Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer
Aim. The work is devoted to the development of less invasive tools for the colorectal cancer (CRC) screening. Methods. Q-PCR and methylation-specific PCR techniques were used in the current work. Results. We have shown that the levels of cell-free plasma DNA are higher in the CRC patients compared w...
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| Cite this: | Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer / A.G. Kondratov, K.A. Nekrasov, L.V. Lototska, G.V. Panasenko, L.A. Stoliar, Y.V. Lapska, O.O. Kolesnyk, I.B. Shchepotin, A.V. Rynditch, V.I. Kashuba // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 129-134. — Бібліогр.: 19 назв. — англ. |
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Kondratov, A.G. Nekrasov, K.A. Lototska, L.V. Panasenko, G.V. Stoliar, L.A. Lapska, Y.V. Kolesnyk, O.O. Shchepotin, I.B. Rynditch, A.V. Kashuba, V.I. 2019-06-14T15:09:33Z 2019-06-14T15:09:33Z 2014 Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer / A.G. Kondratov, K.A. Nekrasov, L.V. Lototska, G.V. Panasenko, L.A. Stoliar, Y.V. Lapska, O.O. Kolesnyk, I.B. Shchepotin, A.V. Rynditch, V.I. Kashuba // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 129-134. — Бібліогр.: 19 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00088B https://nasplib.isofts.kiev.ua/handle/123456789/153751 577.218 + 577.133.4 Aim. The work is devoted to the development of less invasive tools for the colorectal cancer (CRC) screening. Methods. Q-PCR and methylation-specific PCR techniques were used in the current work. Results. We have shown that the levels of cell-free plasma DNA are higher in the CRC patients compared with the healthy donors (p < 0.01). Hypermethylation of APC, FHIT, LRRC3B and HIC1 genes was studied in the tumor and plasma samples of CRC patients. Two-stage verification for CRC screening was proposed. Conclusions. We proposed and tested a novel approach for CRC screening based on the determination of cell-free DNA and methylated DNA fragments in the plasma. Мета. Розробка менш інвазивних методик для скринінгу злоякісних пухлин товстого кишечника (CRC). Методи. Використано кількісну ПЛР і метил-специфічну ПЛР. Результати. Показано, що середнє значення концентрацій вільно циркулюючої ДНК у плазмі крові є статистично достовірно вищим у пацієнтів з CRC порівняно зі здоровими донорами (p < 0,01). Встановлено гіперметилювання генів APC, FHIT, LRRC3B і HIC1 у пухлинах та плазмі хворих на CRC. Висновки. Нами запропоновано і перевірено новітній підхід для скринінгу CRC, який базується на визначенні позаклітинної ДНК і метильованих фрагментів ДНК у плазмі. Цель. Разработка менее инвазивных методик для скрининга злокачественных опухолей толстого кишечника (CRC). Методы. Использована количественная ПЦР и метил-специфическая ПЦР. Результаты. Показано, что среднее значение свободно циркулирующей ДНК в плазме статистически достоверно выше у пациентов с CRC по сравнению со здоровыми донорами (p < 0,01). Установлено гиперметилирование генов APC, FHIT, LRRC3B и HIC1 в опухолях и плазме пациентов с CRC. Выводы. Нами предложен и проверен новейший подход для скрининга CRC, базирующийся на определении внеклеточной ДНК и метилированных фрагментов ДНК в плазме. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Biomedicine Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer Порівняльний аналіз епігенетичних маркерів у плазмі крові пацієнтів, хворих на рак товстого кишечника Сравнительный анализ эпигенетических маркеров в плазме крови пациентов, больных раком толстого кишечника Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| spellingShingle |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer Kondratov, A.G. Nekrasov, K.A. Lototska, L.V. Panasenko, G.V. Stoliar, L.A. Lapska, Y.V. Kolesnyk, O.O. Shchepotin, I.B. Rynditch, A.V. Kashuba, V.I. Biomedicine |
| title_short |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| title_full |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| title_fullStr |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| title_full_unstemmed |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| title_sort |
comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer |
| author |
Kondratov, A.G. Nekrasov, K.A. Lototska, L.V. Panasenko, G.V. Stoliar, L.A. Lapska, Y.V. Kolesnyk, O.O. Shchepotin, I.B. Rynditch, A.V. Kashuba, V.I. |
| author_facet |
Kondratov, A.G. Nekrasov, K.A. Lototska, L.V. Panasenko, G.V. Stoliar, L.A. Lapska, Y.V. Kolesnyk, O.O. Shchepotin, I.B. Rynditch, A.V. Kashuba, V.I. |
| topic |
Biomedicine |
| topic_facet |
Biomedicine |
| publishDate |
2014 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Порівняльний аналіз епігенетичних маркерів у плазмі крові пацієнтів, хворих на рак товстого кишечника Сравнительный анализ эпигенетических маркеров в плазме крови пациентов, больных раком толстого кишечника |
| description |
Aim. The work is devoted to the development of less invasive tools for the colorectal cancer (CRC) screening. Methods. Q-PCR and methylation-specific PCR techniques were used in the current work. Results. We have shown that the levels of cell-free plasma DNA are higher in the CRC patients compared with the healthy donors (p < 0.01). Hypermethylation of APC, FHIT, LRRC3B and HIC1 genes was studied in the tumor and plasma samples of CRC patients. Two-stage verification for CRC screening was proposed. Conclusions. We proposed and tested a novel approach for CRC screening based on the determination of cell-free DNA and methylated DNA fragments in the plasma.
