR
CYTOLOGICAL ABNORMALITIES
S. Andersson, MD, PhD, Professor
Department of Women’s and Children’s Health,
Division of Obstetrics and Gynaecology, Karolinska
Institutet, Karolinska University Hospital 171 76,
Stockholm, Sweden
Sonia.Andersson@ki.se
Human papillomavirus (HPV) L1 capsid protein
is only produced during a productive HPV infection
at the end of the natural viral life cycle and is a major
target of the immune response in women with HPV-
related squamous intraepithelial lesions.
We evaluated the usefulness of L1 detection by im-
munocytochemistry in high-risk (HR) HPV-positive
women with minor cytological abnormalities detected
at organised population-based cervical cancer
130 Experimental Oncology 35, 128–152, 2013 (June)
screening in Sweden, and assessed the relationship
with histological diagnoses. Cytological slides were im-
munocytochemically stained using a HPV L1-specific
monoclonal antibody for all known HPV types. HPV DNA
analysis was performed by Linear Array. Out of 13 L1-
positive women infected with HPV16, only two (15.0%)
progressed to cervical intraepithelial neoplasia grade
2 or worse (CIN2+); compared to four L1-positive
women infected with other HR-HPV types. Among
L1-positive women with CIN2+, 35.7% harboured both
HR and low-risk HPV types, 25.0% harboured HR-HPV
types only, and 13.3% were infected with HPV16. Loss
of L1 expression could be a prognostic marker for the
development of preinvasive cervical lesions. We show
that different HPV types may initiate a parallel onco-
genic process, but only loss of L1 expression predicts
the development of CIN2+, suggesting that HPV typ-
ing in combination with L1 detection could be used
for more focused investigations of women with minor
cytological abnormalities.
CARCINOMA OF THE ENDOMETRIUM:
PREDICTION MARKERS OF TUMOUR
PROGRESSION, AGGRESSIVENESS AND
POTENTIAL TARGETS FOR THERAPY
IN A MITOCHONDRIAL PERSPECTIVE
M. Mintz, MD, PhD, Associated Professor
Department of Women’s and Children’s Health,
Division of Obstetrics and Gynaecology, Karolinska
Institutet, Karolinska University Hospital 171 76,
Stockholm, Sweden
Miriam.Mintz@ki.se
Endometrial cancer (EC) is the most common
gynaecological cancer in the Western world, and its
incidence continues to increase. At present, molecular
markers are not commonly used to detect EC, nor are
there molecular markers that can distinguish between
the more and less aggressive forms of EC. Treatment
is currently based on histological results from a single
pre-surgical endometrial biopsy, which is often quite
different from the biopsy obtained during surgery.
There is a clear need for early molecular markers
that can be used to determine EC diagnosis, as well
as tumour progression and aggressiveness, in order
to improve the prognosis of EC and to reduce recur-
rence. Further investigation of tumour gene mutations
is also needed to find new targeted treatments for EC.
We summarized the known molecular alterations
that occur in EC, with a focus on mitochondrial al-
terations, and the potential to use these alterations
to develop targeted treatments in the future. Studies
concerning mitochondrial dysfunction in carcino-
genesis reported that the LRPPRC protein is highly
expressed in early endometrial carcinogenesis, and
may be considered an early marker of EC. Moreover,
four proteins were reported to be molecular markers
of aggressiveness in patients with EC: 1) the ratio
of progesterone receptor (PR) A/PR B less than 1 was
associated with a shorter disease-free period and
shorter survival in patients with endometrioid EC; 2)
glucose transporter proteins (GLUT 1 and GLUT 3)
were correlated with increased malignancy, invasive-
ness and poor prognosis; and 3) the TKTL1 protein
was overexpressed in low-grade EC.
Currently, there are no tumour markers that can
resolve diagnostic problems in EC. The present review
indicates that molecular markers and tumour char-
acteristics greatly vary. Consequently, a combination
of molecular markers would be necessary to give
information on early diagnosis, tumour progression
and metastatic capability.
EBV-ENCODED EBNA-5 BINDS TO P18ARF
AND PREVENTS P53-INDUCED GROWTH
ARREST AND APOPTOSIS
E. Kashuba, PhD, Associated Professor
Institute of Experimental Pathology, Oncology and
Radiobiology of National Academy of Sciences
of Ukraine, Kyiv, Ukraine
MTC, Karolinska Institute, 171 77, Stockholm,
Sweden
Elena.Kashuba@ki.se
The EBV transformed LCLs maintain wild type
p53 during serial culturing. We addressed the question
why the p53 downstream growth arrest and apoptotic
pathways are non-functional in p53 expressing LCLs,
unless activated by genotoxic agents, by examining
p53 containing protein complexes in LCLs.
We have shown that trimolecular complexes were
formed where MDM2 served as a bridge between
EBNA-5 and p53. All three proteins colocalized in the
nucleus of lymphoblastoid cells with a high p53 ex-
pression.
To explore the functional consequences of the
MDM2–EBNA-5 binding, p53 polyubiquitination and
degradation assays were performed in vitro. GST-
EBNA-5 inhibited MDM2-dependent polyubiquitina-
tion (but not monoubiquitination) of p53, similarly
to GST-p14ARF. The p53 degradation on commer-
cially purified 26S proteasome subunits was inhibited
by EBNA-5 as well.
These findings indicate that the high p53 levels
in LCL are due to EBNA-5 dependent inhibition
of p53 degradation. Chromatin immunoprecipitation
showed that p53 could bind to the p21 promoter
in mitogen stimulated but not the EBV activated
B-cells. Taking together, these findings are con-
sistent with the interpretation that the trimolecular
EBNA-5-MDM-2-p53 complex inhibits the binding
of p53 to DNA.
We have cloned full length of human p14ARF pro-
tein (p18ARF) from the EST clone. We have shown
by GST pull-down and transfections experiments that
p18ARF binds to MDM2, MDMX, and EBNA-5. We have
found that an enhanced expression of p18ARF in cells
bearing wt p53 resulted in the cell death, similarly
to mouse p19ARF. Simultaneous expression of p18ARF
and EBNA-5 rescued these cells. Biological signifi-
cance of the binding between MDMX, p18ARF and
EBNA-5 is currently under investigation.
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 131
TRANSCRIPTIONAL NETWORK IN B CELLS:
IRFS IN THE DEVELOPMENT OF B CELLS AND
LYMPHOMAGENESIS
M. Yurchenko, L.M. Shlapatska,
L.M. Kovalevska, S.P. Sidorenko
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine
myurchenko@hotmail.com
The family of interferon regulatory factors (IRF)
is comprised of 9 members: IRF1, IRF2, IRF3,
IRF4/PIP/LSIRF/ICSAT/MUM1, IRF5, IRF6, IRF7,
IRF8/ICSBP and IRF9/ISGF3γ. All members of this
family of transcription factors contain the homo-
logical DNA-binding domain, which is responsible
for interaction with DNA binding motifs ICS, ISRE,
IRFE, EICE. IRF4 and IRF8 proteins share the most
homology among other IRFs, and their expression
is narrowed to the cells of immune system. For
transcriptional regulation of target genes IRF4 and
IRF8 form complexes with each other or with other
transcription factors and adaptor proteins (Ets
family — PU.1, SpiB, TEL; E47, NFAT, FOXP3, STAT
proteins, MyD88, IBP, TRAF6). IRF1, IRF2, IRF4 and
IRF8 play the central role in the regulation of im-
mune cells differentiation and maturation. IRF4 and
IRF8 are key regulators of multiple steps of B cell
differentiation, starting from stage of pre-B cells.
IRFs 4 and 8 together with other transcription factors
(BCL6, BLIMP1, XBP1, Pax5) control GC formation,
CSR, memory versus plasma cell differentiation.
IRF4 and IRF8 were shown to be deregulated in B cell
malignancies. IRF8 was mainly shown to function
as tumor suppressor protein, but IRF4 was found
to be overexpressed in some tumors (multiple my-
eloma, Hodgkin’s lymphoma, some diffuse large
B cell lymphoma cases, etc), and at the same time
could have tumor suppressor functions in B-acute
lymphoblastic leukemia.
Still there is not so much data on regulation
of IRFs expression by different signals and signaling
pathways in normal and malignant B cells. B cell re-
ceptor and CD40 are the most potent up-regulators
of IRF4 expression. However, nothing is known about
receptor-mediated downregulation of this transcrip-
tion factor. We have shown that CD150 receptor
expression decreased the levels of IRF4 mRNA and
protein in DT40 CD150+ transfectants — the stage
of pre-B cells. Prolonged stimulation of activated
human tonsillar B cells via CD150 resulted in up-
regulation of IRF4 mRNA. Signals via CD150 and
BCR induced the upregulation of IRF4 mRNA ex-
pression by Burkitt’s lymphoma cells BJAB. Overall,
CD150 and BCR had different impact on IRF4 ex-
pression by malignant and normal human B cells.
The identification of surface receptors and signaling
pathways involved in the regulation of IRFs expres-
sion could help to develop new strategies for tumor
cells elimination.
IMMUNOGENIC CANCER CELL DEATH: HOW
IT CAN BE EXPLOITED
L. Skivka
Taras Shevchenko Kyiv National University, Kyiv,
Ukraine
realmed@i.com.ua
When considering normal, non-transformed
cells, it is commonly assumed that the immunologi-
cal consequences of cell death follow a classical
dichotomy of immunogenic necrosis versus non-
immunogenic (or even tolerogenic) apoptosis.
However, recent studies have revealed that apop-
totic tumor cells induced by ionizing irradiation,
anthracyclines and some other cytotoxic agents
are able to induce a potent immune response
in vivo. This led to the hypothesis that cell stress
or death may result in the release of endogenous
danger signals that act as adjuvants to stimulate
an immune response. Some characteristics of the
plasma membrane, acquired at preapoptotic stage,
can cause immune effectors to recognize and at-
tack preapoptotic tumor cells. Depending on the
signal-transduction pathway, tumor cells respond-
ing to chemotherapy or radiotherapy can express
“danger” and “eat me” signals on the cell surface
(such as NKG2D ligands, heat-shock proteins and
calreticulin) or can secrete/release immunostimu-
latory factors (such as cytokines and high-mobility
group box 1) to stimulate innate immune effectors.
These endogenous danger signals (or alarmins)
promote activation of innate immune cells and
recruitment and activation of antigen-presenting
cells engaged in host defense through pattern rec-
ognition receptors such as the TLRs, many of which
have a key role in the detection of pathogens.
In addition, these danger associated molecules
act broadly with many other immunostimulatory
molecules (endogenous and exogenous including
pathogen associated molecular patterns or PAMP)
to amplify their activity in a synergistic manner.
In our investigations we used cytotoxic drug NSC-
631570 in combination with PAMP in the treatment
of melanoma B16 bearing mice. Our results suggest
that bacterial adjuvants synergized with chemo-
therapy to inhibit tumor growth and significantly
augment immune responses. Current evidence
suggests that once in the extracellular milieu,
alarmins can bind to molecules such PAMP and,
depending on its interactions with DNA, can form
complexes of varying structure and with a vary-
ing the number of components. Here we analyse
the current know ledge and our data to discuss
the hypothesis that the formation of extracellular
complexes is an important mechanism for gener-
ating pro-inflammatory signals during cell death
and reverting some of the established immuno-
supressive barriers present within the tumor mi-
croenvironment, ideally recovering the role of the
tumor as an effective immunogenic hub.
132 Experimental Oncology 35, 128–152, 2013 (June)
HUMAN BETA-DEFENSINS IN REGULATION
OF BIOLOGIC PATTERNS OF CANCER CELLS
O. Gerashchenko, E. Zhuravel, P. Pogrebnoy,
M. Soldatkina
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine
pogrebnoy@onconet.kiev.ua
Human beta-defensins (hBDs) belong to a family
of small cationic peptide antibiotics produced by human
epithelial cells and are characterized by wide spectrum
of biologic activities. These molecules exert direct anti-
microbial action and immunomodulatory activity, and are
shown to be involved in many human pathologies including
cancer. Beta-defensins are involved also in wound heal-
ing processes and may affect proliferation of epithelial
cells. To understand the mechanisms of biologic activities
of beta-defensins, we have produced two recombinant
defensins — hBD-2 and hBD-4 expressed in bacterial cells
as GST-hBD-2 or -4 fusion proteins. Analysis of in vitro ac-
tivity of rec-hBD-2 toward different human cancer cell lines
(A341, A549, 3 thyroid cancer cells, 2 melanoma cell lines)
has demonstrated that this defensin is capable to affect
cell proliferation and viability in a concentration-dependent
manner and in nanomolar range may significantly sup-
press cell growth via cell cycle arrest in G1/S checkpoint,
dephosphorylation of pRB and significant suppression
of cyclin D1 expression. The action of hBD-2 is not cell
type specific, at nanomolar concentrations hBD-2 signifi-
cantly suppresses colony forming activity of human cancer
cells and their migration ability in scratch assay. In vitro
hBD-2 may enter tumor cells, could be detected in cell
cytoplasm and also in cell nuclei. Similarly, this defensin
has been detected in cell nuclei in human lung tumor
samples, moreover, in such cases hBD-2 expression levels
positively correlated with human lung adenocarcinoma
differentiation grade, and negatively correlated with PCNA
expression. Hypothetically, concentration-dependent ef-
fects of hBD-2 could be explained by its capability to oligo-
merization. The study of recombinant hBD-4 (presented
in details in current issue of Exp Oncol) has demonstrated
a concentration-dependent bimodal effect of this defensin
on biologic patterns of human cancer cells: in low nano-
molar concentrations rec-hBD-4 significantly stimulates
cancer cell proliferation and viability, promotes cell cycle
progression through G2/M checkpoint, greatly enhances
co lony-forming activity and migration ability of the cells,
but at higher concentrations the effects are of opposite
character — significant suppression of cell proliferation
and viability, cell cycle arrest in G1/S checkpoint, signifi-
cant inhibition of cell migration and colony forming activity.
It’s of interest to analyze further with the use of in vitro
system what mechanism is exploited by beta-defensins
for cancer cell growth suppression — senescence, MET,
other, whether it is reversible or irreversible, and to find ap-
plications of this know ledge in the field of clinical oncology.
PART II. ORAL AND POSTER PRESENTATIONS
NEW MECHANISMS OF CLINICAL
EFFICACY OF LOW-DOSE METRONOMIC
CHEMOTHERAPY TREATMENT OF PATIENTS
WITH METASTATIC COLORECTAL CANCER
Yu.I. Kudryavets1, N.O. Bezdenezhnykh1,
G.I. Maksim̓yak2, V.E. Zhylchuk2,3,
I.M. Adamenko1, N.I. Semesiuk1,
A.L. Vorontsova1, O.E. Sichkoriz3, V.F. Chekhun1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine
2Rivne Region Oncology Hospital, Rivne, Ukraine
3Danylo Halytsky Lviv National Medical
University, Lviv, Ukraine
kudrayvets@mail.ru
Background and Aim: This work describes the
experience of treatment of metastatic colorectal cancer
(mCRC) patients with liver metastases (stages T1–4N0–
2M1) by low-dose chemotherapy (LDCT) in metronomic
regimen including interferon-alpha (IFN), and dem-
onstrates growth behavior and immunophenotypical
changes in CRC cells after influence of LDCT in vitro.
Patients and Methods: Treatment of 254 patients was
carried out using different schemes, which included
cyclophosphamide, cisplatin (CP) and irinotecan (IT)
with IFN, efficiency of therapy was monitored by clini-
cal methods and computer tomography. Cell culture
and immunocytochemical methods were used for
in vitro investigation. Statistical analysis using the
Student’s t-test and Kaplan — Meier survival curves.
Results: Metronomic mode of LDCT in patients with
mCRC demonstrated high performance compared
with conventional treatment using only high dose
of IT. Among the most effective schemes metronomic
mode of application of LDCP and IT combination was
shown: duration of partial clinical effect was nearly 3 fold
higher, duration of the stabilization process and the
overall survival rate increased respectively by 81% and
34.5% in comparison with a high dose IT therapy. Using
of IFN in all the metronomic CT schemes significantly in-
creased their effectiveness. We have found new targets
in the CRC cells for the effects of LDCT in vitro. Cells
of human colorectal adenocarcinoma of COLO 205 line
were cultivated in vitro with LD (10–20 times lower
IC50) of IT, CP, IFN and their combination for 30 days.
