Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 3, 2021, Pages.

98 - 108
Received 16 February 2021; Accepted 08 March 2021.

Diagnostic significance of Serum Clustrin and Heat Shock Protein 70 in

Hepatocellular Carcinoma Egyptian Patients
Hisham Elkhayat 1, Mohamed A. Aboul-Ezz1, Ali Abdel Rahim1, Shereif A. Morsy1,
Ahmed R. Mashaal1, Mohamed A. Elrefaiy1, Mohamed H. Abdelmaksoud3, Marwa S.
Wahdan2, Rabab Fouad2, Omar M. Sabry2
1
Hepato-gastroenterology Department, Theodor Bilharz Research Institute, Giza, Egypt.
2
Hematology Department, Theodor Bilharz Research Institute, Giza, Egypt
3
Radiology department, Theodor Bilharz Research Institute, Giza, Egypt
Emails: [email protected], [email protected], [email protected],
[email protected], [email protected], [email protected],
[email protected], [email protected], [email protected],
[email protected]

Abstract:Background: The most popular primary liver cancer and the second common reason of cancer-
related mortality globally is hepatocellular carcinoma (HCC). In Egypt it is considered the second cause
of cancer related mortality among men. Early detection of HCC increases the success of curative
treatments. Both US and AFP combined usage improves detection rates, yet expenses and false-positive
rates also increase. Increased secreted Clustrin (CLU) expression is correlated with radioresistance,
chemoresistance and resistance to hormones, rendering CLU a promising antitumor therapeutic goal. A
highly conserved stress response protein is heat shock protein 70 (HSP70). It protects the cell and
promotes a range of stimuli to induce repair.
Aim: The objective of this research was to investigate the diagnostic value of CLU and HSP70 in HCC
patients in order to improve the outcome of HCC patients through early diagnosis.
Methods: A total of 120 individuals have been enrolled in this research. They were classified into 3
groups: Group 1 involved 10 healthy individuals who served as a control group. Group 2 included 20
patients diagnosed with liver cirrhosis. Group 3 HCC group was divided into two subgroups: Group (3
A): 60 patients with proved HCC before treatment. Group (3 B): 30 patients with HCC were treated using
interventional radiology and followed up for 3 months post treatment. Serum levels of AFP, Clustrin and
Heat shock protein 70 were assayed using ELISA technique.
Results: The comparison between healthy control group and HCC patients without any intervention
revealed that serum CLU cutoff was 132.2 ng/ml with sensitivity of 96%, specificity of 97 % and
accuracy of 95 %, furthermore, serum HSP 70 cut off was 38.0 ng/ml with sensitivity of 94%, specificity
of 95 % and accuracy of 98 %. While AFP cut off became 114.4 ng/ml with sensitivity of 90 %,
specificity of 92 % and accuracy of 91 %. These results indicate that serum CLU level and serum HSP 70
level are better diagnostic markers for HCC detection than serum AFP level.
Conclusion: CLU and HSP70 are promising and potentially complementary candidate biomarkers for
effective detection of early stageHCC.

Key words: Clustrin, Heat shock protein 70 and HCC.

INTRODUCTION

The most popular primary liver cancer and the second common reason of cancer-related
mortality globally is hepatocellular carcinoma (HCC).1 The high incidence of HCV infection,
accompanied by increasing rates of obesity, alcohol abuse and uncontrolled type II diabetes, is
the risk factor that mainly leads to the increase in HCC.2Egypt has one of the largest burdens of

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worldwide hepatitis C virus (HCV) infection; it is predicted that HCV incidence is about 4.5% to
6.7%.3
Radiological and/or laboratory techniques are used to diagnose HCC. Ultrasonography (US),
triphasic computerized tomography (triphasic CT-scan) and dynamic magnetic resonance
imaging (dynamic MRI) are primarily relays of radiological diagnosis. The US sensitivity of
HCC detection is directly correlated to the size of the tumor. Another main downside of the US
is that it is very reliant on operators.4Laboratory diagnosis of HCC, on the other hand, is
developed either by measuring circulating biomarkers or by fine-needle cytology that is invasive
with intra-or inter-observer variance.5
Surveillance is the only practical approach to improve the management of HCC. Early detection
of HCC increases the success of curative treatments. 6 Serological tests include AFP with a cut
off value 200 ng/ml, is suggested to provide the optimal balance between sensitivity and
specificity. However, its estimated sensitivity is only 60%. Therefore, AFP is considered an
inadequate screening test. 7 US is widely used for surveillance. Its specificity is greater than 90%.
Both US and AFP combined usage improves detection rates, yet expenses and false-positive
rates also increase.
The European Association for the Study of Liver Disease (EASL) and the American Association
for the Study of Liver Disease (AASLD) have recommended guidelines for practice requiring
further analysis of nodules found with dynamic imaging technology during U.S. surveillance,
including contrast-enhanced U.S. or dynamic MRI, to demonstrate the various vascular supplies
of HCC versus non-malignant entities.6 In addition to biannual follow up to determine tumor
doubling time.8
The early diagnosis of HCC is needed as only 44% of the patients are diagnosed at a localized
stage of the disease, leading to limited treatment choices and poor prognosis. The identification
of diagnostic and prognostic biomarkers is a significant problem, as these indicators can make it
easier to diagnose HCC. In addition, these biomarkers can provide possible therapeutic targets
for HCC.9&6
Heat shock protein 70 (HSP70) is a highly conserved stress response protein. It protects the cell
and promotes a range of stimuli to induce repair. HSP70 is expressed under physiological and
stress conditions, including carcinogenesis.10 Hsp70 is an effective inhibitor of apoptosis and is
often constitutively over expressed in tumors.11In cancer patients, serum HSP70 levels have been
shown to be substantially higher relative to healthy people, sufferers without liver diseases and
hepatitis.12
Clusterin (CLU) is a 449-amino acid, heterodimeric glycoprotein which is expressed and found
in the majority of body fluids. Radioresistance, chemoresistance and hormonal resistance are
correlated with increased expression of the secreted CLU, rendering CLU a promising goal for
antitumor therapies.13Two proteins encoded by this gene are present in humans: secretory CLU
protein (sCLU) (75-80 kDa) and nuclear CLU protein (nCLU) (55 kDa).14&15