Мета. Розробка менш інвазивних методик для скринінгу злоякісних пухлин товстого кишечника (CRC). Методи. Використано кількісну ПЛР і метил-специфічну ПЛР. Результати. Показано, що середнє значення концентрацій вільно циркулюючої ДНК у плазмі крові є статистично достовірно вищим у пацієнтів з CRC порівняно зі здоровими донорами (p < 0,01). Встановлено гіперметилювання генів APC, FHIT, LRRC3B і HIC1 у пухлинах та плазмі хворих на CRC. Висновки. Нами запропоновано і перевірено новітній підхід для скринінгу CRC, який базується на визначенні позаклітинної ДНК і метильованих фрагментів ДНК у плазмі.
Цель. Разработка менее инвазивных методик для скрининга злокачественных опухолей толстого кишечника (CRC). Методы. Использована количественная ПЦР и метил-специфическая ПЦР. Результаты. Показано, что среднее значение свободно циркулирующей ДНК в плазме статистически достоверно выше у пациентов с CRC по сравнению со здоровыми донорами (p < 0,01). Установлено гиперметилирование генов APC, FHIT, LRRC3B и HIC1 в опухолях и плазме пациентов с CRC. Выводы. Нами предложен и проверен новейший подход для скрининга CRC, базирующийся на определении внеклеточной ДНК и метилированных фрагментов ДНК в плазме.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153751 |
| citation_txt |
Comparative analysis of epigenetic markers in plasma and tissue of patients with colorectal cancer / A.G. Kondratov, K.A. Nekrasov, L.V. Lototska, G.V. Panasenko, L.A. Stoliar, Y.V. Lapska, O.O. Kolesnyk, I.B. Shchepotin, A.V. Rynditch, V.I. Kashuba // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 129-134. — Бібліогр.: 19 назв. — англ. |
| work_keys_str_mv |
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2025-11-24T04:42:08Z |
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| fulltext |
BIOMEDICINE
UDC 577.218 + 577.133.4
Comparative analysis of epigenetic markers
in plasma and tissue of patients with colorectal cancer
A. G. Kondratov1, K. A. Nekrasov1, L. V. Lototska1, G. V. Panasenko1, L. A. Stoliar1,
Y. V. Lapska1, O. O. Kolesnyk2, I. B. Shchepotin2, A. V. Rynditch1, V. I. Kashuba1, 3
1State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2National Cancer Institute
33/43, Lomonosova Str., Kyiv, Ukraine, 03022
3Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet
Nobels vag 16, Box 280, 171 77-Stockholm,
o.g.kondratov@imbg.org.ua
Aim. The work is devoted to the development of less invasive tools for the colorectal cancer (CRC) screening.
Methods. Q-PCR and methylation-specific PCR techniques were used in the current work. Results. We have
shown that the levels of cell-free plasma DNA are higher in the CRC patients compared with the healthy donors
(p < 0.01). Hypermethylation of APC, FHIT, LRRC3B and HIC1 genes was studied in the tumor and plasma
samples of CRC patients. Two-stage verification for CRC screening was proposed. Conclusions. We proposed
and tested a novel approach for CRC screening based on the determination of cell-free DNA and methylated
DNA fragments in the plasma.