The ability of cells after prolonged exposure by LDCT
to the agar colony formation decreased by 3–5 times,
the maximal effect was observed with the combination
of IT + CP + IFN (from 9.25% to 0,25%). A significant
decrease of number of cells with mesenchymal and
malignant stem cell characteristics was found: num-
ber of N-cadherin-, SLUG- and CD44-positive cells
decreased more than 10 fold for various combina-
tions of drugs. Expression of ERCC1 marker in COLO
205 cells completely disappeared in all combinations
with IFN, but increased somewhat under the influence
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 133
of IT + CP. At the same time sensitivity of this cells
to drugs in IC50 doses didn’t change. Conclusions:
Low-dose metronomic chemotherapy of patients with
mCRC is a new and perspective approach that provides
suppression of the disease progression. LDCT drugs
inhibit signs of epithelial-mesenchymal transition and
malignant phenotype in CRC cells in vitro.
THE COMET ASSAY: APPLICATIONS
TO STUDY CANCER RISK INFLUENCED
BY EXTERNAL EXPOSURE
O. Bilyk, O. Brieieva, L. Buchynska
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
lenabilyk@gmail.com
Introduction: The comet assay is a reliable method
for measuring DNA damage and repair in human cells
such as lymphocytes, epithelial cells, and tumor cells
of different origin and has applications in genotoxicity
studies, bio-monitoring and cancer research. The poten-
tial use of assay as a tool for estimation an individual can-
cer risk in patients with high predisposition to cancer and
clinical management of cancer is widely discussed. Aim:
To study DNA damage and repair capacity in epithelial
cells and peripheral blood lymphocytes (PBL) after expo-
sure to genotoxic agents in healthy and cancer patients
with family history of cancer. Methods: PBL from 45 en-
dometrial cancer (EC) patients were exposed to bleo-
mycin for 30 min. DNA repair capacity was assessed
after 15 min incubation in RPMI 1640 without bleomycin.
Primary and immortalized by HPV16 E6/E7 oncogenes
of ovarian surface epithelial (OSE) cell cultures ob-
tained from 5 women at high risk (HR) of ovarian cancer
were exposed to mitomycin C (MMC) at concentration
50 nmol/L. PBL from 10 healthy volunteers and 5 primary
OSE cell cultures from women without family history
of cancer were evaluated for DNA damage as a control.
DNA damage was assessed by comet assay as a mean
% of DNA in the tail and the comet tail moment (TM).
Results: The level of basal DNA damage in PBL of EC pa-
tients was significantly higher (TM 2.1±0.2) compared
to healthy volunteers (TM 0.2±0.1). Among EC patients
the sensitivity of PBL to genotoxic exposure to bleomy-
cin was higher in patients with family history of cancer
(TM 104.9±1.2) then in patients with sporadic cancer
(TM 97.3±0.6). It has been found that efficiency of DNA
repair in PBL of EC patients depended on family history
of cancer. After 15 min repair PBL restored the damaged
DNA to the level of TM 55.0±2.4 in EC patients with family
history of cancer and up to 22.7±1.1 in patients with spo-
radic EC. Basal damage of DNA was significantly higher
for the primary HR OSE cells than in control OSE cells
(TM 15.1±0.1 and 1.0±0.2, respectively). Cell incubation
with MMC resulted in progressive increasing of the level
of DNA damage of HR OSE cells (TM 25.3±0.2) whereas
normal OSE cell line was resistant to genetoxic expo-
sure (TM 1.1±0.5). After immortalization of OSE cells
by HPV E6/E7 oncogenes the intensity of DNA damage
significantly increased independently of family history
of cancer. HR OSE cells immortalized with HPV E6/E7 on-
cogenes demonstrated increased sensitivity to genotoxic
exposure compared to normal immortalized OSE cells
(TM 88.5±0.1 and 16.0±0.1, respectively). Conclusion:
Enhanced DNA damage of PBL and OSE cells in healthy
and cancer patients with family history of cancer under
genotoxic and viral oncogenes exposure could expand
our understanding of endometrial and ovarian cancer
etiology and biology. Comet assay is a useful tool for
assessment of individual sensitivity to genotoxic and viral
exposure to evaluate the risk of malignancy in patients
with predisposition to cancer.
ANTITUMOR EFFECT OF OXY CORRELATES
WITH ITS ABILITY TO DECREASE THE LEVEL
OF NITROSYL-HEM COMPLEXES IN LEWIS
LUNG CARCINOMA CELLS
I. Boichuk, O. Melnikov, G. Solyanik
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
boichuk_irina@mail.ru
Introduction: It is known that the tumor growth
is accompanied by changes of redox carriers in the
mitochondrial electron transport chain. These changes
may result in decrease of energy metabolism. The
functional state of the mitochondrial electron transport
chain also defines the intensity of production and ac-
cumulation of the oxygen and nitrogen reactive spe-
cies (ROS and NOS), which affect different biological
processes and can promote cell damage. It has already
been observed that non-cytotoxic doses of oxyres-
veratrol (OXY) showed significant antineoplastic and
anti-metastatic effects and prolonged the life of animals
with tumors. Aim: To analyze the influence of OXY on the
mitochondrial iron-sulfur centers and nitrosyl-hem
complexes of tumor cells during Lewis lung carcinoma
growth. Materials and Methods: EPR method was
used to analyze the OXY influence on the content of the
mitochondrial iron-sulfur centers (g=1.94) and nitrosyl-
hem complexes (g=2.007) in tumor tissues. The total
dose of OXY was 0.2 mg/g of animal weight. The agent
was administered per os from the 2nd day after cancer
cell inoculation, 5 times/week, during 3 weeks. Tumor
volume as well as characteristics of tumor mitochondrial
electron transport chain was registered on the 10th, 14th,
16th, 20th, 25th day of the tumor growth. Results: It was
shown that OXY affected the formation of nitrosyl-hem
complexes in mitochondrial electron transport chain.
In OXY free mice the level of nitrosyl-iron complexes
was significantly increased during tumor growth, while
the level of iron-sulfuric centers was progressively
decreased. Treatment of LLC-bearing mice with OXY
resulted in a considerable LLC growth inhibition and
caused the reduction of NO-hem complexes. Its level
at the end of the treatment (20th, day of tumor growth)
was by 64% (р < 0.05) lower than that of control mice.
In 5 days after therapy completion the level of NO-
hem complexes in OXY treated mice was not changed
compared to untreated animals. It was shown that OXY
134 Experimental Oncology 35, 128–152, 2013 (June)
didn’t influence the content of iron-sulfuric centers
in LLC. Conclusions: LLC growth is accompanied
by decrease of iron-sulfur centers’ content and increase
of nitrosyl-hem complexes in mitochondria of cancer
cells. Decrease of the level of nitrosyl-hem complexes
in cancer cells under OXY treatment correlates with
an ability of OXY to inhibit LLC growth.
COMBINED APPLICATION OF AUTOLOGOUS
AND XENOGENEIC HEAT SHOCK PROTEINS
IN VACCINOTHERAPY OF MALIGNANT
TUMORS
I. Boliukh, G. Didenko, G. Potebnya
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
tavrovska_1@mail.ru
Introduction: The use of immunotropic substanc-
es of natural and synthetic origin, including heat shock
proteins (HSP) is one of the successful strategies
to improve the efficiency of existing tumor vaccines.
Aim: Combined application of heat shock proteins
derived from the chicken xenogeneic embryonictissue
and from autologous tumor cells for creating effective
antitumor vaccines. Methods: Protein extracts from
embryonic and tumor tissue were obtained by protein
ammonium sulphate salting-out. Column chromatog-
raphy was used for HSP separations with molecular
weight of 70 kDa. HSP were analyzed by western blot
with anti-HSP-70 monoclonal antibody (Enzo, USA).
Based on HSP fraction, a number of vaccines were
constructed and their efficacy was analyzed in vivo
in Balb/c and C57Bl/6 mice with transplantable Ehrlich
carcinoma and Lewis lung carcinoma (3LL). All animals
were divided on 6 groups consisting of 10 animals
each. Antitumor effect of vaccines was evaluated
by the dynamics of tumor growth, survival rate and an-
timetastatic effect. Results: Immunoblot test demo-
n strated antigenic homology of HSP-70 derived from
embryonic chicken tissues and Ehrlich carcinoma.
It was found that level of HSP-70 was 1.68 times higher
in chicken embryonic tissues compared to Ehrlich
carcinoma. Vaccines enriched with HSPS 70 were
made from tumor samples and chicken embryonic
tissues and their antitumor efficacy, manifested in in-
hibition of tumor growth (IG 24.35%) and the increase
in life expectancy of experimental animals, has been
shown. The purification of HSP-peptide complexes
in tumor extracts and embryonic tissues was made
and on their basis a number of vaccines has been de-
veloped, antitumor activity of which has been tested
in the system in vivo on animals with transplantable
3LL. The effective vaccines stimulated cytotoxic
activity of lymphocytes and macrophages (straight
by 25% and antibody-dependent by 27%), increased
serum cytotoxic activity and decreased the content
of circulating immune complexes. Anticancer activity
of vaccines was mainly reflected in the inhibition of me-
tastasis. Conclusions: Combined use of xenogeneic
and autologous HSP-peptide complexes for design
of vaccines significantly increases their antitumor
activity and immunomodulating effect compared with
the vaccine prepared by the traditional technology.
MISMATCH REPAIR PROTEINS MSH2,
MLH1 EXPRESSION AND DNA DAMAGE
IN TUMOR CELLS OF ENDOMETRIAL
CANCER PATIENTS WITH FAMILY HISTORY
OF CANCER
O. Brieieva, N. Iurchenko, L. Buchynska
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
olha.brie@gmail.com
Introduction: DNA repair deficiency may cause
genetic instability that results in high sensitivity of cells
to exogenous and endogenous DNA damaging agents.
Mismatch repair has important role in the maintenance
of genome integrity in tissues with high proliferative
activity such as endometrium. Genetic and epigen-
etic defects of mismatch repair genes MSH2 and
MLH1 induce accumulation of mutations leading
to malignant transformation. Patients with family his-
tory of cancer may be characterized by inherited DNA
repair deficiency therefore further studies are needed
to assess DNA repair activity in these patients. Aim:
To evaluate the level of DNA damage and mismatch
repair activity in tumor cells of endometrial cancer pa-
tients considering family history of cancer. Methods:
20 endometrial cancer (EC) patients with the mean
age 57.2 years were included in the study. Assess-
ment of DNA damages in endometrial tumor cells was
performed by DNA comet assay. The level of DNA dam-
age was quantified as the mean percentage of DNA
in the tail and the comet tail moment (TM). Mismatch
repair activity was assessed by immunohistochemistry
using primary monoclonal antibodies MSH2 (clone
25D12) and MLH1 (clone G168–15) (“Diagnostic Bio-
Systems”). The mean percantage of immunopositive
cells was calculated (LI, %). Results: 30% of EC pa-
tients had family history of gastrointestinal tract and
female reproductive system cancers. Assessment
of DNA damage in endometrial tumor cells revealed
that TM equaled to 32.2±1.2 and the % DNA in comet
tail — 51.7± 1.3. It was found that level of DNA dam-
age in tumor cells of EC patients with family history
of cancer was almost two times higher (TM 50.4±2.9;
% DNA in comet tail 75.72±1.96) compared to sporadic
EC (TM 27.3±1.2; % DNA in comet tail 45.2±1.5).
Immunohistochemical study didn’t reveal significant
difference of MSH2 expression in EC of patients with
and without family history of cancer (LI 40.0±3.4% and
40.5±7.7%, respectively). Expression of MLH1 was
higher in sporadic EC (LI 45.8±2.3%) compared
to EC of patients with family history of cancer
(LI 37.8±4.9%). Conclusions: Endometrial cancer
cells of patients with family history of cancer are char-
acterized by increased level of DNA damage that may
be caused by low level of MLH1 protein expression
indicating abnormalities of mismatch repair capacity.
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 135
THE INFLUENCE OF THE PELVIC PLEXUS
PRESERVATION UNDER RADICAL
HYSTERECTOMY IN PATIENTS WITH
INFILTRATIVE CERVICAL CANCER ON THE
CONTRACTILE FUNCTION OF THE URINARY
BLADDER
T. Dermenzhy, V. Svintsitsky, I. Schepotin,
O. Iatsyna, O. Kabanov
National Cancer Institute, Kyiv, Ukraine
nacluf@mail.ru
Aim: To justify the need to preserve pelvic plexus un-
der radical hysterectomy (RH) in patients with infiltrative
cervical carcinoma (CC). Patients and Methods: 30 pa-
tients with infiltrative CC (mean age 32.7±4.9 years) which
underwent RH were enrolled into study: in 15 patients
(group I) RH was performed with preservation of the pelvic
plexus and in 15 patients (group II) RH was performed
by standard method without preservation of the
pelvic nerve plexus. The study protocol was approved
by Ethical committee permission of National Cancer
Institute (Kyiv, Ukraine). Cytomanometric assessment
of urinary bladder’s contractile function after RH with
preserving of the pelvic plexus in infiltrative CC patients
was performed by using urodynamic stand URO-PRO
according to the standard procedure. Results: Accord-
ing to cytomanometric data contractile function of urinary
bladder was fully restored in 2–3 days after RH in 80%
of group I patients and only in 20% of group II patients.
In patients of group II the function of the lower urinary
tract restored within 7 days after RH, in 20% of these
patients persistent paresis of the urinary bladder detrusor
was observed, which partially restored from 7 to 21 days.
It indicates strong neurological disorders of contractile
function of urinary bladder under surgical trauma of pel-
vic plexus during standard RH. Conclusion: Recovery
of the contractile function of urinary bladder in 24 hours
after RH justifies necessity of the pelvic plexus preserva-
tion during RH in patients with infiltrative CC.
ALTERATIONS IN FUNCTIONAL PROPERTIES
OF BONE MARROW STEM CELLS
IN CHRONIC MYELOID LEUKEMIA PATIENTS
WITH DIFFERENT RESPONSE TO TYROSINE
KINASE INHIBITORS TREATMENT
M. Diachenko1, N. Bilko1, I. Dyagil2
1National University of Kyiv-Mohyla Academy,
Center of Molecular and Cell Research, Kyiv,
Ukraine
2National Research Center for Radiation Medicine
NAMS of Ukraine, Kyiv, Ukraine
diachenko.m@gmail.com
Introduction: It has been revealed the relative qui-
escence and resultant therapeutic resistance of primitive
chronic myeloid leukemia (CML) progenitor cells providing
a basis for identifying the hematopoietic developmental
stage of chronic phase initiating events, such as altered
stem cell differentiation and survival. This survival advan-
tage is largely mediated by leukemic stem cells (LSCs)
microenvironment. Aim: The aim of this study was to de-
termine the functional characteristics of hemopoietic and
mesenchymal stem cells from CML patients with different
response to the tyrosine kinase inhibitor (TKi) Imatinib (No-
vartis). Patients and Methods: All 32 patients who were
treated with TKi between 0 and 41 month have undergone
cytogenetic analysis for the Philadelphia chromosome
as well as in vitro assays: CFU-A in the semisolid agar
and suspension cultures. According to the TKi therapy
response patients were divided into groups: with optimal
response (no Ph+ cells), suboptimal response (Ph+ >
0%) and the group of patients who received hydroxyurea
before TKi treatment. Results: The functional activity
of bone marrow progenitor cells of patients with an opti-
mal response to therapy was significantly lower (p <0.05)
compared to patients with resistance to the therapy (the
average CFU-GM numbers 29.3 and 79.3, respectively).
Leukemic progenitor cells from CML patients were able
to survive in stroma-free suspension culture without
any additional growth factors supplementation during
21 day (median survival of 10 days). Such autonomous
growth, not typical for normal cells, could be explained
by autocrine mechanisms in primitive leukemia cells.
Mesenchymal stem cells (MSCs) from CML patients
with suboptimal response to TKi therapy showed 6 fold
increases in proliferation rate compared to MSCs from
bone marrow of patients with optimal response, sug-
gesting that stem cell microenvironment is also involved
in transformation process. Conclusions: Our data indi-
cate the presence of individual response to the TKi therapy
in CML’s patients that is probably associated with specific
changes in the population of stem and progenitor cells due
to alterations of their functional activity. Further researches
of leukemic stem cells and their niche would allow better
understanding of mechanisms of drug resistance and
disease progression.
