International Journal of Medical Sciences And Clinical Research
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VOLUME
Vol.05 Issue02 2025
PAGE NO.
33-42
10.37547/ijmscr/Volume05Issue02-07
Results of immunogystochemic study in primary liver
cancer developed on the basis of chronic viral hepatitis
Musaboev E.I
Republican Specialized Scientific and Practical Medical Center of Virology, Tashkent, Uzbekistan
Abdikhakimov A.N
Tashkent Regional Branch of the Republican Specialized Scientific and Practical Medical Center of Oncology and Radiology, Tashkent,
Uzbekistan
Mirzaev Kh.M
Center for the Development of Professional Qualifications of Medical Workers, Tashkent, Uzbekistan
Gafur-Akhunov M.A
Center for the Development of Professional Qualifications of Medical Workers, Tashkent, Uzbekistan
Nishanov D.A
Republican Specialized Scientific and Practical Medical Center of Pathological Anatomy, Tashkent, Uzbekistan
Yigitaliyev A.B
Fergana Medical Institute of Public Health, Fergana, Uzbekistan
Received:
14 December 2024;
Accepted:
16 January 2025;
Published:
18 February 2025
Abstract:
This article is devoted to immunohistochemical studies associated with primary liver cancer
(hepatocellular carcinoma - HCC) developed against the background of chronic viral hepatitis B, C. The topic is
relevant, since HCC is one of the aggressive tumors with a high mortality rate, arising as a result of chronic hepatitis
B and C, alcoholic or non-alcoholic fatty liver diseases. The study included 30 patients examined in 2020-2023, all
of whom were diagnosed with viral hepatitis B or C. In the immunohistochemical study, molecular genetic markers
(Ki-67, Bcl-2, VEGF, and p53) were studied. According to the results, in cases of HCC associated with viral hepatitis,
high proliferative activity (Ki-67 >20%), apoptosis index (Bcl-2), angiogenesis activity (VEGF), and p53 gene
suppressor mutations were observed more often. Molecular-genetic markers in primary liver cancer are
important in assessing the aggressiveness of the tumor, the degree of metastasis, and the response to treatment.
The research results open up new possibilities for improving the diagnosis, prognosis, and personalized therapy
strategies for HCC.
Keywords:
Hepatocellular carcinoma, Ki-67, Bcl-2, VEGF, p53 gene suppressor, immunohistochemical
examination. Chronic hepatitis, liver cancer, proliferative activity, apoptosis, angiogenesis mutation.
Introduction:
Relevance of the problem. Primary liver
cancer (hepatocellular carcinoma, HCC) is one of the
most aggressive and fatal malignant tumors. The
development of this tumor is associated with chronic
liver diseases, including: viral hepatitis B and C
infections, alcoholic liver disease, non-alcoholic fatty
liver disease [1,2].
In recent years, molecular genetic markers that allow
International Journal of Medical Sciences And Clinical Research
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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)
predicting the course of the disease, the likelihood of
metastasis, and the response to therapy have been
actively studied. The most important markers in
primary liver cancer are Ki-67, Bcl-2, VEGF, and p53.
These biomarkers play an important role in the
mechanisms of: tumor cell proliferation, formation of
new blood vessels (angiogenesis), cell death
(apoptosis), which is very important for determining
their diagnosis, prognosis, and treatment tactics
[3,4,5].
Ki-67 - a marker of tumor proliferation activity - is a
protein expressed in all phases of the cell cycle (except
for the G0 phase) and is an important marker of the
degree of cell proliferation. High Ki-67 expression is
associated with tumor aggressiveness, rapid growth,
and a poor prognosis [6]. Studies show that high Ki-67
expression in HCC is associated with: high mitotic
activity, rapid progression (development), low-grade
differentiated tumors, shortened patient lifespan [7.8],
high Ki-67 levels can be an independent prognostic
marker in the late stages of the disease [9]. Patients are
classified based on Ki-67 expression: patients with high
Ki-67 levels require aggressive treatment methods,
including targeted therapy and systemic treatment
[10].
Bcl-2 - a protein that regulates apoptosis (B-cell
lymphoma 2) - is an anti-apoptotic protein that
prevents apoptosis (natural cell death). Disruption of
apoptosis leads to the survival of cancer cells [11].
Studies show that Bcl-2 expression was detected in 40-
60% of HCC cases. Its high level is associated with:
resistance of cancer cells to apoptosis, low sensitivity
to chemotherapy, and a high risk of relapse [12,13].