Keywords: colorectal cancer, cell-free DNA, DNA methylation, APC, FHIT, LRRC3B, HIC1.
Introduction. Colorectal cancer (CRC) is the third com-
monly diagnosed cancer that causes more than 600 000
deaths per year worldwide [1]. In most cases, CRC tu-
mors grow slowly – approximately 1 cm per year with-
out noticeable symptoms. The most sensitive modern
diagnostic tool for the CRC detection is a colonoscopy.
It allows detecting the tumors of less than 1 cm. How-
ever, the colonoscopy procedure is painful and in some
cases is not recommended to the patients with heart di-
seases, because of possible adverse cardiopulmonary re-
actions that are usually related to the sedation [2] as the
colonoscopy is performed under narcosis. Also, the co-
lonoscopy meets some difficulties in case of altered to-
pography of a colon [3]. Thus, the development of less
invasive tools for screening CRC is a relevant problem
of the modern oncology.
DNA methylation is a stable epigenetic mark which
is associated with gene silencing in the case of promo-
ter localization in CpG-island [4]. The gene hyperme-
thylation frequently targets the potential tumor-sup-
pressor genes (TSG), inactivation of which promotes
the tumor development. According to Toyota et al., the
colon cancer can be ascribed to the tumors with a high
frequency of gene hypermethylation. Approximately
17 % of CpG-islands are hypermethylated in CRC [5].
The circulating cancer cells and cell-free DNA (cf
DNA) are frequently detected in the patients with diffe-
rent types of the malignant disease. Moreover, a level
of cfDNA is elevated in the cancer patients in compa-
rison with the healthy individuals [6].
In the proposed paper DNA hypermethylation of
the well-known tumor associated genes like LRRC3B,
FHIT, APC and HIC1 was detected by methylation-
specific PCR (MSP) in the plasma and tumor samples
129
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 2. P. 129–134 doi: http://dx.doi.org/10.7124/bc.00088B
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
from the CRC patients [7–10]. Adjacent to the tumor
non-malignant tissues of bowel were used as a control.
Additionally, the levels of cfDNA in plasma from the
CRC patients and healthy donors were mesuared by
quantitative PCR (Q-PCR). All experiments were car-
ried out with the tumors, adjacent non-malignant tis-
sues and plasma samples of the same patients.
Material and methods. Ethics statements. The samp-
les were collected in accordance with the Declaration
of Helsinki and approved by the guidelines issued by
the Ethic Committee of the National Cancer Institute of
the Academy of Medical Sciences, Kyiv, Ukraine.
Sample collection, genomic and cell-free DNA iso-
lation. Twenty surgically excised tissue samples of CRC
were used in the present study (Table 1). All tumor
samples were paired with non-malignant tissues, which
were taken as the normal tissue samples. Immediately
after surgery, the tissue samples were frozen in liquid
nitrogen and stored at –70 oC. Neither chemotherapy
nor radiotherapy was conducted for any patients prior to
surgery. Each tissue sample was accompanied by a cor-
responding blood sample. All tissue samples were cha-
racterized histologically. Blood of 21 healthy donors
was used as a control group.
Genomic DNA was purified by GenElute Mamma-
lian Genomic DNA Miniprep Kit («Sigma-Aldrich»,
USA) according to the manufacturer’s recommen-
dations. Briefly, 50 mg of tissue were homogenized in
liquid nitrogen, subjected to lysis and purified with
GenElute Miniprep Binding Columns. The quality and
size of genomic DNA were assessed by gel electro-
phoresis («Sigma-Aldrich»).
Plasma from the cancer patients and the healthy
donors was obtained by the multistage centrifugation
of the blood in range from 1000 to 3000 g, using EDTA
as an anticoagulant. The cell free circulating DNA from
200 �l of plasma was isolated by the Proba NK DNA
(«DNA Technology», Russia) isolation kit, according
to the manufacturer’s instructions.
Determination of concentrations of cell free DNA.
To detect the concentration of cell free circulating
DNA in the plasma, the Q-PCR was used. The Q-PCR
was performed with SYBR Green mixture («Thermo
Scientific», USA) and primers for the GAPDH gene:
gRef For 5'-GGCTCCCACCTTTCTCATC-3' and
gRef Rev 5'-AGCGTACTCCCCACATCA-3', using
the following reaction conditions: 95 oC – 4 min, then
35 cycles at 95 oC – 15 s, 60 oC – 20 s and 72 oC – 30 s.