THE FREQUENCY OF HORMONE-RELATED
TUMORS IN BREAST CANCER PATIENTS
AFTER SURGICAL TREATMENT
I.B. Djakiv, A.E. Kryzhanivska
Ivano-Frankivsk National Medical University,
Ivano-Frankivsk, Ukraine
irynadjakiv@ukr.net
Introduction: The frequency of hormone-related tu-
mors is about 20%, among which 10.8% belong to breast
cancer (BC). Women with BC have increased risk of de-
veloping a new primary cancer in the opposite breast and
second tumors of different localization that can be linked
to genetics and hormonal risk factors. However, there
is no data about the frequency, character and preventive
measures of origin of the second hormone-related tumors
in BC patients. Aim: To study the frequency of malignant
tumors in women who were treated for BC. Methods:
We have analyzed clinical data of 51 BC patients with stage
I–IV. The patients’ age varied from 26 to 76 years (the mean
age 54.3). Immunohistochemical staining of BC samples
was performed using the primary monoclonal antibo dies
against progesterone (PR) (clon 1A6, “Dako”, Denmark),
estrogene (ER) (clon 1D5, “Dako”, Denmark) receptors
and HER2/neu (cerv2 oncoprotein, “Dako”, Denmark).
136 Experimental Oncology 35, 128–152, 2013 (June)
28 (54.9%) BC patients received neoadjuvant treatment
and all patients received complex treatment including
surgery. The study was approved by the local Institutional
Ethic Committee. Results: BC of stage I was diagnosed
in 8 (15.7%), stage II — 33 (64.8%), stage III/IV —
10 (18.6%) patients. In BC patients second oncological
pathology has been developed: in 19 (37.3%) patients —
endometrial cancer, in 10 (19.6%) patients — ovarian can-
cer, in 7 (13.7%) — cervical cancer, in 2 (3.9%) — cancer
of vulva, in 9 (17.6%) — colorectal cancer, in 1 (1.9%) —
stomach cancer, in 1 (1.9%) — thyroid gland cancer and
in 1 (1.9%) — kidney cancer. Family history of cancer
with prevalence of BC was observed in 32 (62.7%)
patients. Immunohistochemical phenotype ER+HER2/
neu+ was detected in 45 (88.2%), PR+ER+HER2/neu++ —
in 10 (19.6%) and PR+ ER+ HER2/neu+++ — in 3 (5.9%)
BC samples. Conclusions: Oncogynaecological tumors
occupy the first place (74.5%) among hormone-related
tumors in BC patients. The further study of risk factors for
developing second malignant tumors in BC patients after
treatment is needed.
EVALUATION OF INTRAPERITONEAL
HYPERTHERMIC CHEMOPERFUSION
EFFICIENCY FOR CHEMORESISTANT
OVARIAN CANCER
U. Dutchak, M. Fetsych
Danylo Galytskyj Lviv National Medical
University, Lviv, Ukraine
Uliasia3@rambler.ru
Introduction: Intraoperative hyperthermic intra-
peritoneal chemoperfusion (HIPEC) is a new method
of combined treatment of chemoresistant ovarian
cancer (OC) with peritoneal carcinomatosis, which ef-
ficiency was not fully determined. Aim: To improve the
efficiency of treatment of patients with chemoresistant
recurrent ovarian cancer after cytoreductive operation
with HIPEC. Patients and Methods: We conducted
a full clinical examination and treatment of 181 patients
with recurrent OC, including 72 patients with chemore-
sistant recurrent OC. Depending on the treatment of OC,
patients were divided into three groups. The first group —
72 patients who received systemic chemotherapy: 1A
group — 17 patients with chemoresistant recurrent OC,
1B group — 55 patients with chemosensitive recurrent
OC. The second group — 61 patients who underwent
cytoreductive operation and systemic chemotherapy: 2A
group — 29 patients with chemoresistant recurrent OC,
2B group — 32 patients with chemosensitive recurrent
OC. The third group — 48 patients who underwent cyto-
reductive operation, HIPEC and systemic chemotherapy:
3A group — 26 patients with chemoresistant recurrent
OC, 3B group — 22 patients with chemosensitive recur-
rent OC. Results: Median of survival for patients, who
received combined treatment including cytoreductive
operation and HIPEC with systemic chemotherapy,
was 27 months and median of survival for patients with
chemoresistant recurrent OC, who received treatment
including cytoreductive operation with systemic chemo-
therapy without HIPEC, was 18 months, median of sur-
vival for patients, who received treatment with systemic
chemotherapy, was 10 months. Procedure of HIPEC
didn’t increase the frequency of surgical complications
and postoperative lethality in OC patients. Surgical com-
plications after cytoreductive operation and HIPEC was
observed in 10 (21%) patients, after cytoreductive opera-
tion without HIPEC — in 14 (22.9%) patients. Postopera-
tive lethality after the procedure of HIPEC was observed
in 2 (4.2%) patients, and in 3 (4.9%) patients after the
procedure of HIPEC. Conclusions: Application of HIPEC
in combined treatment of patients with chemoresistant
recurrent OC evidently increases survival of patients.
PROGNOSTIC VALUE OF SCCA TUMOR
MARKER IN PATIENTS WITH CERVICAL
CANCER
L. Vorobijva, I. Goncharuk, S. Nespryadko
National Cancer Institute, Kyiv, Ukraine
goncharuk_irina@i.ua
Squamous cell carcinoma antigen (SCCA) level
in blood serum is proven to be valuable for diagnosis
and monitoring of patients with cervical carcinoma.
Since cervical carcinoma as a rule originates from
squamous epithelium, SCCA estimation in blood se-
rum is significant for the study of the disease develop-
ment. The aim of the study was to monitor SCCA level
in patients with cervical carcinoma with primary and
metastatic disease for determination of recurrence
probability value. Patients and Methods: Changes
in SCCA level in blood serum and clinical data were
analyzed in 224 patients with cervical carcinoma
of I-IV stage. Primary cancer was diagnosed in 171 pa-
tients; recurrence was confirmed in 53 patients. SCCA
concentration in blood serum was determined
by chemiluminescent immunoassay at micro particles
(HIAM). Statistical analysis was performed with the
use of Statistica 6.0 software. The study protocol was
approved by Ethical Committee permission of Na-
tional Cancer Institute (Kyiv, Ukraine). Results: It was
shown that SCCA values different reliably in groups
of patients with different development of the disease
(p<0.05). SCCA level in patients with Ca in situ and
IA stage was equal to 0.6 ± 0.1 and 0.9 ± 0.1 ng/mL,
respectively. At the higher stage of the disease, the
higher SCCA level in blood serum was detected (l 5.9 ±
1.4 ng/mL in IIIB stage). It was confirmed that SCCA
level in patients with primary cervical cancer with
metastases in regional lymph nodes was higher com-
pared to SCCA level in patients without metastases
(5.6 ±1.4 ng/mL vs 2.3 ± 0.1 ng/mL, p < 0.05). SCCA
level in blood serum increased up to 86.4% of patients
with the recurrence and equaled 19.4 ± 3.7 ng/mL
(from 0.5 to 70.0 ng/mL). SCCA value in blood serum
increased up to 60.4% of patients with cervical carci-
noma of IB-III stage. Test-sensitivity increased from
7.8% (I stage) to 77.8% (III stage). Conclusion: SCCA
level in blood serum can be considered as a sensitive
method in prognostic study and determination of dis-
ease stage in patients with cervical carcinoma.
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 137
OVARIAN TISSUE CRYOCONSERVATION
IN PATIENTS WITH GYNECOLOGICAL
CANCER
L. Vorobijva1, V. Svintsitskiy1, S. Nespryadko1,
I. Goncharuk1, P. Nemtinov2, V. Shablij2
1National Cancer Institute, Kyiv, Ukraine
2Institute of Cell Therapy, Kyiv, Ukraine
goncharuk_irina@i.ua
Introduction: During recent years the increase
in incidence rate of gynecological malignancies in re-
productive age patients, who had no time to implement
reproductive function, has been registered. In majority
of cases special treatment results in partial or full depri-
vation of fertility as a result of the high ovarian sensitivity
to chemotherapy and radiation therapy. Cryopreservation
and autotransplantation of ovarian tissue appear to be-
come the method of fertility preservation. Aim: To evalu-
ate the ovarian reserve and develop policy of treatment
in patients with gynecological malignancies with the
purpose of cryoconservation of ovarian tissue for further
realization of reproductive function. Methods: 10 pa-
tients of reproductive age (< 35 years) have been treated
in National Cancer Institute from 2010 to 2012 year with
diagnosis IB–IIB stage cervical cancer. Panhysterectomy
type 3 has been executed in 9 patients, radical abdominal
trachelectomy — in 1 patient. Ovarian tissue obtained after
laparotomy was placed in aseptic terms in container with
special medium with temperature +20–25 °C and after that
it was transported to Institute of Cell Therapy for following
investigation, cryopreservation and storage. Currently all
the patients are placed under dynamic surveillance. There
is no evidence of the disease. The study protocol was ap-
proved by the Ethical Committee permission of national
Cancer institute (Kyiv, Ukraine). Results: As a result, cryo-
conservation of ovarian tissue can be fulfilled with the aim
to realize the delayed reproductive function. Conclusions:
This method does not require the delay of the treatment,
ovarian stimulation and can be accepted for the patients
with gynecological malignancies.
MODERN CHEMOIMMUNOTHERAPY
APPROACH BASED ON DENDRITIC CELLS
AND LOW-DOSE CISPLATIN IN EXPERIMENT
A. Gorbach, N. Khranovska, O. Skachkova,
R. Sydor, N. Svergun
National Cancer Institute, Kyiv, Ukraine
horbach.alex@gmail.com
Introduction: Nowadays, combined therapy
based on dendritic cells (DC) and low-dose chemo-
therapy is intensively investigated worldwide. There are
no commonly accepted regimens of combined therapy
and development of efficient chemoimmunotherapy
schemes remains an actual problem. Aim: To develop
scheme of combined chemoimmunotherapy based
on DC and low-dose cisplatin. Materials and Methods:
120 CBA mice have been involved in the experiment.
Sarcoma-37 was injected intramuscularly at lethal dose
(2x106 cells per animal). Cisplatin was injected intra-
peritoneally 5 times in metronomic regimen according
to the two schemes: 0.2 or 2 mg/kg on the 7th day after
tumor transplantation with interval of 1 day and 3 days,
respectively. DC vaccines were administered intrave-
nously 3 times on the 4 day after the chemotherapy.
All experiments were approved by Ethical Committee
permission of National Cancer Institute (Kyiv, Ukraine).
Results: We have found that both chemoimmunotherapy
schemes had significant antitumor and immunomodula-
tion effect. The most pronounced effect was observed
with DC-vaccine and cisplatin at concentration of 2 mg/
kg in combination. Combination of DC vaccine and low-
dose cisplatin had a synergistic effect on the reduction
of primary tumor. Significantly decrease of primary tumor
volume in animals was received in combined therapy
compared to the control (p = 0.001) and DC-vaccine
(p = 0.007) groups have been found. We showed that
administration of combined therapy improves survival
by 20% in comparison with the control group. A signifi-
cant increase of cytotoxic activity of splenocytes in re-
sponse to allogeneic stimulation to 42.33 ± 4.8% in the
combined therapy group compared to 29.67 ± 4.1%
in the control group had been observed, p < 0.05. We also
found that phagocytic activity of splenocytes in this group
increased up to 52.33 ± 4.67% compared to 37 ± 5.29%
in the control group, p < 0.05. The coefficient of reactive
oxygen species induction was statistically significant
and increased to 1.61 ± 0.23 in comparison with 1.05 ±
0.13 in the control group. Conclusions: Low-dose che-
motherapeutics enhance the antitumor effect of DC-
based immunotherapy. These investigations form the
basis to a new multimodality treatment of cancer patients.
THE ROLE OF CD150 SURFACE RECEPTOR
IN REGULATION OF TOPOLOGY
AND EXPRESSION OF P50 AND
P65 NF-KB IN NORMAL B CELLS
I.M. Gordiienko, L.M. Kovalevska,
L.M. Shlapatska, S.P. Sidorenko
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine
hordiyenko24@mail.ru
Introduction: CD150 receptor is widely expressed
in hematopoietic cell lineage and is involved in lym-
phocyte activation, differentiation and apoptosis via
regulation of signal transduction pathways. Moreover,
CD150 is expressed on malignant cells at Hodg-
kin’s lymphoma and diffuse large B cell lymphoma that
are characterized by enhanced level of NF-kB activa-
tion. CD150-mediated signal transduction pathways are
not fully understood. Particularly, it is not clear whether
CD150 is involved in regulation of NF-kB transcription
factors that play a key role in immune system. Aim of our
study was to find the potential role of CD150 in regula-
tion of NF-kB transcription factors function in normal
human B-cells. Methods: Dense human tonsillar B cells
were obtained in Percoll gradient, were stimulated via
CD150 cell surface receptor, followed by immunofluo-
rescence staining with 3D deconvolution and Western
blot analysis. Results: In dense B cells that comprise
138 Experimental Oncology 35, 128–152, 2013 (June)
IgD+ naive cells p65 was predominantly localized in cy-
toplasm and p50 — in the nucleus, while several p65/
p50 heterodimers were observed in nucleus, but not
in cytoplasm. After 90 min of CD150 ligation p50 was
also detected in cytoplasm. Stimulation of dense B cells
via CD150 for 16 h resulted in complete translocation
of p50 and p65 from nucleus to the cytoplasm. Further
cell stimulation via CD150 up to 24 h resulted in return-
ing only p50 back to the nucleus, although, a high level
of p50 was still observed in cytoplasm where it was co-
localized with p65. Western blot analysis has shown that
after 24 hours of CD150 crosslinking on dense B cells, the
expression level of p50 did not change, however, p65 lev-
el was slightly enhanced. The expression level of IkBα
was significantly increased after 24 hour of CD150 liga-
tion that points on the role of IkBα in p65 and p50 subunits
re-exportation from the nucleus and sequestering in the
cytoplasm. Conclusions: CD150 is involved in regulation
of gene expression in B cells via intracellular relocalization
of both p50 and p65 and minor upregulation of p65 ex-
pression level. Thus CD150 may also contribute to the
regulation of NF-kB1 activation in CD150 expressing
malignant B lymphocytes.
MOLECULAR-GENETIC MARKERS FOR
DIAGNOSIS AND PROGNOSIS IN PEDIATRIC
SOLID MALIGNANCIES
N.M. Khranovska, N.V. Ionkina, N.M. Svergun,
M.V. Inomistova, G.I. Klymnyuk
National Cancer Institute, Kyiv, Ukraine
nkhranovska@ukr.net
Introduction: Molecular-genetic markers are very
important tools for diagnosis and prognosis of solid
tumors in children. Aim: To study chromosomal trans-
locations in patients with pediatric solid malignancies.
Patients and Methods: Study has been performed
on 279 tumor specimens, which were collected
at diagnosis, and 90 specimens of bone marrow (BM)
puncture biopsy from 200 patients with neuroblas-
toma (NB), 21 — Ewing sarcoma family tumors (ESFT),
29 — alveolar rhabdomyosarcoma (ARMS), 29 — em- (ARMS), 29 — em-(ARMS), 29 — em-ARMS), 29 — em-), 29 — em-
bryonal RMS (ERMS). The study protocol was approved
by Ethical Committee permission of National Cancer
Institute (Kyiv, Ukraine). Chromosomal translocations
were detected by fluorescence in situ hybridization
(FISH) or real-time PCR on fresh or paraffin-embedded
tissue samples. Results: Chromosomal translocation
t(2;13)(q35;q14) exhibiting chimeric PAX3-FKHR and
PAX7-FKHR gene products are the hallmarks of the
ARMS. PAX-FKHR fusions were detected in 58 RMS
samples: 10 (17,3%) had PAX3-FKHR and had 24 (41,4%)
PAX7-FKHR fusions, 24 (41,3%) cases were fusion-
negative. PAX3-FKHR or PAX7-FKHR fusions were found
in all ARMS. PA3/7-FKHR was detected in 5 of 29 patients
with eRMS; it indicates the type of mixed type RMS
and requires a change of tactics on the protocol for
the treatment of ARMS. In addition, PAX3-FKHR fusion
products were detected in biopsy tissue and BM from
3 patients. ESFT is associated with chromosomal
translocation t(11;22)(q24;q12). Out of the 21 samples
with translocations detected, 16 (76.3%) had EWS-
FLI1 type 1, 1 (4.7%) had EWS-FLI1 type 2, 2 (9.5%) had
EWS-ERG, 2 (9.5%) had EWS-ETV4 translocation. Fusion
transcript EWS-FLI1 type 1 was found in BM of 6 patients.