Determination of the Bcl-2 level allows predicting the
sensitivity of the tumor to treatment. Bcl-2 inhibitors
(for example, venetoclax) are considered promising
drugs in the treatment of HCC [14].
VEGF - angiogenesis factor (vascular endothelial
growth factor) - the main protein regulating
angiogenesis, stimulating the formation of new blood
vessels for the blood supply of the tumor [15]. High
VEGF
expression
is
associated
with:
active
angiogenesis, high vascularization (blood supply) of the
tumor, a high risk of metastasis, and a shorter patient
lifespan [16,17]. Anti-VEGF therapy (bevatsizumab,
ramutsirumab) is used in the treatment of HCC and
improves the prognosis in patients with high levels of
VEGF [18].
p53 - tumor-suspending protein - is a protective protein
against tumors that controls the cell cycle and regulates
apoptosis. p53 mutations lead to uncontrolled cell
proliferation and cancer development [19]. p53
mutations have been detected in 25-50% of HCC cases.
impaired p53 function is associated with tumor
aggressiveness, therapy resistance, and the risk of
metastasis [20]. determining p53 levels helps predict
treatment response. MDM2 inhibitors (negative
regulators of p53) are considered as promising targeted
therapy for HCC [21].
Thus, molecular genetic markers Ki-67, Bcl-2, VEGF, and
p53 play an important role in the pathogenesis and
development of hepatocellular carcinoma (HC). Their
study will allow improving the diagnosis of the disease
and predicting its course, choosing personalized
therapy, and improving the development of new
targeted drugs for HCC [18,19,20]. Further study of
these biomarkers is necessary for the early detection of
primary liver cancer and the development of effective
treatment strategies.
METHODS
In the study, 30 patients with primary liver cancer
examined and treated at the Republican Specialized
Scientific and Practical Medical Center of Oncology and
Radiology and its Tashkent regional branch underwent
retrospective and prospective immunohistochemical
examination in 2020-2023. In this case, all patients had
a history of viral hepatitis B and C and received
treatment. Later, after chronic hepatitis, he was
diagnosed with primary hepatocellular carcinoma and
hospitalized. In these patients, the results of
histological examination and immunohistochemical
studies were studied. Pathomorphologically, the use of
the immunohistochemical method in hepatocellular
liver cancer, in which the study of molecular genetic
markers is carried out for the first time, reveals its
deeper morphologically significant features in
hepatocellular liver cancer developed against the
background of chronic viral hepatitis B,C, plays a special
role in determining treatment tactics and predicting
the disease.
Biopsies (trepan-biopsy, surgical material) obtained
from all patients were taken from paraffin blocks,
processed for immunohistochemical examination, and
examined by taking sections on a slide. The Ki67, bcl 2,
VHFR, and w p53 genes were studied by
immunohistochemical examination. The conducted
immunohistochemical examination method was
technically carried out as follows (Table No1).
Table No. 1
Stages of immunohistochemical (IHC) examination.
№
Procedure
Reagents
Duration
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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)
1
Prepare sections 4 µm
thick
Polylysine-coated slides
2
Drying sections
Room
temperature
24
hours
3
Drying in a thermostat
T: 55-60°C, 60
minutes
4
Deparaffinization
Ortho-xylene
10 minutes x 3
times
5
Dehydration
96% ethanol
3 minutes x 3
times
6
Rehydration
Distilled water
10 minutes
7
Antigen
retrieval
(Demasking)
Demasking buffer, T:
98°C
30-40 minutes
8
Washing
Tris-buffer solution (pH
7.5)
5 minutes
9
Blocking
endogenous
peroxidase activity
3% hydrogen peroxide
5 minutes
10
Washing
Distilled water
3 minutes
11
Incubate
with
primary
antibodies
Specific antibodies
20-30 minutes
12
Washing
Tris-buffer solution (pH
7.5)
5 minutes
13
Incubate with secondary
antibodies (detection)
Visualization system
20-30 minutes
14
Washing
Tris-buffer solution (pH
7.5)
5 minutes
15
Visualization with DAB
DAB-chromogen
5 minutes
16
Washing
Distilled water
3 minutes
17
Counterstaining
Mayer's hematoxylin
5 minutes
18
Washing
Tap water
1 minute
19
Dehydration
96% ethanol
2
times
x
5
minutes
20
Clearing (Despiriting)
Ortho-xylene
2
times
x
5
minutes
21
Mounting
Balsam, cover slip
For immunohistochemical examination, the expression
of Ki67, Vcl2, VGFR and w p53 monoclonal antibodies
in cells was studied using an immunohistoprocessor
"Bond Leica Australia" (Australia).