To carry out Q-PCR the CFX real-time PCR detection
system («BioRad», USA) was used. To determine the
concentration of DNA in the plasma the calibration cur-
ve in coordinates of genomic DNA concentrations and
Ct was plotted. The range of genomic DNA concent-
ration was 2.44–2440 pg. The range of concentrations
was created by DNA dilution. The initial concentration
of DNA for a calibration curve was determined by
means of ND-2000 («Thermo Scientific»).
DNA bisulfite conversion and determination of me-
thylated DNA fragments. The bisulfite treatment of ge-
130
KONDRATOV A. G. ET AL.
N, CRC
patients
cfDNA concentration for
CRC patients, ng/ml
N, healthy
donors
cfDNA concentration for
healthy donors, ng/ml
100 14.0 1 6.1
102 4.5 2 7.8
103 8.6 3 5.3
104 7.1 4 7.9
108 10.3 5 10.0
109 11.9 6 5.4
110 25.5 7 6.8
116 3.2 8 11.0
117 2.7 9 5.3
118 12.6 10 6.7
119 10.7 11 2.7
120 23.8 12 3.2
121 12.1 13 6.3
122 69.9 14 4.8
124 15.1 15 3.9
126 18.2 16 6.0
127 21.1 17 18.8
129 36.5 18 8.2
130 10.8 19 8.3
131 32.8 – –
132 252.7 – –
Table 1
Concentrations of cfDNA in plasma samples of CRC patients and
healthy donors
nomic DNA was performed, using the EZ DNA Me-
thylation kit («ZYMO Research», USA), according to
the manufacturer’s protocol. For the bisulfite treatment
of the plasma DNA 44 �l of solution of the dissolved
DNA sediments with co-precipitator were used. To pro-
tect the plasma DNA during the reaction, 450 ng of Lam-
bda DNA were added per a reaction sample of bisulfite
treatment.The bisulfite treated DNA was dissolved in
20 �l of Elution buffer.
To determine the methylation status of the LRRC3B,
APC, FHIT, and HIC1 genes in tissue samples, the me-
thylation-specific PCR (MSP) was used. MSP was per-
formed using primers that were described previously
[11–14]. Amplification of the bisulfite converted sequ-
ence of the COL2A1 gene was used as a control of the
DNA input [14]. The PCR mixture of MSP reactions
contained 10 � PCR Dream buffer («Thermo Scien-
tific»), 0.2 mM deoxyribonucleotide triphosphates
(dNTPs), 0.3 µM primers, 50 ng of modified DNA and
1 U of DreamTaq polymerase («Thermo Scientific»).
Amplification was performed for 40 cycles (30 s at
95 oC, 30 s at 63 oCand 30 s at 72 oC), initiated with
DNA denaturation at 95 oC for 4 min. The final exten-
sion was at 72 oC for 5 min. Amplified products were
detected by electrophoresis in 12 % polyacrylamide gel
with subsequent ethidium bromide staining.
To determine methylation status of the APC, FHIT,
LRRC3B, and HIC1 genes in the cell free circulating
DNA, the real-time MSP (RT-MSP) was used. RT-
MSP was performed with the above-mentioned primer
set for MSP. Each sample of the amplification reaction
contained 1 � SYBR Green mixture («Thermo Scien-
tific»), 0.4 µM of each primer and 4�l of bisulfite trea-
ted DNA. The conditions of RT-MSP were 95 oC for 10
min, for 50 cycles; 95 oC for 15 s, 62–65 oC for 20 s and
72 oC for 30 s. It was considered the positive methy-
lation status of the sample if Ct of reaction was < 45
cycles. The quality of amplified products was checked
by electrophoresis in 2.5 % agarose gel and melting
curve analysis with the CFX real-time PCR detection
system («BioRad»). To verify RT-MSP data, the MSP
sequencing assay was performed.
Statistical analysis. Statistical analysis was perfor-
med using STATISTICA 7.0 program («StatSoft Inc»,
USA). If the p-value was < 0.05 the results were consi-
dered statistically significant. The nonparametric Mann-
Whitney U Test was used to calculate the difference bet-
ween concentration of plasma cfDNA in CRC patients
and healthy donors.
Results and discussion. The levels of cell free cir-
culating DNA in the plasma from CRC patients are hi-
gher than in healthy donors. Using Q-PCR, the level of
cell free circulating DNA was determined in the plasma
of CRC patients and healthy donors (Table 1, Figure).