The most significant abnormality in NB is amplification
of proto-oncogene MYC; its locus lies on the short arm
of chromosome 1. In 44 (22%) cases of primary NBs
amplification of MYCN gene is present. MYCN gene
is a factor of unfavourable prognosis and influences the
choice of treatment schedules. One sample with MYCN
amp pos and 1p36 del pos, and 3 samples in MYCN amp
neg and 1p36 del pos were detected. Loss of hetero-
zygosity (LOH) at 1p36 was independently associated
with a worse outcome in NB patients. NB expresses
the tyrosine hydroxylase (TH) mRNA, the first enzyme
in the catecholamine pathway. TH expression was found
in BM of 74 (37%) patients with NB. Molecular-genetic
testing is more accurate than standard morphological
methods; it enables detection of the presence of tumor
cells in BM at diagnosis and during treatment, allows
optimization of the treatment strategy. Conclusion:
Molecular-genetic analysis helps to identify specific
genes in children with solid malignancies in order
to specify the diagnosis, prognosis; it also influences the
selection of at-risk patients and treatment monitoring.
NUCLEIC ACID MARKERS IN BLOOD PLASMA
OF PATIENTS WITH COLORECTAL CANCER
A.G. Kondratov1, K.A. Nekrasov1, L.V. Lototska1,
G.V. Panasenko1, L.A. Stoliar1, O.O. Kolesnyk2,
I.B. Shchepotin2, A.V. Rynditch1, V.I. Kashuba1
1Institute of Molecular Biology and Genetics, NAS
of Ukraine, Kyiv, Ukraine
2National Cancer Institute, Kyiv, Ukraine
o.g.kondratov@imbg.org.ua
Introduction: Colorectal cancer (CRC) is the third
commonly diagnosed cancer that causes 400 000 deaths
worldwide. The most sensitive modern diagnostic tool
of CRC is colonoscopy, which is a painful procedure
and can not be recommended for patients with altered
topography of colon. Aim: Development of less invasive
tools for CRC screening based on detection of nucleic
acids in blood plasma. Methods: Concentration of cell-
free circulating DNA in blood plasma was analyzed
by qPCR. Methylation status of LRRC3B, APC, FHIT
and HIC1 genes in cell-free circulating DNA of samples
was determined by using methyl-specific PCR (MSP)
with subsequent melting curve analysis. Results: It was
shown that mean-value of cell-free blood plasma DNA
is statistically higher in CRC patients than in healthy do-
nors (p < 0.01). Thus the mean-value of concentration
of cell-free circulating DNA in blood plasma of CRC pa-
tients was 17.57 ± 3.43 ng/mL and 7.07 ± 0.84 ng/mL —
in healthy donors. We have revealed hypermethylation
of APC, FHIT and LRRC3B genes in 45% (10/22), 73%
(16/22) and 68% (15/22) of tumor samples, respectively.
Altogether hypermethylation of at least one of the se-
lected genes was detected in 95% (21/22) of samples.
Using MSP with subsequent melting curve analysis
we have detected methylated fragments of APC, FHIT
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 139
and LRRC3B genes in blood plasma of 29% (6/21), 19%
(4/21) and 14% (3/21) of CRC patients, respectively.
We have identified hypermethylation at least in one of the
selected genes in 48% (10/21) of blood plasma samples
of CRC patients. Additionally we have registered high
frequency of HIC1 hypermethylation in blood plasma
of CRC patients (8/10). We have suggested that two
stage verification might be applied for CRC screening,
which includes measurement of cell-free circulating DNA
concentration with following detection of methylated
fragments of APC, FHIT and LRRC3B genes in blood
plasma of CRC patients. Overlapping of the above
mentioned approaches allowed to increase sensitivity
of studied panel up to 71% (15/21) in CRC detection.
Conclusion: We have deve loped approach for screen-
ing of CRC which is based on determination of cell-free
DNA and methylated fragment of well-known tumor
related genes APC, FHIT, LRRC3B in blood plasma. The
sensitivity of CRC detection might be increased by using
of additional perspective genes like HIC1.
COMPLEX INFORMATION SYSTEM WITH
WEB-INTERFACE CONCERNING BREAST
CANCER
T.V. Pyatchanina, A.O. Koval, O.S. Dvorshchenko,
M.B. Narolsky
R.E. Kavetsky Institute of Experimental Pathology,
Oncology, and Radiobiology of the National
Academy of Sciences of Ukraine, Kyiv, Ukraine
koval_anet@mail.ru
Introduction: Breast cancer (BC) is the most
frequent cancer among women with an estimated
17232 new cancer cases diagnosed in 2010 in Ukraine
(19.9% of all cancers among women). As a result
BC ranks as the first cause of death from cancer among
women. BC is an actual social economical problem
in Ukraine due to high rates of mortality and disability
of women of active working age and requires significant
funding. Economic situation in Ukraine gives priority
to the prevention of BC, its most important component —
increasing awareness of the public. Today a significant
amount of information about BC has been accumu-
lated and solution of the problem can be accomplished
through the use of up-to-date tools of information, such
as electronic information technologies that offer quick
access to large amounts of information. The primary
tool of obtaining this information is a global Internet net-
work, but a significant amount of different information
makes the problem of choosing the right materials more
complicated. So there is a need for systematization and
generalization of data to create a complex information
system concerning BC. Aim: To develop the Ukrainian
language information system about prevention, diag-
nosis and treatment of BC aimed on informing and self-
education of Ukrainian population as well as specialists
in the medical field. Results: Technical specification for
creation of a web-portal (conceptual, logical and physi-
cal design), software development, portal and template
design, is developed. The site is filled up with thematic
material content according to the designed structure.
Created web-resource contains information concern-
ing BC, which uses brunched structure of the site and
satisfies the information needs of the target audience —
specialists in the field of medicine and ordinary citizens.
Section of the site target audience, which defines the
general population of Ukraine, is devoted to various
aspects of BC in women such as questions disclosing
BC risk factors, symptoms and diagnosis, primary pre-
vention, clinical classification of tumors and treatment.
Module that contains data appointed to provide informa-
tion to medical specialists reflects the modern aspects
of the pathogenesis of hormonal and molecular-genetic
features of BC as a result of new post-genomic research.
Conclusion: Complex Ukrainian language information
system concerning nature, early symptoms, preven-
tion, diagnosis and treatment of BC as well as providing
medical specialists with the latest structured scientific
data about molecular biology of BC, has been created.
SIGNIFICANCE OF COX-2 AND VEGF
EXPRESSION IN TUMOR FOR ESTIMATION
OF TREATMENT RESULTS IN CERVICAL
CANCER STAGE IIB
A.Y. Kryzhanivska1, A.V. Andriyiv1,
N.Yu. Lukianova2
1Ivano-Frankivsk National Medical University,,
Ivano-Frankivsk, Ukraine
2R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
anna.nivska@gmail.com
Introduction: One of the important tasks of mod-
ern oncogynecology is the estimation of biological
characteristics of tumors. Among the biological mark-
ers studied in relation to cervical cancer, cyclooxygen-
ase-2 (COX-2) and vascular endothelium growth factor
(VEGF) play an important role in the processes of the
tumor invasion and neoanginogenesis. Aim: To deter-
mine the COX-2 and VEGF expression in tumors and
their connection with clinico-morphological indicators
of the efficiency of treatment in patients with cervical
cancer. Patients and Methods: Operation materi-
als of 48 patients with cervical cancer stage IIB after
chemotherapy was used. The immunohistochemical
analysis of COX-2 and VEGF expression was per-
formed using primary monoclonal antibodies (MCAB)
of the “Diagnostic BioSystems” company, USA (clone
4H12) and “DakoCytomation”, Denmark (clone VG1)
on the paraffin sections of the tumors. Results: A posi-
tive immunohistochemical reaction of specific MCAB
to COX-2 was determined in 44 (92%) tumors, and
to VEGF — in 42 (88%). It was shown that tumors with
partial regression (PR), stabilization of the tumor pro-
cess degree II of pathomorphosis after the treatment
and the disease recurrence are characterized by high
levels of the COX-2 and VEGF expression. Tumors
with PR and with degree III of pathomorphosis after
the treatment and disease recurrence are character-
ized by high levels of the COX-2 and VEGF expression
140 Experimental Oncology 35, 128–152, 2013 (June)
but of low intensity. In patients with complete tumor
regression after the treatment and without cervical
cancer recurrence for more than 5 years no COX
-2 and VEGF expressions were found. Conclusion:
The results indicate that determining of the COX-2 and
VEGF expression in tumor cells after treatment of pa-
tients could be considered as additional indexes for
estimation of treatment efficiency and the prognosis
of the cervical cancer course.
A COMPARISON OF MULTIPLEX SHORT
TANDEM REPEAT PCR AND REAL-TIME PCR
INSERTION/DELETION POLYMORPHISMS
METHODS FOR QUANTIFICATION
OF CHIMERISM AFTER HEMATOPOIETIC
STEM CELL TRANSPLANTATION
V. Lavrinenko, T. Savitskaya, Y. Mareika
Belarusian Research Сenter for Pediatric Oncology,
Hematology and Immunology, Minsk, Belarus.
viallav@mail.ru
Introduction: Quantitative monitoring of chime-
rism after allogeneic hematopoietic stem cell trans-
plantation (HSCT) by molecular methods has be-
come an indispensable diagnostic tool in detection
of engraftment/graft failure, predicting rejection and
disease relapse. Despite the great utility of chimerism
analysis there is no unique standard method for its
quantification. Aim: The objective of the present inves-
tigation was to compare the sensitivity (detection limit)
and the quantification accuracy of two perspective
methods: multiplex short tandem repeat polymerase
chain reaction (STR-PCR) and real-time PCR insertion/
deletion polymorphisms (InDel-PCR) for the quanti-
fication of chimerism after stem cell transplantation.
Methods: We performed a perspective study analyz-
ing the chimerism status in 46 patients by STR-PCR
using AmpFlSTR SGM Plus PCR Amplification Kit (ABI,
UK) with capillary electrophoresis and by InDel-PCR
with primers and probes to 20 allele-specific markers
and reference gene albumin. Results: Recipient-
donor discrimination was possible with STR-PCR
in all patient-donor pairs (100%), whereas informative
alleles for recipient were found in 94% pairs with InDel-
PCR. The sensitivity (detection limit) of STR-PCR was
1–5% donor DNA with variation among STR markers.
The sensitivity of InDel-PCR was more than 0.01%
donor cells. The accuracy of quantification was higher
for InDel-PCR than for InDel-PCR, when level of donor
chimerism was <5% or >95% as it had higher sensi-
tivity. The accuracy of quantification was higher for
STR-PCR than for InDel-PCR, when level of chimerism
was >10%, because there was 0.5 threshold cycle er-
ror for InDel-PCR and this corresponded to a variation
in DNA of 50%. The results obtained by two methods
showed a good agreement and correlated well (r=0.98;
p<0.0001). Conclusions: These methods can be suc-
cessfully used to determine chimerism after allogeneic
HSCT. Considering the higher sensitivity and quanti-
fication accuracy of InDel-PCR it should be chosen
if donor chimerism level is less than 5–10% or more
90–95% (i.e. after myeloablative conditioning) and
in other cases STR-PCR should be chosen.
MATRIX METALLOPROTEINASES 2 AND
9 ACTIVITY AND TUMOR-ASSOCIATED
MACROPHAGES IN HUMAN GASTRIC
CANCER: CORRELATION WITH METASTASIS
L.A. Mamontova1, L.D. Gumenyuk1,
S.P. Merentsev2, I.I. Ganusevich1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
2City Clinical Oncological Center, Kyiv, Ukraine
mamontova.lesya@bk.ru, osion@onconet.kiev.ua
Introduction: Matrix metalloproteinases-2 and
9 (MMP-2 and -9) participate in the degradation
of extracellular matrix (ECM) in tumor that results
in the invasion, neoangiogenesis and metastasis.
It is also known that tumor-associated macrophages
(TAM) are important source of MMPs. Aim: To study
the interrelationship between TAM number (CD68-
positive cells) and concentration of MMP-2 and
-9 active forms in tumor tissue as well as their link
with clinicopathologic characteristics, metastasis into
lymph node (LN) and survival of patients with gastric
cancer (GC). Material and Methods: 112 resected
specimens of primary gastric cancer were used.
CD68 (tumor-associated macrophages) expression
was evaluated using immunohistochemistry, and
MMP-2 and MMP-9 activity, zymography. Statistical
analysis was conducted using Spearmans’s test,
Kaplan — Meier survival analysis, log-rank test and
Cox proportional hazards model. All patients were
thoroughly informed about the study, which was ap-
proved by the local Ethics Committee. Results: It was
shown that TAM number in GC categorized as N1–2 and
M1 was significantly higher than that in GC categorized
as N0 and M0 (p < 0.05). Correlation of concentration
of MMPs active forms in GC categorized as N0 and
N1–2 was not statistically significant. At the same
time MMP-2 activity was higher by 1.5-fold in tu-
mors categorized as N1 in comparison with those
in tumors categorized as N0 (p < 0.05). Moreover,
the inverse correlation between tumor concentration
of MMP-2 active form and M category was shown:
concentration of MMP-2 active form in tumor was
higher by 2-fold in patients without distant metas-
tasis (p < 0.05). The positive correlation between
TAM number and tumor concentration of active
form of both MMP-2 and MMP-9 was observed
(rho = 0.4 and rho = 0.51, respectively; p < 0.05).
Overall survival (OS) of patients with concentration
of active form of MMP-2 (< 2 μg/g) was signifi-
cantly better than that in patients with higher activity
of MMP-2 in tumor (p=0.004). It was also shown that
risk of unfavorable outcome increased by more than
a factor of 3.5 (hazard ratio 3.8, (95% CI 0.78–8.2;
p < 0.05) in patients with high concentration of ac-
tive MMP-2 in tumor. OS of patients with concentra-
tion of active form of MMP-9 (< 4.5 μg/g) was also
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 141
significantly better than that in patients with higher
activity of MMP-9 in tumor (p=0.015). Hazard ratio
for patients with MMP-9 activity of tumor > 4.5 μg/g
(HR 4.7, 95% CI 0.83–7.7; p<0.05) was significantly
higher than that for patients with MMP-9 activity
< 4.5 μg/g. The survival rate of patients with high
number of TAM in tumor tissue (number of CD68-
positive cells > 23%) was significantly lower than
that of those with a low TAM number (p = 0.003).
Risk of unfavorable outcome in patients with TAM
number higher than the median value (> 23%) in-
creased by more than a factor of 2 (HR=2.4; 95%
CI 0.86–7.25; p < 0.05). Conclusion: It was sug-
gested that TAM number and concentration of active
form of MMP-2 and MMP-9 in gastric cancer may
be used to the disease prognosis.
GENE EXPRESSION OF ACTIVATING
TRANSCRIPTION FACTOR 5 AS A PREDICTOR
OF SURVIVAL IN GERMINAL CELL DIFFUSE
LARGE B-CELL LYMPHOMA
A. Martynchyk, I. Kriachok
National Cancer Institute, Kyiv, Ukraine
hematology@unci.org.ua
Introduction: Prognostic models based on cur-
rently used methods of prediction of the outcome
in diffuse large B-cell lymphoma (DLBCL) do not
identify the molecular basis of clinical heterogeneity.
Aim: To identify new independent predictor of survival
in germinal B-cell like subtype (GCB) of DLBCL. Pa-
tients and Methods: Analysis was performed on RAW
data from 203 DLBCL samples from a previously
stu died cohort of patients treated with CHOP (Se-
ries 11318) using Partek Genomics Suite software
(methods of regression analysis and Cox-model for
prognostic significance of studied parameters). Study
was conducted according to bioethical standards
of National Cancer Institute (Kyiv, Ukraine). Only
GCB samples were choosing for the next analysis.