In our study, the study of the expression of molecular
structures in liver cancer cells led to the development
of viral hepatitis B, C in hepatocellular carcinoma.
RESULTS
The Ki67 indicator morphologically characterized the
staining of the cell nucleus in
hepatocellular liver cancer as follows: the obtained
results were assessed as a mild, moderate, and severe
positive reaction. The results of the study showed that
out of 30 patients, 5 (16.6%) had a mild positive
reaction, 10 (33%) had a moderate positive reaction,
and 15 (50%) had a high positive reaction. No negative
reaction processes were observed (Table No2).
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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)
Table No 2
Ki67 indicators in hepatocellular carcinoma associated with viral hepatitis B, C
Microscopically, liver cells with hyperplasia and
polymorphism of hepatocytes, trabeculae are not
preserved, malignant tumor cells form tumor islands of
polymorphic hepatocytes with pathological mitosis and
cell necrobiosis. The nuclei of tumor cells are stained
dark brown (Fig. 1).
Figure 1. A high degree of positive response to the Ki 67 marker in patients diagnosed with
hepatocellular carcinoma, viral hepatitis B (80%). IHC - Dab chromogen. Ob10. Ok40.
№
Level
Patients (N=30)
1
<10% low activity
5 (16.6%)
2
10-20% moderate activity
10 (33%)
3
>20% high proliferative activity
15 (50%)
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Figure 2. Degree of proliferative activity of tumor cells when hepatocellular carcinoma
develops against the background of viral hepatitis B.
To determine tumor apoptosis in hepatocellular
carcinoma with viral hepatitis B, the Vcl2 indicator was
studied in 30 patients. In patients, the Vcl2 marker was
used to determine tumor apoptosis, which regulates
cell death by controlling the permeability of the
mitochondrial membrane. The obtained results were
evaluated using the ALLRED method. It looks at how
many percent of the system's cells are positive for their
receptors and how well the receptors appear after
staining. These data are then combined to evaluate the
sample on a scale from 1 to 3. In this case, the minimum
score is 0 (negative), 1 point (low positive 10-30%), 2
points (medium positive 30-60%), 3 points (high
positive 60-100%). Of the 30 selected patients, 4
(13.3%) had a mild positive reaction, 12 (40%) had a
moderate positive reaction, and 14 (46%) had a high
positive reaction (Table 3).
Table No. 3
Positive reaction of the Bcl-2 marker in patients with viral hepatitis B,C in
hepatocellular carcinoma
Microscopically: liver cells with hyperplasia and
polymorphism of hepatocytes, trabeculae are not
preserved, malignant tumor cells form tumor islands of
polymorphic hepatocytes with pathological mitosis and
cell necrobiosis. The cell membranes of malignant
tumors are stained dark brown.
№
Level
Patients (N=30)
1
<10% low activity
4 (13.3 %)
2
10-20% moderate activity
12 (40 %)
3
>20% high proliferative activity
14(46 %)
0
2
4
6
8
10
12
14
16
18
20
Negative reaction
Low positive reaction
Moderate positive
reaction
High positive
reaction
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Figure 3. High positive reaction of the Bcl-2 marker in patients diagnosed with viral hepatitis
B in hepatocellular carcinoma. (70%). IHC - Dab chromogen. Ob10. Ok40.
Figure 4. Level of Bcl-2 marker expression in patients diagnosed with viral hepatitis B in
hepatocellular carcinoma.
The results of w p53 expression in hepatocellular
carcinoma with viral hepatitis B were assessed using
the ALLRED method. It considers what percentage of
system cells are positive for receptors and how well
receptors appear after staining. Then these data were
combined for a sample assessment on a scale from 1 to
3. The minimum score was 0 (negative), 1 point (low
positive 10-30%), 2 points (medium positive 30-60%), 3
points (high positive 60-100%). Of the 30 selected
patients, 7 (23.3%) had a mild positive reaction, 10
(13.3%) had a moderate positive reaction, and 13
(43.3%) had a high positive reaction (Table 4).
0
2
4
6
8
10
12
14
16
Negative reaction
Low positive
reaction
Moderate positive
reaction
High positive
reaction
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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)
Table No4.