It was shown that the mean value of concentration of
plasma cfDNA was significantly higher in CRC pati-
ents, compared with the healthy donors (p < 0.01). Thus,
the mean value (MV) of concentration of cell-free cir-
culating DNA in the plasma from CRC patients was
29.45 ± 12.24 ng/ml (MV ± St. Error), whereas it was
7.07 ± 0.82 (MV ± St. Error) in healthy donors. In order
to generate the upper cut-off value of the cell free circu-
lating DNA concentration in plasma we used the highest
permissible concentration in the healthy donors. It was
defined as a mean value concentration of cfDNA of
healthy donors and three standard deviations. So, the
upper cut-off value of the free-circulating DNA concent-
ration was 17.7 ng/ml of the plasma in healthy donors
(Figure).
Therefore, there were 8 out of 20 samples of CRC
which fell into criteria as samples with an abnormally
increased DNA level.
Hypermethylation of tumor suppressor genes in the
cancer tissues and plasma samples from CRC patients.
APC, FHIT, LRRC3B and HIC1 are the colon cancer as-
131
COMPARATIVE ANALYSIS OF EPIGENETIC MARKERS OF PATIENTS WITH COLORECTAL CANCER
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ng
/m
L
1 2
0
10
20
30
40
50
60
70
CfDNA concentration in plasma of 20 CRC patients (1) and 19 healthy
donors (2) was detected by Q-PCR. The upper cut-off value of the
free-circulating DNA concentration of healthy donors is depicted as a
dashed line at 17.7 ng/ml level of plasma. To prevent merger of data
points one CRC (N 132) concentration was not included here, it equals
252,7 ng/ml
sociated genes. Their alterations due to the promoter
CpG-island methylation were described previously.
Thus, hypermethylation of APC, FHIT, LRRC3B
and HIC1 was detected in 45, 37, 77 and 42 % of CRC
samples correspondingly [15–18]. Therefore, the MSP-
based detection of the methylated fragments of these ge-
nes was used for further development of screening pa-
nel for CRC. In the current research we have found that
the APC, FHIT, and LRRC3B genes were hypermethy-
lated in 50 % (10/20), 70 % (14/20) and 65 % (13/20) of
tumor samples, correspondingly. Altogether, the hyper-
methylation of at least one of the selected genes was de-
tected in 90 % (18/20) of samples.
Using MSP and the subsequent melting curve ana-
lysis, the methylated fragments of APC, FHIT and
LRRC3B genes were detected in 30 % (6/20), 20 %
(4/20) and 15 % (3/20) of the plasma of CRC patients,
respectively. The hypermethylation of at least one se-
lected gene was found in 50 % (10/20) of the plasma
samples from CRC patients.
132
KONDRATOV A. G. ET AL.
Tissue Plasma
N/Genes LRRC3B APC FHIT Totally LRRC3B APC FHIT cfDNA Totally
100 M M M + ND M ND L A
102 U M U + ND M ND L A
103 U U U – ND ND ND L NC
104 M U M + M ND ND L A
108 M U U + ND ND ND L NC
109 M U U + ND ND ND L NC
110 M M M + ND ND M H A
116 M M U + ND ND ND L NC
117 M M M + ND ND ND L NC
118 M M M + ND M ND L A
119 U M M + ND M M L A
120 U U M + ND ND ND H A
121 M U M + ND ND M L A
122 M M M + ND ND ND H A
126 U U M + ND ND ND H A
127 M M M + ND ND ND H A
129 M U M + M ND M H A
130 U U U – ND M ND L A
131 U U M + ND ND ND H A
132 M M M + M M ND H A
Frequency
65 %
(13/20)
50 %
(10/20)
70 %
(14/20)
90 %
(18/20)
15 %
(3/20)
30 %
(6/20)
20 %
(4/20)
40 %
(8/20)
75 %
(15/20)
N o t e . M – methylated DNA was detected; U – unmethylated DNA was detected; H – cfDNA concentration is higher than upper cut-off value; L –
cfDNA concentration is lower than upper cut-off value; ND – methylated DNA was not detected; «+» – methylation was detected in one or more
genes for the tissue; «–» – no methylation was detected for at least one genes for the tissue; A – abnormal plasma; NC – no changes have been de-
tected in the plasma.
Table 2
Total data of cfDNA concentration and methylation of APC, FHIT and LRRC3B genes from plasma of CRC patients.