According to the IPI score 71 GCB samples were
divided into two groups: with “high” IPI (3–4 risk
factors, 14 samples) and “low” IPI (0–2 risk factors,
50 samples). Detection of differentially expressed
genes was performed (IPI high vs low IPI). Gene list
was created (fold changes < -1.3, > 1.3, p-value with
FDR < 0.05). 37 differentially expressed genes were
detected. Highest fold-change was in gene activating
transcription factor 5 (ATF5). The medium expression
of this gene in GCB subtype of DLBCL was 5.42 and
the expression was higher in high IPI samples vs low
IPI (7.5 vs. 4.9 respectively). GCB samples were di-
vided into two groups depending on APF5 expression.
In Kaplan — Meier survival analysis the probability
of survival was better for patients with low expression
of ATF5 compared to high expression. Results: Higher
expression of ATF5 was observed in patients from high
risk group (IPI score 3–4) (p-value with FDR 0.05)
and the proba bility of survival was less for this group
of patients treated with CHOP, especially after one
year of follow up. Conclusions: ATF5 could be a useful
prognostic factor for DLBCL, but its significance need
to be verified in further studies.
ABERRANT EXPRESSION OF IKZF1 GENE
IN BLAST CELLS OF CHILDHOOD ACUTE
LYMPHOBLASTIC LEUKEMIA
A. Meleshko, I. Prakharenia, S. Doronina
Belarusian Research Center for Pediatric Oncology,
Hematology and Immunology, Minsk, Belarus
meleshko@tut.by
Introduction: Modern molecular-genetic methods
of acute lymphoblastic leukemia (ALL) research re-
vealed alterations of a number of novel genes involved
in pathogenesis. The most frequent are transcription
factors of lymphoid differentiation, such as pax5,
EBF-1, IKZF1 (Ikaros), IKZF2 (Helios), IKZF3 (Aiolos).
Ikaros, coded by IKZF1 gene, is a key factor of early
lymphocyte development. Knock-out of IKZF1 leads
to high frequency of lymphoid malignances and
autoimmune disorders. The Ikaros protein contains
4 DNA-binding zinc fingers forming DNA-binding do-
main near the N-terminus. The IKZF1 gene contains
8 exons and is transcribed as at least 16 isoforms due
to alternative splicing. Long isoforms (Ik1–3) have
at least three zinc fingers, which are able to bind DNA
and are considered to be functional. Short isoforms
(Ik6,8,9,0) lack two or more zinc-finger domains and
impair the function of Ikaros proteins in a dominant-
negative manner (DN-Ik). Aim: Estimation of Ikaros
isoforms expression by RQ-PCR in bone marrow
(BM) blast cells of primary and relapsed childhood
ALL patients in comparison with mononuclear cells
of healthy donors’ BM. Methods: We developed panel
of primers and probes for independent quantitative as-
sessment of all isoforms expression. Deletion of exon
3–6 was examined at the genomic level by PCR. Re-
sults: 132 BM samples from 104 ALL patients were
analyzed, including 43 relapses and 27 paired cases.
9 BM from healthy donors composed a control group.
Profile of Ikaros expression was similar in controls and
most patients. Levels of Ikx, Ik2, Ik4 and Ik8 isoforms
were significantly lower in patients. In 18 (27.3%),
3 (4.5%) and 1 (1.5%) out of 66 cases aberrant
overexpression of Ik6, Ik9 and Ik0, respectively, was
found. In 13 of 18 (72%) cases DN-Ik overexpression
was detected in relapsed and primary patients who
finally relapsed. Deletion in IKZF1 gene was found
in 11 of 103 (10.7%) cases. In 24 cases both deletion
and Ik-DN expression was absent, and in 4 both fea-
tures were present. In 9 cases Ik6 was overexpressed
in the absence of deletion. Pair cases at diagnosis
and relapse displayed the same Ikaros status. Con-
clusions: Expression of DN-Ik is a relatively frequent
event for ALL. It may be detected by RQ-PCR analysis
of cDNA apart from genomic analysis and possibly
associated with poor prognosis. Ik6 expression
may be caused not only by the exon 3–6 deletion
in IKZF1 gene. Ik6 overexpression is hardly the factor
of relapse, but rather it is an initial event at the time
of de novo diagnosis.
142 Experimental Oncology 35, 128–152, 2013 (June)
CREATION OF NEW COMBINATIONS
OF ANTITUMOR REMEDIES (EXPERIMENTAL
STUDY)
O. Khavych, N. Sharykina, T. Grygorieva,
N. Meshkova, V. Zakharenko
State Institution “Institute of Pharmacology and
Toxicology of NAMS of Ukraine”, Kyiv, Ukraine
Hamedall@bigmir.net
Introduction: Development of a new combination
of antitumor remedies replacing one or two compo-
nents in well-known combinations by preparations with
more selective antitumor activity is one of approaches
to improve effectiveness of cancer pharmacotherapy.
Aim: To create new combinations of antitumor drugs
on a basis of initial combinations from standard treatment
protocols for cancer replacing cyclophosphamide, fluo-
rouracil and bonefos by chlofiden, fludinat and mebifon,
which were developed in SI “IPT NAMSU”. Materials
and Methods: Toxicological and histological meth-
ods and method of tumor allografts were used. Mela-
noma B16 and Lewis lung carcinoma (3LL) were used
as tumor models. Drugs were injected intraperitonially
24 hours after tumor implantation. Drug dosage for mice
based on single dose for human was calculated using
rapid extrapolation method. Results: The substitution
of phosphorylated chlorethylamine chlofiden and fludi-
nat for traditional combinations of cyclophosphamide
and fluorouracil restored high antitumor activity of initial
combinations but reduced their toxicity (chlofiden does
not induce nephro- and hepatotoxicity, unlike cyclophos-
phamide; toxicity of fludinat is 3.3 fold lower than that
of fluorouracyl). Thus, new remedies have more selec-
tive antitumor action. In melanoma B16, combination
of doxorubicine, 3.0 mg/kg, cisplatine, 3.0 mg/kg and
cyclophosphamide, 30.0 mg/kg, caused 80.5% of tumor
growth inhibition, while replacement of cyclophospha-
mide by chlofiden, 30.0 mg/kg, in this combination
enhanced tumor inhibition up to 85.9%. Doxorubicine,
3.0 mg/kg, cyclophosphamide, 30.0 mg/kg and fluoro-
uracil, 37.0 mg/kg caused 90.2% of 3LL growth inhibition,
meanwhile the same combination with fludinat, 37.0 mg/
kg, instead of fluorouracil showed 85.9% of 3LL growth
inhibition. Toxicity of mebifon and bonefos is similar, but
mebifon showed higher antitumor activity than bonefos
(72.0% and 37.4% of B16 growth inhibition respectively).
Combination of dacarbazine, 60.1 mg/kg, cisplatin,
1.5 mg/kg, vinblastine, 0.2 mg/kg and bonefos, 14.0 mg/
kg inhibited growth of melanoma B16 by 81.7%, the
substitution of mebifon, 14.0 mg/kg, for bonefos resulted
in 91.6% of B16 growth inhibition (10% higher than initial
combination). Conclusions: On the basis of combina-
tions from standard cancer treatment protocols of NCI
of Ukraine, there were developed combinations of an-
ticancer remedies with replacement of cyclophospha-
mide, fluorouracil and bonefos by chlofiden, fludinat and
mebifon. Combinations of chlofiden and fludinat had the
same antitumor activity and were less toxic than standard
combinations. The replacement of bonefos by mebifon
enhanced antitumor activity of initial combination.
THE ROLE OF PRO- AND ANTIOXIDANT
PROCESSES IN DNA DAMAGE
ORIGINATING FROM COMBINED EFFECTS
OF ENVIRONMENTAL FACTORS AND TUMOR
GROWTH
I. Muzalov, O. Ganzha, V. Mikhailenko
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
imuzalov@mail.ru
Introduction: Influence of environmental factors
plays a crucial role in the formation and development
of cancer. The peculiarity of combined action of adverse
factors on the human organism is the ability of each
factor to modify the overall pathogenic effect. Nitro-
gen oxides (NOx) and low doses of ionizing radiation
(LDIR) are genotoxic factors. Their interaction can lead
to increased levels of DNA damage, changes in cell
response to stressors, alterations in microenvironment
of cells in tissues, the development of nitrosative and
oxidative stress, resulting in increase of carcinogenic
risk. At present, genotoxic effect of combined influ-
ence of environmental factors on organism upon tumor
growth is studied insufficiently. In this regard, investiga-
tion of the antioxidant system status and the develop-
ment of genetic instability upon the tumor growth and
combined action of exogenous NOx and LDIR is impor-
tant. Aim: To investigate changes in DNA damage and
dynamics of the activity of superoxide dismutase (SOD)
and catalase (CAT) in the process of tumor growth
in the conditions of the individual and combined effect
of exogenous NO and LDIR. Methods: The formation
of DNA strand breaks was determined using horizontal
gel electrophoresis of isolated rat’s peripheral blood
lymphocytes (PBL). The object of study were PBL of rats
exposed to NO inhalation (150 mg/m3 of air) for 30 days
and LDIR (10-fold by 0.1 Gy) in the conditions of indi-
vidual and combined treatment. Changes in CAT and
SOD activity in rat peripheral blood were investigated
in parallel. Guerin carcinoma (GC) was used as a tu-
mor model. Study was approved by Ethical Committee
permission of IEPOR NASU (Kyiv, Ukraine). Results:
GC growth was accompanied by decrease of CAT ac-
tivity in the blood of rats after 12 days of tumor growth.
CAT activity increased both upon single LDIR and joint
influence of NO+LDIR and reached its maximum upon
combined action. The activity of SOD in blood increased
in the conditions of the action of LDIR and NO+LDIR,
and also upon the GC development. GC inoculation
after treatment with NO+LDIR significantly increased
the SOD activity on the 12 day followed by normalization
on the 18 day of tumor growth. However, the increased
activity of antioxidant enzymes was insufficient in the
conditions of oxidative and nitrosative stress. Individual
action of NO and LDIR resulted in DNA damage gradual
increase during tumor growth. Maximal genotoxic effect
was observed in the case of NO and LDIR combination
(4-fold excess of the control value). Conclusions: Pre-
liminary impact of NO and/or LDIR resulted in persistant
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 143
additive genotoxic effect accompanied by the increase
of genetic damage on background of GC growth after
the direct action of these factors. Tumor development
and combined effect of environmental factors of differ-
ent nature were accompanied by complex interactions
between the main enzymes of antioxidant protection —
CAT and SOD. An imbalance of free radical processes
under oxidative and nitrosative stresses lead to the
development of genetic instability, which in turn is a fac-
tor of increased carcinogenic risk and implemented
as accelerated tumor growth
THE CLINICAL EFFICACY OF DRUGS OF
“ERBISOL” CLASS IN THE TREATMENT
OF COLORECTAL CANCER PATIENTS WITH
LIVER METASTASES
O. Nikolaenko1, V. Zhylchuk2,3, G. Maksim’yak2,
Yu. Chyshkevich2, O. Sichkoriz3,
N. Bezdenezhnykh4, Yu. Kudryavets4
1Scientific-Production Center “Erbis”, Kyiv,
Ukraine
2Rivne Region Oncology Hospital, Rivne, Ukraine
3Danylo Halytsky Lviv National Medical
University, Lviv, Ukraine
4R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine
kudryavets@mail.ru
Introduction: In recent years the incidence
of colorectal cancer (CRC) is significantly increased
in all countries, including Ukraine. The problem is CRC
metastasis in the liver at the time of diagnosis in 25–30%
of patients. Aim: To improve the efficiency of treatment
of CRC patients with liver metastases using drugs of
“ERBISOL” class (Kyiv, Ukraine). Patients and Meth-
ods: The study included 111 CRC patients with liver
metastases at the disease stage T1–4N0–2M1. All pa-
tients underwent surgical treatment. The patients were
divided into 2 groups: group 1 (control, 54 patients) who
underwent treatment by Roswell-Park scheme; group
2 (57 patients), which, on chemotherapy Roswell-Park
scheme held 18 cycles of treatment with Erbisol Ultra-
pharm.. The study protocol was approved by Ethical
committee. Results: According to preliminary research,
Erbisol Ultrapharm is a metabolic antimutagen, its use
helped to reduce the number of multiaberrant and an-
euploid cells, also Erbisol Ultrapharm exhibits properties
of natural cytostatic agent that inhibits the abnormal cell
growth. The analysis of the clinical course of the disease
showed that in patients of group 2 general condition has
been improved without local and general side events,
and allergic reactions. In group 2, stabilization of the
disease occurred in almost half of cases, compared with
control patients where the progression of the disease was
recorded in two thirds of patients. Additional biotherapy
with Erbisol showed improvement in clinical response and
duration of stable disease (13.5 months ± 1.0 in the main
group, 7.4 ± 1.1 months in the control group, p <0.05);
and the median duration of effect (13.3 ± 1.0 months
in the main group, 7.0 ± 1.1 months in the control group,
p < 0.05). The trend for improved clinical effect was
observed in patient survival rate. Conclusion: The use
of drugs of “Erbisol” class in the combined treatment
of CRC patients is a new and promising approach improv-
ing treatment effectiveness. Combined therapy of CRC
patients with liver metastases using 5-FU and drugs of
“Erbisol” class prolongs duration of disease stabilization
and increases patient survival (p < 0.05).
MOLECULAR GENETIC STUDIES IN PATIENTS
WITH CANCER AND BENIGN TUMOR OF THE
FEMALE REPRODUCTIVE SYSTEM FROM THE
FAMILIES WITH HISTORY OF CANCER
O. Paliychuk1, V. Chekhun1, N. Gorovenko2,
Z. Rossokha3, L. Polischuk1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
2P.L. Shupik National Medical Academy of Post-
Graduate Education, Kyiv, Ukraine
3Reference-Centre for Molecular Diagnostic, Ministry
of Public Health of Ukraine, Kyiv, Ukraine
oncology@2upost.com
Introduction: Screening of single nucleotide poly-
morphisms (SNP) allows determining the genetic pre-
disposition to various multifactorial diseases and may
help to predict individual sensitivity to pharmacological
agents. Aim: To conduct clinical-genealogic, molecu-
lar-genetic studies in patients with ovarian cancer (OC)
and / or breast cancer (BC), benign tumor of the female
reproductive system from the families with cancer his-
tory. Patients and Methods: The clinical-genealogic
study of 45 patients aged 25–72 years was carried out.
19 (42.2%) patients had cancer of the female repro-
ductive organs (OC, BC, stage I-II), 26 (57.8%) — be-
nign tumor of the uterus, ovary and breast. The genetic
analysis was used to identify BRCA1 (185delAG and
5382insC) and BRCA2 (6174delT) genes mutations
and ESR1 (T-397C, A-351G), Cyp2D6*4 (G1846A)
genes polymorphism in peripheral blood DNA of the
patients. Results: Among 19 OC and BC patients with
familial cancer 5382insC mutation in BRCA1 gene
was detected in 2 (10.5%) patients. Other mutations
in BRCA1/2 genes were not found. The -397C allele
(CC and CT genotypes) of ESR1 gene was detected
with the same frequency in patients with cancer and
benign tumor, respectively, 50.0 and 45.7% (p > 0.05).
The frequency of -351G allele (GG and AG genotypes)
of ESR1 gene was significantly higher in cancer
patients compared to patients with benign tumor,
respectively, 71.4 and 25.7% (p < 0.05). However,
the frequency of 1846A allele (GA and AA genotypes)
of Cyp2D6*4 gene was significantly higher in women
with benign tumor than in cancer patients, respectively,
17.1% and 0.05% (p < 0.05). Among cancer patients
we found more individuals (13–68.4%) with combi-
nation of two polymorphisms in investigated genes
in comparison with benign tumor patients (12–46.2%).
144 Experimental Oncology 35, 128–152, 2013 (June)
Conclusion: Analysis of BRCA1/2 genes mutations
and polymorphic variants of ESR1 gene among healthy
patients with familial cancer can be useful tool for ge-
netic risk estimation of cancer development.