Level of expression of the p53 marker in hepatocellular carcinoma against the
background of viral hepatitis C.
When viewed under a microscope, liver cells with
hyperplasia and polymorphism of hepatocytes,
trabeculae are not preserved, malignant tumor cells
form
tumor
islands
of
polymorphic hepatocytes with pathological mitosis and
cell necrobiosis. The nuclei of malignant tumor cells are
stained dark brown (Fig. 3).
Figure 5. High positive reaction of the p53 marker in patients diagnosed with viral hepatitis B
in hepatocellular carcinoma. (90%). IHC - Dab chromogen. Ob10. Ok40.
№
Level
Patients (N=30)
1
1 point low position reaction
(10-30%)
7 (23.3 %)
2
2 points average positive reaction
(30-60%)
10 (33.3 %)
3
3 points high positive reaction
(60-100%)
13 (43.3 %)
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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)
Figure 6. Level of the p53 marker in patients diagnosed with viral hepatitis C in
hepatocellular carcinoma.
In detected hepatocellular carcinoma of viral hepatitis
C. The obtained results were considered in what
percentage of cells the markers were positive and how
expressive they were after staining. These data were
then combined to evaluate the sample on a scale from
1 to 3. The minimum score is 0 (negative), 1 point (low
positive 10-30%), 2 points (medium positive 30-60%), 3
points (high positive 60-100%). Of the 30 selected
patients, 8 (26.6%) had a mild positive reaction, 12
(40%) had a moderate positive reaction, and 10 (33.3%)
had a high positive reaction (Table 5).
Table 5.
Level of expression of the VGFR marker in patients with viral hepatitis C in
hepatocellular carcinoma
Microscopically: liver cells with hyperplasia and
polymorphism of hepatocytes, trabeculae not
preserved, malignant tumor cells form tumor islands
with
pathological mitosis of polymorphic hepatocytes and
cell necrobiosis. The endothelium of blood vessels is
stained dark brown.
№
Level
Patients
(N=30)
1
1 point low position reaction
(10-30%)
8 (26.6 %)
2
2 points average positive reaction
(30-60%)
12 (40 %)
3
3 points high positive reaction
(60-100%)
10 (33.3 %)
0
2
4
6
8
10
12
14
Negative reaction
Low positive
reaction
Moderate
positive reaction
High positive
reaction
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Figure 7. High positive reaction of the VHFR marker in patients diagnosed with viral
hepatitis B in the light cell type of hepatocellular carcinoma. (90%). IHC - Dab chromogen. Ob10.
Ok40.
Figure 8. The level of the VHFR marker in patients with viral hepatitis C in hepatocellular
carcinoma.
CONCLUSION
In our study, 30 patients were selected for
immunohistochemical examination of patients with
viral hepatitis B, C diagnosed with hepatocellular
carcinoma. The results obtained in all patients were
evaluated as a percentage of the marker of
proliferative activity of Ki 67 tumor cells.
The obtained results were assessed as mild, moderate,
and severe positive reactions. Of the 30 observed
patients, 5 (16.6%) had a mild positive reaction, 10
(33%) had a moderate positive reaction, and 15 (50%)
had a high positive reaction. No negative reaction
processes were observed.
For the purpose of detecting tumor apoptosis Bcl-2 in
hepatocellular carcinoma with identified viral hepatitis
B, C. Of the 30 selected patients, 4 (13.3%) had a mild
positive reaction, 12 (40%) had a moderate positive
reaction, and 14 (46%) had a high positive reaction.
0
2
4
6
8
10
12
14
Negative reaction
Low positive
reaction
Moderate
positive reaction
High positive
reaction
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In hepatocellular carcinoma with viral hepatitis B, out
of 30 patients selected for the gene suppressor - p53, 7
(23.3%) had a mild positive reaction, 10 (33.3%) had a
moderate positive reaction, and 13 (43.3%) had a high
positive reaction.
VHFR, a signaling protein produced by cells to stimulate
angiogenesis in hepatocellular carcinoma with viral
hepatitis C, was studied.
Of the 30 selected patients, 8 (26.6%) had a mild
positive reaction, 12 (40%) had a moderate positive
reaction, and 10 (33.3%) had a high positive reaction.
The results of the study showed that in hepatocellular
carcinoma developed against the background of viral
hepatitis B, C, the indicators of molecular genetic
markers Ki 67, Bcl-2 gene-suppressor p53 and VEGF are
high, which are considered factors negatively affecting
the tactics and prognosis of its progression.
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