No amplification was found in reactions with MSP
primers and bisulfite treated Lambda DNA which did
not include the human DNA. No methylated fragments
of the selected genes were identified in plasma of heal-
thy donors.
Therefore, the hypermethylated fragments of APC,
FHIT, and LRRC3B genes in the plasma were detected
in 50 % (5/10), 31 % (4/13) and 23 % (3/13) of tumors
which were positive for hypermethylated fragments of
the abovementioned genes, respectively. To verify speci-
ficity of MSP, the PCR products of the APC and LRRC3B
genes were sequenced after amplification with the pri-
mers for methylated DNA.
To study specificity of the gene panel, hypermethy-
lation of the APC, FHIT and LRRC3B genes was stu-
died in the plasma of healthy donors. Moreover, the me-
thylation of APC, FHIT and LRRC3B in plasma was de-
tected in 92 % (12/13) of the samples with pre-detected
methylation of these genes in tumor tissue. Initially we
had shown the same tendency in hypermethylation of
the APC, FHIT and LRRC3B genes in the plasma of
CRC patients [19].
Additionally, we have registered a high frequency
of the HIC1 hypermethylation in the plasma of CRC pa-
tients – 80 % (8/10). Similar study of the healthy do-
nor’s plasma has revealed the hypermethylation of HIC1
at the similar level – 80 % (12/15). No difference in the
HIC1 and COL2A1 hypermethylation was observed in
CRC patients and healthy donors: mean values of CtCRC
32.7 – HIC1 and 28.6 – COL2A1 and Cthealthy donors 35.0 –
HIC1 and 30.5 – COL2A1.
This indicates a low selectivity of the HIC1 hyper-
methylation in CRC patients. Therefore, we concluded
that the HIC1 hypermethylation is not a valuable mar-
ker for the prediction of CRC.
We proposed that a two-stage verification must be
applied for CRC screening. These stages include the
measurement of the cell-free circulating DNA and the
following detection of the methylated fragments of
APC, FHIT, and LRRC3B genes in the CRC patient plas-
ma. This allows us to achieve a sensitivity of the panel
in CRC detection up to 75 % (Table 2).
However, our research has not resulted in the sen-
sitivity of 100 % for the CRC registration that is essen-
tial for the prevention of this disease. We hope that a hi-
gher sensitivity might be achieved by further extension
of the gene panel for the identification of methylated
DNA in the plasma of CRC patients.
Conclusions. In the present work we have charac-
terized hypermethylation of the APC, FHIT, LRRC3B,
and HIC1 genes in the patients with CRC in compari-
son with the healthy donors. We have found that hyper-
methylation of the APC, FHIT, and LRRC3B gene frag-
ments in the plasma fully corresponds to hypermethy-
lation of these genes in the tumors.
We have proposed and tested the novel approach
for CRC screening, based on the detection of cell-free
DNA and methylated fragments of the well-known tu-
mor associated genes, such as APC, FHIT, LRRC3B, and
HIC1 in the blood plasma. With such approach 75 % of
sensitivity could be achieved. The sensitivity for CRC
detection might be increased by the analysis of additio-
nal tumor-associated genes.
Funding. This work was supported by a grant from
the Ukrainian Academy of Sciences (41/12).