ABERRANTLY METHYLATED CIRCULATING
DNA AS A TOOL FOR DIAGNOSTICS AND
EVALUATION OF THERAPEUTIC EFFECT
IN LUNG CANCER
A. Ponomaryova1, Е. Rykova2, N. Cherdyntseva1,
T. Skvortsova2, А. Dobrodeev1, A. Zav’yalov1,
S. Tuzikov1, V. Vlassov2, P. Laktionov2
1Cancer Research Institute of SB of the RAMS,
Tomsk, Russia
2Institute of Chemical Biology and Fundamental
Medicine of SB of the RAS, Novosibirsk, Russia
anastasia-ponomaryova@rambler.ru
Introduction: Cancer cell-specific aberrantly
methylated DNA was found in blood plasma from
cancer patients, indicating that cell-free DNA cir-
culating in blood (cirDNA) is a convenient source
of tumor-associated DNA markers for the minimally
invasive diagnostic tests. Aim: Estimation of the di-
agnostic and prognostic significance of methylation
changes in RARB2, RASSF1A tumor suppressor genes
detected in the cirDNA from lung cancer patients
before and after combined treatment. Methods:
Blood samples were taken from 33 healthy subjects
(HS) and 60 patients with non-small cell lung can-
cer (NSCLC) before treatment, after neoadjuvant
chemotherapy, surgery and during post-treatment
follow-up. CirDNA was extracted from blood plasma
and cell-surface-bound cirDNA (csb-cirDNA) fraction
which was obtained by successive treatment of blood
cells with PBS/EDTA and trypsin solutions. Concentra-
tion of methylated and unmethylated RASSF1A and
RARB2 tumor suppressor genes circulating in blood
was quantified by methylation-specific PCR and meth-
ylation index (IM) was calculated as IM = 100 х [copy
number of methylated /(copy number of methylated
+ unmethylated gene)] for plasma cirDNA and csb-
cirDNA. Results: Methylation index for RASSF1A,
RARB2 genes was elevated 2–3 fold in plasma cirDNA
and csb-cirDNA from lung cancer patients versus
HS (Mann — Whitney U-test, p<0.05). Exceeding
of at least one of RASSF1A or RARB2 IM values over
the selected cut-offs discriminates NSCLC patients
from healthy subjects with 90% sensitivity and 82%
specificity when plasma cirDNA and csb-cirDNA
were analyzed. The mean RARВ2 IM in csb-cirDNA
was higher for stage III patients compared with stage
I–II patients (p < 0.05). In post-surgery period most
patients had more pronounced decrease of RASSF1A,
RARB2 IM in both plasma cirDNA and csb-cirDNA
compared with IM values detected before treat-
ment and after chemotherapy. The increased level
of methylation markers was found in blood of patients
with recurrent NSCLC after treatment. Conclusions:
Detection of RARB2, RASSF1A methylation changes
in csb-cirDNA and plasma cirDNA of NSCLC patients’
blood appeared to be more informative in comparison
with analysis of plasma cirDNA only. The obtained data
provides evidence that RARB2, RASSF1A IM analysis
in the cirDNA is valuable for lung cancer diagnostics,
evaluation of cancer treatment efficiency and post-
treatment monitoring.
PECULIARITIES OF MODIFYING EFFECTS
OF CHEMICAL AGENTS ON THE FORMATION
OF RADIATION-INDUCED DAMAGE
IN GENETIC APPARATUS OF HUMAN CELLS
O. Pylypchuk, O. Demchenko, E. Domina
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
lena.pylypchuk@ukr.net
Introduction: Accumulation of chromosomal
aberrations in cells is potentially oncogenic; thus,
ionizing radiation (IR), which induces such changes,
is a carcinogenic factor. Combination of chemical
agents, IR with different mechanisms of action on hu-
man genome still has to be elucidated. Aim: To deter-
mine and provide a comparative analysis of combined
action of irradiation, co-mutagens, and mutagens
using cell culture of human peripheral blood lym-
phocytes (LPB). Materials and Methods: Culture
of LPB, metaphase chromosomes cytogenetic analy-
sis after ionizing radiation in the range of 0.3–2.0 Gy;
co-mutagen verapamil (V) treatment in different con-
centrations (1.5–4.0 μg/ml); mutagen — nitrozotiol
(GSNO) treatment as a transport form of nitrogen
oxide (0.25–1.0 mM/ml) (experiments in vitro,
two cell generations after irradiation). Results:
A phenomenon of co-mutagenesis, which stands for
combined action of radiation exposure (0.3–2.0 Gy)
and V treatment (1.5–4.0 μg/ml of blood), on the
level of chromosomal aberrations in PBL has been
established. It has been shown that V at concentra-
tion of 4.0 μg per ml of blood increased the damag-
ing effect of low doses of radiation up to 1.5-fold.
Frequency of chromosomal aberrations (radiation
markers) became higher with the increase of concen-
tration of co-mutagen V (1.5–4.0 μg/ml). Combined
treatment of PBL with IR and mutagen GSNO (two
cell generations after irradiation) resulted in syn-
ergistic effect which enhanced the additive effect
due to chromosomal aberrations, which are known
to be the genetic markers of chemical agents’ action.
Proliferative potential of PBL decreased more than
60% under IR and GSNO treatment. Level of aberra-
tions of the chromatid type rose with increasing con-
centrations of GSNO (0.5 mM to 1.0 mM) by 3-fold.
Combination of low doses of radiation and V inhibited
cell proliferative potential, but increasing of exposure
dose did not alter this effect. Conclusions: Increase
in concentration of co-mutagens in combination with
ionized radiation enhances chromosomal aberra-
tions in PBL. Moreover, proliferative potential of PBL
decreases under IR and GSNO treatment. Low doses
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 145
of radiation and V inhibit cell proliferative potential;
nevertheless, it remained unchanged when the doses
of radiation were increased.
COMPARATIVE ANALYSIS OF POLYMERASE
CHAIN REACTION AND CONVENTIONAL
CYTOLOGICAL EXAMINATION FOR
PERITONEAL RECURRENCE RISK
ASSESSMENT IN PATIENTS UNDERGOING
SURGERY FOR COLORECTAL CANCER
A. Revura, M. Fetsych
Department of Oncology and Radiology, Danylo
Halytskyy Lviv National Medical University, Lviv,
Ukraine
vezalij@gmail.com
Introduction: Peritoneal recurrence is a major
cause of surgical treatment failure in patients with
colorectal cancer (CRC). Diffuse metachronous
peritoneal carcinomatosis is associated with poor
prognosis and median survival of 5–6 months. There
is a chance for prolongation of survival in patients
with limited peritoneal carcinomatosis who are eli-
gible for aggressive combined treatment. Evaluation
of peritoneal relapse risk at a time of surgery for pri-
mary colorectal tumor is essential for early diagnosis
and treatment of metastasis. The presence of free
cancerous cells (FCC) within the peritoneal cavity
is believed to be a powerful predictor of peritoneal
carcinomatosis. A method of real-time polymerase
chain reaction (PCR) was proposed recently to detect
FCC in peritoneal washes. Aim: To evaluate the feasi-
bility of real-time PCR for detection of FCC within the
peritoneal cavity of patients with CRC who underwent
curative surgery and to compare the results with those
of conventional cytology. Patients and Methods:
Twenty-two patients were enrolled in this study with
ethical considerations. Patients were divided into
three groups. The main group consisted of 16 patients
radically operated for CRC. Positive control group
included three patients with macroscopic peritoneal
carcinomatosis diagnosed at a time of surgery for
CRC. Negative control group consisted of three pa-
tients with benign colorectal tumors. A laparotomy
and subsequent peritoneal lavage were performed
in all 22 patients. Peritoneal wash samples were ana-
lyzed microscopically by Giemsa staining and by PCR
analysis with primers for carcinoembryonic antigen
(CEA) gene as a molecular target of cancerous cells.
Real-time thermal cycler “Bio-Rad iCycler iQ5” (USA),
PCR reagent kits “Fermentas” (Lithuania) and oligo-
nucleotide primers “Metabion” (Germany) were used.
Cases with CEA signal were defined as PCR-positive.
Results: PCR reaction was positive in three (19%)
of 16 patients in the main group, while cytology was
negative in all samples. No positive results of PCR
and cytology in negative control group was detected,
i.e. both methods yielded no false-positive results.
Cytological examination was positive only in one
(33%) of three patients of positive control group
showing lack of sensitivity, whereas all (100%) of them
were PCR-positive. Conclusions: CEA identification
by PCR appears to be reliable method of FCC detec-
tion. It can be useful for identifying patients at high
risk of peritoneal relapses of colon cancer following
primary tumor resection. PCR is a more sensitive
method for FCC detection in peritoneal washes com-
pared to conventional cytology.
THE PREDICTIVE MODEL
OF CHEMOTHERAPY RESPONSE
IN MULTIPLE MYELOMA PATIENTS
N. Kostukova1, Z. Rossokha2, S. Kiryachenko2,
S. Vudyborets1, N. Gorovenko1
1P.L. Shupik National Medical Academy of Post-
Graduate Education, Kyiv, Ukraine
2Reference-Centre for Molecular Diagnostic,
Ministry of Public Health of Ukraine, Kyiv,
Ukraine
rossokhazoya@mail.ru
Introduction: Multiple myeloma (MM) is plasma
cell neoplasm with different sensitivity to drug treat-
ment. The drug resistance worsens the individual
prognosis for MM patients. Aim: To determine the
predictive markers of refractory forms in MM pa-
tients. Methods: 130 patients with first time diag-
nosed ММ from different regions of Ukraine were
examined during the period of 2007–2011. 26 (20%)
of 130 patients underwent treatment according
to the scheme МР, 55 (42.31%) — according to the
scheme М2, 49 (37.69%) — according to the scheme
VAD. Patients were divided into two groups: the 1st
group — 52 patients, who had no response to treat-
ment, the 2nd group — 78 patients with response
to treatment. Patients of both groups were scruti-
nized closely with a help of 68 clinical-laboratory
indexes and molecular-genetic tests, and GSTT1,
GSTM1 genes deletion polymorphism, GSTP1 gene
А313G polymorphism, MDR1 gene C3435T polymor-
phism were characterized. Binary logistic regression
with consequent including of predictors (program
SPSS 16.0) was implemented to create a predictive
model. Results: The patients of the 1st group had
significantly lower frequency of GSTM1 gene dele-
tion polymorphism as compared to patients of the 2nd
group (32.20% and 62.82%, respectively; χ2 = 11.33,
OR = 0.29 (0.14–0.60), p < 0.001). The GSTM1 gene
allele polymorphism correlated with elevated risk
of development of refractory forms in MM patients
(OR = 3.48 (1.66–7.29)). There was no difference
in the frequency of others genes polymorphism.
We have found that the higher prognostic value had
GSTM1 gene polymorphism (69.90%), while calcium
and α2-globulin level in blood serum before treatment
had lower prognostic value (62.30%). The better
prognostic value was obtained using statistical model
which includes GSTM1 gene polymorphism, α2-
globulin and calcium levels in blood serum (73.60%).
Conclusion: Implementation of predictive model
which considers GSTM1 polymorphism, α2-globulin
146 Experimental Oncology 35, 128–152, 2013 (June)
and calcium levels in blood serum from the beginning
of treatment increases the accuracy of prognosis
of response to treatment for each individual patient.
EXPRESSION OF CD44 AND Е-CADHERIN
IN THE SEROUS OVARIAN CANCER AND ITS
CLINICAL SIGNIFICANCE
N.Yu. Lukianova, O.D. Ryabtseva,
L.Z. Polishchuk, V.F. Chekhun
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
lu_na_u@rambler.ru
Introduction: Molecules of cell adhesion
(CD44 and E-cagherin) are significant for tumor
growth and are connected to changes of cell prolif-
eration and migration. Recently, it was hypothesized
that cancer stem cells (CSCs) express CD44 which
regulates cancer cell survival, metastatic potential,
resistance to conventional radio-chemotherapy,
disease relapse and prognosis. Aim: To evaluate
the interrelations of CD44 and E-cadherin expres-
sion with clinical and cytomorphological features
of serous ovarian cancer and its clinical significance.
Patients and Methods: Immunohistochemical and
morphological of CD44 and E-cadherin expres-
sion analysis was applied to pathological material
obtained after surgery of 72 patients with ovarian
cancer of I-III stage (FIGO classification) before
neoadjuvant chemotherapy. Monoclonal antibod-
ies against СD44 (clone DF 1485) and E-cagherin
(clone NCH-38) were used. Results of immunohis-
tochemical analysis were evaluated by semi-quan-
titative assessment, the percentage of CD44 (+)
and presence/absence of E-cadherin (+) cells were
estimated. Survival of patients was analyzed by Ka-
plan — Mayer method. Results: Variability in the
number, distribution, and location of CD44 (+) and
E-cadherin (+) tumor cells was observed in ovarian
cancer patients. Expression of CD44 was defined
in solid structures, and also in clusters of tumor
cells (budding) located in stroma, in cells of tumor
papilles, in adenomatous structures, and in gland
lumen which in total provides the evidence of cel-
lular discohesion and active invasion. The expres-
sion of CD44 (+) (over 10%) was observed in 58.3%
patients. The negative correlation between CD44 (+)
and E-cadherin (-) expression was determined
(r = -0.38 p < 0.05). Survival analysis of ovarian can-.05). Survival analysis of ovarian can-05). Survival analysis of ovarian can-Survival analysis of ovarian can-
cer patients showed that 5-year survival of patients
with tumor phenotype СD44 (+), Е-cadherin (-) was
lower compared to survival of patients with tumor
phenotype СD44 (-), Е-cadherin (+). Conclusion:
In serous ovarian cancer variability of СD44 and
E-cadherin expression was determined. СD44 ex- expression was determined. СD44 ex-expression was determined. СD44 ex-
pression in zones of cellular discohesion and active
invasion of tumor cells was shown. We demonstrated
the correlation between the percentage of CD44 (+)
cells and survival of ovarian cancer patients. The
results from this study suggest that quantification
of CD44 (+) and E-cadherin (-) expression and their
localization in ovarian cancer can be helpful as pre-
dictors of tumor progression.
THE ROLE OF SOME CYTOKINES AS A LOCAL
MICROENVIRONMENT IN BREAST CANCER
PROGRESSION
N. Semesiuk, N. Bezdenezhnykh, A. Lykhova,
Yu. Kudryavets
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
kudryavets@mail.ru, nadijka@inbox.ru
Introduction: Breast cancer (BC) is a leading
cause of cancer death among women. In most cases,
death results from the dissemination of cancer cells
and the development of distant metastases. Approxi-
mately 65–75% of patients with advanced breast
cancer will develop bone metastases, which result
in bone destruction. The presence of disseminated
tumor cells (DTC) in bone marrow (BM) of BC pa-
tients has been proven to have clinical relevance.
Despite the clinical significance of DTCs in the BM,
the biological relevance remains controversial.
Microenvironment plays an important role in tumor
progression both within primary tumor and distant
metastases in secondary sites. BM is a depot of vari-
ous growth factors. It is not excluded that increased
level of some cytokines may serve as the additional
factor of prediction progression of tumor growth,
because certain cytokines create the microenviron-
mental conditions for the establishment of distant
metastases from a primary BC. Therefore, a search
of new comprehensive prognostic algorithm, based
on simultaneous detection of DTC in BM and cytokine
profile in BC patients, should promote the develop-
ment of personalized therapeutic approaches. Aim:
The aim of this study was to detect DTC in BM and
determine an impact of cytokine status of peripheral
blood (PB) and BM of primary BC patients to can-
cer prognosis. Methods: Immunocytochemistry,
bioassay tests, ELISA, cultivation tests and statisti-
cal methods. Results: In our study DTC in samples
of BM was revealed in 50% breast cancer patients
of progression group. The level of TNF, CSF-1, IL-6,
TGF-β1, VEGF and IFN was analysed. It was found
that most significant additional markers of tumor pro-
gression with detection of DTC in BM are the levels
of TNF in BM and PB, of CSF-1 and IL-6 in PB and
of endogenous IFN in BM and PB of BC patients.
In patients of disease progression group in com-
parison with patients in remission group the levels
of TNF in BM were increased by 44% (p < 0.01),
of CSF-1 and IL-6 in PB — by more than on 40–60%
(p < 0.05). Conclusion: Comprehensive detection
of DTC in BM and identification of the levels of TNF,
IFN, CSF-1, VEGF, IL-6 and TGF- β1 in PB and
BM of BC patients could be one of the ways to predict
metastatic process and correct antitumor individual-
ized therapy.
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 147
CONNECTIONS OF SERUM AND TUMOR
FERRITIN LEVELS WITH SENSITIVITY
TO NEOADJUVANT CHEMOTHERAPY
IN BREAST CANCER PATIENTS
A.V. Shepil, N.Yu. Lukianova, V.F. Chekhun
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
shepilalex@gmail.com
Introduction: Design of new strategies for breast
cancer (BC) treatment is still one of the actual prob-
lems in oncology. The main phase of complex therapy
of locally disseminated BC stage II–III is neoadjuvant
chemotherapy. Nowadays different prognostic values
are being used in clinical practice, but objective criteria
of BC sensitivity to neoadjuvant chemotherapy, which
could give data about answer to chemotherapy on or-
ganism and tumor levels, are still unclear. Aim: To study
connections between ferritin levels in serum and tumor
tissue with sensitivity to neoadjuvant chemotherapy
in BC patients. Patients and Methods: We studied
168 women with BC stage IIА–IIIB. Age of patients ranged
from 28 to 69 years. Serum ferritin was measured by solid-
phase immunoenzyme assay after histological verifica- immunoenzyme assay after histological verifica-immunoenzyme assay after histological verifica- assay after histological verifica-assay after histological verifica- after histological verifica-after histological verifica-
tion of diagnosis before the first cycle of neoadjuvant
chemotherapy. Patients received FAC, AC chemotherapy
(2–6 cycles) every 21 days. Neoadjuvant chemotherapy
efficacy was evaluated every 2 cycles by mammography
according to RECIST criteria. Ferritin expression studies
were performed on operation material of BC patients
by standard immunohistochemical assay with specific
monoclonal antibodies. Statistical analysis was performed
using STATISTICA 6.0 software. Results: Patients were
divided into 2 groups according to the degree of the
clinical effect after neoadjuvant chemotherapy. The first
group consisted of 79 BC patients with positive reaction
on chemotherapy: complete tumor regression was ob-: complete tumor regression was ob-complete tumor regression was ob-
served in 8, partial — in 71 patients from this group. The
second group consisted of 89 women with tumors resis-89 women with tumors resis-women with tumors resis-
tant to chemotherapy. Among them 61 patients showed
stabilization of tumor growth and 28 — tumor progression
after neoadjuvant chemotherapy. The highest levels of se-. The highest levels of se-The highest levels of se-
rum and tumor ferritin were registered in group of patients
with tumors resistant to neoadjuvant chemotherapy.
In particular at the beginning of the therapy before the first
cycle of neoadjuvant treatment average ferritin concen-
tration in serum of these patients was 226.2 ± 5.7 ng/ml
(213.4 ± 4.8 ng/ml — in patients with stabilization and
254.2 ±6.2 ng/ml — in patients with progression of tumor
growth). At the end of treatment patients with resistant
tumors showed significant (p < 0.05) elevation of serum
ferritin levels (to 294.7 ± 7.3 ng/ml in patients with stabi-(to 294.7 ± 7.3 ng/ml in patients with stabi-to 294.7 ± 7.3 ng/ml in patients with stabi-294.7 ± 7.3 ng/ml in patients with stabi-.7 ± 7.3 ng/ml in patients with stabi-7 ± 7.3 ng/ml in patients with stabi-7.3 ng/ml in patients with stabi-/ml in patients with stabi-ml in patients with stabi-
lization and to 351.9 ± 5.5 ng/ml — in patients with tumor
progression). Most part (87%) of tumors from the resistant
group of patients showed elevated levels of ferritin expres-
sion in operation material. The highest levels of expres-. The highest levels of expres-The highest levels of expres-
sion of this protein (more then 92% of positive cells) was
observed in tumors with progression after neoadjuvant
treatment. Patients with tumors sensitive to neoadjuvant
therapy had slightly elevated average concentration
of serum ferritin before the first cycle of neoadjuvant che- before the first cycle of neoadjuvant che-before the first cycle of neoadjuvant che- the first cycle of neoadjuvant che-the first cycle of neoadjuvant che- first cycle of neoadjuvant che-first cycle of neoadjuvant che- cycle of neoadjuvant che-cycle of neoadjuvant che- of neoadjuvant che-of neoadjuvant che- neoadjuvant che-neoadjuvant che- che-che-
motherapy (93.1 ± 2.8 ng/ml: 56.8 ± 4.0 ng/ml in patients
with full and 97.2 ± 3.1 ng/ml in patients with partial tumor
regression). After treatment patients with sensitive tumors
showed significantly (p < 0.05) lower serum ferritin levels
(4.3 and 3.2 times for patients with full and partial tumor
regression, respectively). Average and low levels of fer-, respectively). Average and low levels of fer-respectively). Average and low levels of fer-). Average and low levels of fer- Average and low levels of fer-
ritin expression were observed only in 18% of tumors with
partial regression, although tumors with full regression
showed absence of this protein expression. Correla-. Correla-Correla-
tion analysis showed reverse correlation between levels
of serum and tumor ferritin and sensitivity of BC patients
to FAC and AC neoadjuvant chemotherapy (r = -0.39;
p < 0.05). Conclusions: So, we found relations between
levels of ferritinin in serum and tumor tissue with sensitivity
to neoadjuvant chemotherapy of BC patients. High serum
and tumor ferritin levels point on resistance of BC patients
to neoadjuvant chemotherapy. The acquired data showed
that ferritin level should be used as objective criteria for
determination of BC sensitivity to neoadjuvant chemo-
therapy on both organism and tumor levels.
GOLD NANOPRISMS AS POTENTIAL
DELIVERY AGENTS FOR PHOTODYNAMIC
THERAPY PHOTOSENSITIZERS
I. Shton1, P. Yermak1, A. Chevichalova2,
V. Estrela-Llopis2, N. Gamaleia1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
2F.D. Ovcharenko Institute of Biocolloidal
Chemistry, NAS of Ukraine, Kyiv, Ukraine
gamaleia@onconet.kiev.ua
victorio.estrela@gmail.com
Introduction: It is well known, that properties
of nanomaterial strongly depend on particle shape
and size. Gold nanospheres have already become
an object for targeted drug delivery research. How-. How-How-
ever, recently more attention is drawn to nanoparticles
with different geometrical parameters, particularly
nanoprisms. Such nanocarriers can be successfully
applied as delivery agents for photodynamic therapy
(PDT). One of the most questionable aspects of this
approach is nanoparticle interaction with different
cell types. Aim: To assess the gold nanoprism toxic- assess the gold nanoprism toxic-assess the gold nanoprism toxic-
ity and ability to accumulate in malignant and normal
cells in vitro and to evaluate the effectiveness of PDT
in vivo using Fotolon-nanoprism composite as a pho-
tosensitizer. Methods: Human glioblastoma cell line
A172 (kindly provided by Dr. S.P. Sydorenko), human
Burkitt’s lymphoma cell line Namalwa, peritoneal
mouse macrophages, normal human monocytes and
lymphocytes were used for studies in vitro. Mice
C57Bl/6 with transplanted Lewis lung carcinoma were
used in PDT experiments. Nanoprisms with average
diameter of 88.3 nm (determined by DLS analyzer
«Analysette 12 DynaSizer”, «Fritsch”, Germany) were
stabilized with chlorella polysaccharides. Chlorine e6-
148 Experimental Oncology 35, 128–152, 2013 (June)
based drug Fotolon (RUE Belmedpreparaty, Belarus)
was used as a photosensitizer. Accumulation of nano- used as a photosensitizer. Accumulation of nano-used as a photosensitizer. Accumulation of nano- as a photosensitizer. Accumulation of nano-as a photosensitizer. Accumulation of nano- photosensitizer. Accumulation of nano-photosensitizer. Accumulation of nano-. Accumulation of nano- Accumulation of nano-
prisms in target cells was studied using dark-field
microscopy. Cell viability in toxicity tests was assessed
by trypan blue dye exclusion method. In vivo effective-
ness of gold nanoprisms was studied by assessment
of tumor growth inhibition. Results: Cultured cells
were incubated with nanoprisms at concentrations
of 10 or 100 μg/ml for 1 hour in accumulation stud-μg/ml for 1 hour in accumulation stud- for 1 hour in accumulation stud-
ies and 1 or 24 hours in toxicity stu dies. Dark-field
microscopy studies of Namalwa cells showed that the
nanoparticles accumulate mostly on the cell surface.
Apparently, they do not penetrate cell membrane.
Normal human lymphocytes do not accumulate
nanoprisms even at concentration 100 μg/ml. On the
contrary, treatment of peritoneal mouse macrophages
and human blood monocytes, as well as human
glioblastoma cells A172 with gold nanoprisms leads
to nanoprism accumulation in cytoplasm. Gold nano-
prisms were not toxic to normal human lymphocytes
in both concentrations tested (10 and 100 μg/ml).
As to Namalwa cell line, viability of the cells, treated
with nanoprisms, decreased slightly (by 6–8%) only
after their incubation with maximal nanoparticle
concentration. Evaluation of Fotolon-nanoprism
composite activity in PDT tests in vivo showed inhibi-
tion of Lewis lung carcinoma growth to 70% versus
32% in the group of animals treated with free Foto-
lon. Conclusions: 1. Gold nanoprisms are relatively
nontoxic to cells. 2. Nanoprisms aggregated on the
surface of transformed lymphocytes and did not ag-
gregate on the surface of normal cells. 3. The nanopar-
ticles were absorbed mostly by phagocytic cells. 4.
Antitumor photodynamic activity of Fotolon-nanoprism
composite exceeded such of the free Fotolon more
than twice.
CHARACTERISTIC OF HLA-GENOTYPE
IN CHILDREN WITH ALL AND AML
K. Takun, T. Savitskaya, O. Aleinikova
National Research Center for Pediatric Oncology,
Hematology and Immunology, Minsk, Belarus
kira.takun@ymail.com
Introduction: The immunogenetic study has dis-
closed the correlation between HLA alleles and diseas-
es. Investigated alleles could be used as genetic mark-
ers in the diagnosis of malignant diseases. However,
the results of an immunogenetic research obtained
in any population can not be automatically transferred
to other ethnic groups. The HLA-distribution specific-
ity analysis in acute leukemia patients is of primary
importance. This information can help practitioners
to reveal individuals with a high probability of ma-
lignant diseases. Aim: The HLA-genotype analysis
in children suffering from acute leukemia and in chil-
dren with non-malignant diseases. Marker specificy
detection of high and low risk of leukemia progres-
sion. Methods: 25 children with acute lymphoblastic
leukemia (ALL), 16 children with acute myeloblastic
leukemia (AML) and 16 children with non-malignant
diseases were included in the study. HLA-genotyping
of leucocytes was performed by PCR-SSO (sequence
specific oligonucleotide) and PCR-SSP (sequence
specific primers) with reagent kits (Invitrogen, USA).
The significance of discrepancy between groups was
assessed by using chi-squared test. Results: The
distribution pattern of HLA-alleles in two subgroups
of patients with acute leukemia has been analyzed. The
most frequent alleles in ALL patients were A*02 (15 pa-
tients, 34%), A*24 (9 patients, 18%), B*35 (9 patients,
18%), B*07 (6 patients, 12%), C*07 (9 patients, 20%),
C*04 (6 patients, 12%), DRB1*01 (9 patients, 18%),
DRB1*04 (8 patients, 16%), DQB1*05 (11 patients,
50%), DQB1*03 (15 patients, 36%). The most fre-
quent alleles in AML patients were A*02 (8 patients,
34%), A*24 (4 patients, 16%), B*35 (5 patients,
16%), B*51 (3 patients, 13%), C*07 (6 patients, 22%),
C*06 (6 patients, 22%), DRB1*07 (6 patients, 22%),
DRB1*11 (5 patients, 19%), DQB1*03 (7 patients,
30%), DQB1*02 (7 patients, 27%). In patients with
non-malignant diseases HLA-alleles distribution was
the following: A*02 (8 patients, 31%), A*03 (5 patients,
19%), B*44 (8 patients, 28%), B*08 (4 patients, 13%),
C*07 (10 patients, 31%), C*04 (7 patients, 25%),
DRB1*03 (5 patients, 19%), DRB1*15 (6 patients,
19%), DQB1*02 (9 patients, 31%), DQB1*06 (9 pa-
tients, 28%). We displayed that differences between
groups of ALL patients and patients with non-malignant
diseases on НLА-DRB1*03 are statistically significant
(p = 0.04). Conclusion: Analysis of HLA-DRB*03 locus
has revealed the significant difference in ALL patients
when compared with the group of children with non-
malignant diseases. Obtained results are of certain
interest and require further investigation.
PHOTODYNAMIC THERAPY
OF TUMORS WITH CONTROLLED DELIVERY
OF PHOTOSENSITIZER HEMATOPORPHYRIN
L. Taranets1, O. Chepurna2, V. Cholin2, N. Gamaleia1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
2SME “Fotonika Plus”, Cherkasy, Ukraine
ltaranec@gmail.com
Introduction: Photodynamic therapy (PDT) is a tu-
mor treatment modality, which is grounded on applica-
tion of a photosensitizing drug (PS) and light of appro-
priate wavelength which activates PS leading to tumor
cell destruction. To enhance PDT antitumor effect,
a number of approaches are now under investigation,
including elaboration of targeting techniques, such
as use of metal or polymeric nanoparticles, and de-
velopment of novel regimens of light and PS delivery.
One of the promising approaches, which we exploited
in this study, may be called metronomic or chronic
PDT. It is based on continuous low level photoirra-
diation of a tumor and delivery of PS over extended
period of time — from several hours to a few days.
Aim: To establish a chronic regimen of PDT, provid-
ing controlled release and prolonged accumulation
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 149
of PS in tumors by employment of natural polymeric
materials with simultaneous low-intensity light illumi-
nation, and to evaluate PDT effects on murine Lewis
lung carcinoma (LLC) tumor growth and metastatic
dissemination. Methods: C57Bl6 female mice (6 per
qroup) with intracutaneous transplanted LLC were
used. A special photodiode panel was developed
for continuous low-intensity blue-light irradiation
of tumor-bearing animals. Photosensitizer hemato-
porphyrin was loaded into 4% hyaluronic acid hydrogel
which was inoculated intratumorally twice: on the 1st
and 3rd days of light treatment. On the whole, mice were
illuminated for 6 days. Before the start of experiments,
kinetics of hematoporphyrin release from hyaluronic
acid gel, introduced to mice, was determined by re-
cording its fluorescence in tumors for 5 days using
USB 4000 fiber optic spectrometer (“Ocean Optics”,
USA). All procedures involving animals were performed
in accordance with the Institutional Animal Care and
Use Committee. Results: Hyaluronic acid hydrogel
during three days provided controlled release and
higher accumulation (up to 7 times) of hematoporphy-
rin compared to free hematoporphyrin. This enabled
us to use the gel-carrier for controlled release of hema-
toporphyrin in PDT experiments. For accomplishment
of this chronic PDT, six-day illumination of LLC-bearing
mice with hematoporhyrin-containing hydrogel was
carried out. Remarkable inhibition up to 80% of tumor
growth was observed in a group of treated animals
compared to untreated ones. Moreover, the number
of lung metastases in treated animals was 24-fold
lower than in control group, indicating clear antimeta-
static effect of chronic PDT. Conclusion: A feasibility
of controlled continuous release of hematoporphyrin
photosensitizer from hyaluronic acid hydrogel was
demonstrated. Six-day blue-light low-level illumination
of tumors concomitant to controlled hematoporphyrin
release resulted in remarkable antitumor and antimeta-
static effect, indicating significance of this approach
to PDT of tumors.
THE PROGNOSTIC VALUE OF LEUKEMIC
STEM CELLS PERCENTAGE IN B-LINEAGE
CHILDHOOD ACUTE LYMPHOBLASTIC
LEUKEMIA
A.V. Tarasova, T.V. Shman, L.V. Movchan
Belarusian Research Сenter for Pediatric Oncology,
Hematology and Immunology, Minsk, Belarus
tarasova_alinav@mail.ru
One generally accepted theory is that leukemia
is maintained by leukemia stem cells (LSCs), which
play the central role in drug resistance and metasta-
sis. It would be of great interest to study the quantity
of LSCs during follow-up and their prognostic impact
on different types of leukemia. Moreover, developing
LSCs-targeted therapy may provide, in combination
with standard therapy, a way to eradicate the leuke-
mic process. The aim of our study was to evaluate
LSCs percentage and their prognostic meaning for
childhood B-precursor acute lymphoblastic leukemia.