Ïîð³âíÿëüíèé àíàë³ç åï³ãåíåòè÷íèõ ìàðêåð³â ó ïëàçì³ êðîâ³
ïàö³ºíò³â, õâîðèõ íà ðàê òîâñòîãî êèøå÷íèêà
Î. Ã. Êîíäðàòîâ, Ê. À. Íåêðàñîâ, Ë. Â. Ëîòîöüêà,
Ã. Â. Ïàíàñåíêî, Ë. À. Ñòîëÿð, Þ. Â. Ëàïñüêà,
Î. Î. Êîëåñíèê, ². Á. Ùåïîò³í, À. Â. Ðèíäè÷, Â. ². Êàøóáà
Ðåçþìå
Ìåòà. Ðîçðîáêà ìåíø ³íâàçèâíèõ ìåòîäèê äëÿ ñêðèí³íãó çëîÿê³ñ-
íèõ ïóõëèí òîâñòîãî êèøå÷íèêà (CRC). Ìåòîäè. Âèêîðèñòàíî
ê³ëüê³ñíó ÏËÐ ³ ìåòèë-ñïåöèô³÷íó ÏËÐ. Ðåçóëüòàòè. Ïîêàçàíî,
ùî ñåðåäíº çíà÷åííÿ êîíöåíòðàö³é â³ëüíî öèðêóëþþ÷î¿ ÄÍÊ ó
ïëàçì³ êðîâ³ º ñòàòèñòè÷íî äîñòîâ³ðíî âèùèì ó ïàö³ºíò³â ç CRC
ïîð³âíÿíî ç³ çäîðîâèìè äîíîðàìè (p < 0,01). Âñòàíîâëåíî ã³ïåðìå-
òèëþâàííÿ ãåí³â APC, FHIT, LRRC3B ³ HIC1 ó ïóõëèíàõ òà ïëàçì³
õâîðèõ íà CRC. Âèñíîâêè. Íàìè çàïðîïîíîâàíî ³ ïåðåâ³ðåíî íîâ³ò-
í³é ï³äõ³ä äëÿ ñêðèí³íãó CRC, ÿêèé áàçóºòüñÿ íà âèçíà÷åíí³ ïîçà-
êë³òèííî¿ ÄÍÊ ³ ìåòèëüîâàíèõ ôðàãìåíò³â ÄÍÊ ó ïëàçì³.
Êëþ÷îâ³ ñëîâà: çëîÿê³ñí³ ïóõëèíè òîâñòîãî êèøå÷íèêà, â³ëüíî
öèðêóëþþ÷à ÄÍÊ, ìåòèëþâàííÿ ÄÍÊ, APC, FHIT, LRRC3B, HIC1.
Ñðàâíèòåëüíûé àíàëèç ýïèãåíåòè÷åñêèõ ìàðêåðîâ â ïëàçìå êðîâè
ïàöèåíòîâ, áîëüíûõ ðàêîì òîëñòîãî êèøå÷íèêà
À. Ã. Êîíäðàòîâ, Ê. À. Íåêðàñîâ, Ë. Â. Ëîòîöêàÿ,
Ã. Â. Ïàíàñåíêî, Ë. À. Ñòîëÿð, Þ. Â. Ëàïñêàÿ,
Î. Î. Êîëåñíèê, È. Á. Ùåïîòèí, À. Â. Ðûíäè÷, Â. È. Êàøóáà
Ðåçþìå
Öåëü. Ðàçðàáîòêà ìåíåå èíâàçèâíûõ ìåòîäèê äëÿ ñêðèíèíãà çëîêà-
÷åñòâåííûõ îïóõîëåé òîëñòîãî êèøå÷íèêà (CRC). Ìåòîäû. Èñ-
ïîëüçîâàíà êîëè÷åñòâåííàÿ ÏÖÐ è ìåòèë-ñïåöèôè÷åñêàÿ ÏÖÐ.
Ðåçóëüòàòû. Ïîêàçàíî, ÷òî ñðåäíåå çíà÷åíèå ñâîáîäíî öèðêóëè-
ðóþùåé ÄÍÊ â ïëàçìå ñòàòèñòè÷åñêè äîñòîâåðíî âûøå ó ïàöè-
133
COMPARATIVE ANALYSIS OF EPIGENETIC MARKERS OF PATIENTS WITH COLORECTAL CANCER
åíòîâ ñ CRC ïî ñðàâíåíèþ ñî çäîðîâûìè äîíîðàìè (p < 0,01).
Óñòàíîâëåíî ãèïåðìåòèëèðîâàíèå ãåíîâ APC, FHIT, LRRC3B è
HIC1â îïóõîëÿõ è ïëàçìå ïàöèåíòîâ ñ CRC. Âûâîäû. Íàìè ïðåäëî-
æåí è ïðîâåðåí íîâåéøèé ïîäõîä äëÿ ñêðèíèíãà CRC, áàçèðóþ-
ùèéñÿ íà îïðåäåëåíèè âíåêëåòî÷íîé ÄÍÊ è ìåòèëèðîâàííûõ ôðàã-
ìåíòîâ ÄÍÊ â ïëàçìå.
Êëþ÷åâûå ñëîâà: çëîêà÷åñòâåííûå îïóõîëè òîëñòîãî êèøå÷-
íèêà, ñâîáîäíî öèðêóëèðóþùàÿ ÄÍÊ, ìåòèëèðîâàííàÿ ÄÍÊ, APC,
FHIT, LRRC3B, HIC1.
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