Patients and Methods: We investigated cord blood
samples from 87 children with B-ALL. The percentage
of LSCs with phenotype CD34+CD38-CD19+ (Wil-Wil-
son K., 2010) in the blast population was determined
at diagnosis. Minimal residual disease (MRD) was
assessed by detection of aberrant immunophenotype
using flow cytometry (FC 500, “Beckman-Coulter”).
Comparisons of patient’s characteristics were per-
formed using the Mann — Whitney U-test. Data were
presented as median and percentiles (25–75) and ac-
cepted as reliable at p < 0.05. Results: We observed
that initial increased level of CD34+CD38-CD19+ cells
correlates with a poor response to induction chemo-
therapy. The median content of CD34+CD38-CD19+
was 0.13% (0.01–3.7) for MRD-negative (<0.01%
of blasts) and 2.1% (0.2–5.7) for MRD-positive
(> 0.01% of blasts) patients at 15th day of induction
chemotherapy (p <0.05). We compared the proportion
of the LSCs within the blast population in both diag-
nostic groups at day 36 and found that MRD-positive
patients have higher content of LSC at diagnosis than
MRD-negative: 3.3% (1.4–5.7) and 0.6% (0.06–3.7)
respectively (p < 0.05). Conclusion: Thus, increased
LSCs percentage correlates significantly with a lack
of complete response in patients with childhood B-
ALL. CD34+CD38-CD19+ compartment at diagnosis
was higher for MRD-positive patients at 15 and 36 days
of chemotherapy. The prognostic impact of the MRD
stem cell quantity might improve the already strong im-
pact of total MRD quantity on outcome. Identification
of new therapeutic targets based on LSCs biological
features is the aim of the future studies.
ROLE OF ENDOMETRIAL HYPERPLASIA
IN PROGNOSIS OF SURVIVAL OF PATIENTS
WITH OVARIAN CANCER
J.G. Tkalya, L.I. Vorobyova, V.S. Svintsitsky
National Cancer Institute, Kyiv, Ukraine
j.tkalya@gmail.com
Introduction: In ovarian cancer (OC) patients
endometrial pathology is diagnosed, that is explained
by the common risk factors and pathogenesis of the
diseases. The aim: To study the relationship be-
tween general and recurrence-free survival rate
in OC patients and presence/absence of associated
endometrial hyperplasia (EH). Patients and Meth-
ods: Retrospective analysis of history of diseases
in 303 OC patients with stages I-IV who underwent
standard treatment according to the radical program
in National Cancer Institute within 2001–2008 years.
The study protocol was approved by Ethical Committee
permission of National Cancer Institute (Kyiv, Ukraine).
Results: Group of OC patients with EH composed
59.1% (among those — reproductive age patients
made up 46.9%, menopausal period patients —
53.1%), without EH — 40.9% (among those — repro-
ductive age patients made up 30.5%, menopausal
period patients — 69.5%). The morphology of ovarian
tumor of patients with associated EH were allocated
as follows: papillary adenocarcinoma — 69.8%,
150 Experimental Oncology 35, 128–152, 2013 (June)
stromal tumors — 11.2%, mucinous cancer — 6.1%,
germinogenic tumors — 3.3%, non-differentiated ad-
enocarcinoma — 1.7%, endometrioid cancer — 1.1%.
Correlation relationship of general and recurrence-free
survival rates was defined in the investigated patients
due to presence or absence of associated EH. It was
shown that the median of recurrence-free survival
of OC patients with EH made up 60 months, while
in absence of EH — 24 months (p < 0.05). The five-
year survival rate in OC patients with EH composed
53%, in absence of EH — 32% (p < 0.05). It is worth
to note that early recurrences of the disease (pro-
longatio morbi) was observed in 24% of OC patients
in presence of EH and in 76% patients with its absence
(p < 0.05). Conclusions: Thus, the presence of benign
endometrial changes is a favorable prognostic sign
that is associated with OC prognosis. The obtained
data have practical significance as predictive factors
for estimation of OC prognosis.
THE PHOSPHO-C-JUN CONTENT IN GASTRIC
MUCOSA CELLS UNDER EXPERIMENTAL
GASTRIC CARCINOGENESIS
M. Tymoshenko, O. Kravchenko, L. Ostapchenko
Taras Shevchenko Kyiv National University,
Educational and Scientific Center “Institute
of Biology”, Kyiv, Ukraine
maria.bulavka@gmail.com
Introduction: The protein c-Jun belongs to the
subgroup of DNA binding transcription factors that
form AP-1 dimers. c-Jun is unique in its ability to posi-
tively regulate cell proliferation through the repression
of tumor suppressor gene expression and function,
and induction of cyclin D1 transcription. c-Jun is phos-
phorylated on Ser63 and Ser73 following activation
of signalling cascades, which made c-Jun maximally
effective in stimulating transcription. However, the
exact role of c-Jun in gastric oncogenesis is un-
known. Aim: To determine the phosphorylated form
of c-Jun content in the lysate of rat gastric mucosa
cells in chemically induced gastric cancer develop-
ment. Methods: The content of phospho-c-Jun was
measured by Sandwich ELISA method using the assay
kit PathScan Phospho-c-Jun (Ser63) (Cell Signaling
Technology, USA) and represented in conventional
unit of absorbancy on mg of the protein. The protein
concentration was registered by Bradford’s method.
Gastric carcinogenesis was initiated by 10-week
replacement of drinking water by 0.01% solution
of carcinogen N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG), at the same time the rats were redefined
on diet containing 5% NaCl. The samplings of mate-
rial were performed at the end of 4th, 6th, 8th, 10th, 12th,
18th and 24th week. The study protocol was approved
by Ethical Committee permission of Educational and
Scientific Center “Institute of Biology” (Kyiv, Ukraine).
Results: At the end of the 4th week of MNNG and NaCl
consumption the control reference of phospho-c-Jun
content was observed. The gastric mucosa cells were
characterized by increased content of phospho-c-
Jun at 4 and 6.3 fold over the control at the end of 6th
and 8th weeks, respectively, of the of MNNG and NaCl
treatment. Also it was established that the MNNG and
NaCl treatment for 10 weeks causes 1.9-fold increase
in phospho-c-Jun content compared to the control.
At terminal stages (12, 18 and 24 weeks) of the gas-
tric carcinogenesis study there was a stable increase
of phospho-c-Jun content on the average at 3.6-fold
in comparison with reference values. Conclusions:
The increased level of the phosphorylated form
of c-Jun from 6th to 24th week of gastric cancer devel-
opment was probably caused by activation of MAP-
kinase cascade and inactivation of phosphatases
that would lead to the intensification of target genes
transcription responsible for cell proliferation.
EPIDEMIOLOGICAL AND EXPERIMENTAL
APPROACHES FOR RISK ASSESSMENT
OF LOW INTENSITY RADIOFREQUENCY
RADIATION ON THE PUBLIC HEALTH
EFFECTS
E. Sidorik1, I. Yakimenko2, A. Burlaka1,
O. Tsybulin2, S. Lukin1, Y. Kolesnik3
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
2Bila Tserkva National Agrarian University, Bila
Tserkva, Ukraine
3Bila Tserkva Regional Oncology Center, Bila
Tserkva, Ukraine
yakymenko@btsau.net.ua
Introduction: Global development of wireless
technologies, including cellular telephony and Wi-
Fi, leads to a tremendous growth of radiofrequency
electromagnetic radiation (RF-EMR) level in human
environment. Today some studies revealed both epi-
demiological and experimental evidence on potential
carcinogenicity of long-term exposure to the low
intensity RF-EMR from the modern wireless devices.
Aim: To demonstrate the effectiveness of epidemio-
logical approach (a public survey) as well as the model
biological experiments in RF-EMR risk assessment.
Materials and Methods: 1) We interviewed stu-
dents of Kyiv region on a specificity of cellular phones
use and self-assessment of their health, including
a headache, an earache, and a physical discomfort
manifestation. 2) We used a model of Japanese quail
embryos in ovo for the assessment of metabolic
changes in living cells under the low intensity RF-EMR
exposure. Results: The survey of Ukrainian students
(17–21 years old) revealed that about a half of them
used cellular phones for talking more than 1 h per
day, and about 25% of them — more than 2 h per day.
And 42% of students surveyed had a headache and/
or an earache during the long-term cellular-phone
conversations. A strong correlation between headache
prevalence and the duration of everyday cell-phone
conversations was revealed. We could not exclude
at least partial effect of RF-EMR from the cellular
Experimental Oncology 35, 128–152, 2013 (June)35, 128–152, 2013 (June) (June) 151
phones in the phenomena revealed. On the other hand,
the exposure of quail embryos in ovo to extremely low
intensity RF-EMR of GSM 900 MHz standard during
at least 158 h led to significantly increased levels of su-
peroxide and nitrogen oxide, TBA-reactive substances
and 8-oxo-dG in embryo cells. This was accompanied
by a depression of antioxidant enzymes activity in the
cells. Previously the distinctive mutagenic effects
of this regimen of RF-EMR exposure on the quail em-
bryos were demonstrated by alkaline comet assay.
Conclusions: Both epidemiological and experimental
approaches used in this study were effective in the risk
assessment of the low intensity RF-EMR. Our findings
indicate the necessity of at least wide implementa-
tion of the precautionary principle as for uncontrolled
exposure of the public to RF-EMR from the modern
wireless devices.
DECREASE OF POSTNEUROLYTIC PAIN
IN PATIENTS WITH ADVANCED PANCREATIC
CANCER AFTER PARAVERTEBRAL
NEUROLYTIC BLOCK
S. Zemskov1, O. Dronov1, V. Ramazanov2
1Bogomolets National Medical University, General
Surgery #1, Kyiv, Ukraine
2Hospital of Ministry of Internal Affairs,
Anesthesiology Department, Kyiv, Ukraine
szemskov@yahoo.com
Introduction: More than 60% of advanced
pancreatic cancer (APC) patients suffer from
resistant pain syndrome whether they were previ-
ously resected or not. One of effective and minimal
invasive pain control techniques is paravertebral
neurolytic block (PNB). The most frequent adverse
effect of PNB is postneurolytic pain (PP) persist-
ing up to 7 days and more after the procedure.
Aim: To decrease PP in the patients after PNB and
to study the effectiveness of PNB in combination
with prolonged ropivacaine epidural infusion (PREI)
in APC patients. Patients and Methods: Group
I consisted of 21 sequential patients matched
to inclusion criteria and treated by PNB in our clinic
before we have started PREI application in postneu-
rolytic period. Group II consisted of 21 sequential
patients matched to inclusion criteria and treated
with PNB and PREI. Neural roots were identified
at Th5–Th10 level by rhythmic eliciting paresthesia
and/or pain radiating to corresponding body seg-
ment according to frequency of Stimuplex® DIG elec-
trostimulation after puncture. 0.2% ropivacaine was
used for PREI. Numeric Pain Rating Scale (NPRS)
from 0 to 10 and morphine equivalent dose (MED)
for tramadol daily intake were used for evaluation
of pain. The study protocol was approved by Ethi-
cal Committee permission of Bogomolets National
Medical University (Kyiv, Ukraine). Results: Pain
assessment in postneurolytic period is represented
in Table. Average duration of PREI application was
6.4 ± 1.3 days. 14 patients from group I and 11 pa-
tients from group II completely refused opioids.
No severe side effects of PREI as pronounced
arterial hypotension or paresis of lower extremities
were recorded.
Table. Pain assessment in patients with advanced pancreatic cancer.
Pa-
tients
Initial level Day 3rd after PNB Day 7th after PNB
NPRS MED NPRS MED NPRS MED
Group I 7.11±0.23 38.20±0.90 8.44±1.21 54.60±5.30 4.85±0.86 15.70±2.40
Group II 6.98±0.29 39.10±1.20 3.73±0.45 12.40±0.80 3.96±0.58 14.90±1.90
p value >0.05 >0.05 <0.05 <0.05 >0.05 >0.05
Conclusion: PREI application significantly reduces
PP in early postneurolytic period. PNB in combination
with PREI is effective and safe technique to reduce pain
and improve quality of life in APC patients.
FEATURES OF TREATMENT OF THE BREAST
CANCER PATIENTS WITH DISSEMINATED
TUMOR CELLS AND OVERPRODUCTION
OF TUMOR NECROSIS FACTOR IN BONE
MARROW
A. Zhylchuk1, Yu. Kudryavets2
1Rivne Regional Oncological Hospital, Rivne,
Ukraine
2R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv,
Ukraine
kudryavets@mail.ru
Introduction: One of the main problems of oncolo-
gy is the low efficiency of treatment against dissemi-
nated forms of neoplastic process — recurrence and
metastasis, which is the main cause of high mortality
among cancer patients. The important characteristic
of disseminated cancer is the presence of tumor cells
in the bone marrow (BM), as in many cases the use
of high-dose chemotherapy by accepted protocols
is inefficient. The goal was to prevent cancer progres-
sion and bone metastasis in breast cancer patients
with disseminated tumor cells (DTC) in BM and with
elevated levels of TNF in BM using modified schemes
of polychemotherapy with bisphosphonates. Pa-
tients and Methods: This research was carried out
during the therapy of 119 patients with breast cancer
in a disease stage T1–4N1–2M0. DTCs and TNF were
analyzed in BM of all patients before therapy. The
patients with DTCs in BM were divided into 2 groups.
Group 1 (n = 20) underwent preoperative therapy
according to AC (doxorubicin + cyclophosphamide)
scheme. Group 2 (n = 27) received preoperative ther-
apy in compliance with AC — Paclitaxel scheme and +
Zoledronic acid (Zometa) in the postoperative period.
Patients with high level (more than 150 pg/ml, n = 53)
TNF in BM were divided into the same two therapeu-
tic groups (n = 27 and n = 26). Results: It was found
that DTC had been present in 47 patients (39.4%).
Proportion of patients who were in 36 months remis-
sion was about the same in both groups — with DTC
in BM or without it (59.5% and 60.2% respectively).
The efficacy of treatment schemes AC → P + Zometa
schemes vs. 48.9% in AC group. A similar pattern
is evident while comparing the same therapeutic pat-
terns for patients with high level of TNF in BM. Disease
152 Experimental Oncology 35, 128–152, 2013 (June)
progression was observed in 25.9% of patients with
AC → P + Zometa schemes vs. 73.1% in AC group.
Conclusion: Presence of DTC and TNF in the bone
marrow indicates a high risk of disease progression for
patients with breast cancer. Preoperative polychemo-
therapy according to the scheme of AC → P followed
by Zometa in the postoperative period for patients with
DTC in BM and high TNF levels in BM is more effective
in comparison with AC scheme.
EFFECT OF A FENUGREEK ON LIFETIME
OF EXPERIMENTAL RODENTS WITH
TRANSPLANTED TUMORS
V. Zhylenko
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology
NAS of Ukraine, Kyiv, Ukraine
gilenkovv@yandex.ru
Introduction: Recent studies show that Trigonella
foenum graecum (Leguminosae family), commonly
known as fenugreek, has growth-inhibiting activ-
ity against some kinds of experimental tumors. Aim:
To study the effect of fenugreek on lifespan of tumor
bearing animals. Materials and Methods: Experi-
ments were carried out on the adult C57Bl/6 female
mice with subcutaneously grafted Ca755 mammary
carcinoma, and adult C57Bl/6 male and female mice
transplanted with Lewis lung carcinoma (3LL). The
animals in the experimental group were administered
with the mix of fenugreek powder (250 mg/kg of body
weight) and standard mash from the day of tumor graft-
ing up to the end of experiments. All experiments were
carried out according to the rules of local Ethic Com-
mittee and were approved by the Ethic Board of IEPOR
NASU. Results: The fenugreek consumption increased
the lifetime of Ca755 carcinoma mice by 19%, and
3LL-bearing male and female mice by 9% and 26%,
respectively. Moreover, it was shown that fenugreek
consumption has resulted in the inhibition of Ca755 ad-
enocarcinoma growth by 48%. Anti-metastatic effect
of mentioned agent was also observed: an average
volume of the metastases per animal was decreased
by 86% and 18% in the female and male mice, re-
spectively. Conclusions: The results have shown that
fenugreek can be considered a potential anticancer
agent with more significant effect in the female animals.
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