PATIENTS AND METHODS

Study population and demographic information

This study was conducted at Hepato-Gasteroenterology department, Theodor Bilharz Research
Institute and written informed consent was given by all participants. Within the ethical guidelines
of the Helsinki Declaration of 1975.A total of 120 individuals were enrolled in this research.
They have been categorized into 3 groups:

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Group I involved 10 healthy individuals who served as a control group (6 males/ 4 females) and
whose age ranged from 32 to 70 years (mean ± SD =53.2±16.7).
Group II included 20 patients diagnosed with liver cirrhosis (12 males/ 9 females), and their age
ranged from 43 to 58 years (mean± SD=50.5±7.5). Group III HCC group, which divided into
two subgroups:
- Subgroup (3A): 60 patients with proved HCC with no history of any radiological intervention
or any intervention for HCC (Before treatment), (50 males/ 10 females), and their age ranged
from 46 to 66.5 years (mean± SD= 56.7±9.8).
- Subgroup (3B): 30 patients with HCC in group 3A were continue follow up after 3 months of
treatment (interventional radiology) (25 males/ 5 females) whose age ranged from 53 to 62 years
(mean ± SD =57.7±4.6).
Exclusion criteria for study and for interventional radiology included malignancy other than
HCC, hepatic metastatic lesion , Patients receiving antitumor treatment, Patients with history of
ischemic cardiac disease in the preceding six months, Uncontrolled hypertension, unstable
angina, inadequate kidney functions (S.creatinine >1.5 mg/dl), Obesity and overweight.
In complicated cases, all patients included in this research underwent full medical history and
complete clinical assessment with special liver and splenic stress (hepatosplenomegly), presence
of ascites, symptoms of failure of liver cells such as jaundice, palmer erythema, lower limb
edema or encephalopathy.

Laboratory investigations including:

 Complete blood picture was performed using automated cell counter, Medonic, Boule
Diagnostics, Sweden. Prothrombin time (PT), Prothrombin concentration (PC) and
international normalized ratio (INR) was performed using fully automated haemostasis
analyser STA Compact®, Diagnostica Stago, Spain. Blood Chemistry including aspartate
aminotransferase (AST), alanine aminotransferase (ALT), total/ direct serum bilirubin, total
protein, serum albumin and alk.phosphatase (ALP), serum creatinine and serum urea were
performed on Olympus AU480 Chemistry Analyzer, Beckman Coulter, USA. Viral markers
including HCV immunoglobulin G (HCV IgG) and hepatitis B surface antigen (HBsAg) were
performed on ADVIA Centaur CP Immunoassay System, Siemens, Germany.
 Serum AFP concentrations were determined using Human Alfa–feto protein (α-FP)
ELISA kit, Cat. No. ELH-AFP, RayBio® Inc., Georgia, USA. Serum Clustrin and
HSP70 concentrations were assayed using Human Clusterin (CLU), ELISA Kit, Cat. No.
In-Hu3203 INOVA® andHuman Heat Shock Protein 70 (HSP70) ELISA Kit, Cat. No.
In-Hu3299 INOVA®, both from Innova Biotech Co Limited, Beijing, China. The optical
densities of standards and samples were determined using microplate reader (Tecan,
Sunrise™Japan). The standard curve was created by decreasing the data using
Magellan™ softwarethat can produce a log/log curve-fit.

Imaging studies:
Abdominal ultra-sonography (U/S) and Tri-phasic CT (Triphasic abdominal CT scan was
performed on HCC group only. Evaluation of the severity of liver cirrhosis was obtained for all
cirrhotic patients with Child Pugh Score.

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Statistics analysis methods:

Using an unpaired (t) test, the data is expressed as a mean ± SD or number (%) comparison
among the mean values of the different parameters in the two groups. Comparison among
categorical data was carried out using (Chi Square test) correlation between different variables
was performed using (pearson correlation), version of the SPSS software (12) was being used in
data analysis (p) value less than or equal to 0.05 was regarded to be significant and < 0.01 was
regarded to be highly significant.

RESULTS

Table (1): Clinical presentations of patients of the studying groups.

G3 P. value
G2( N=20)
A(N=60) B(N=30) A B C
Asymptomatic 12 (60%) 6 (10.0%) 6 (20%) 0.001** 0.09 0.001**
Rt hypochondrial pain 3 (15%) 42 (70.0%) 28 (93.3%) 0.001** 0.001** 0.001**
Anorexia & weight loss 4 (20%) 48 (80.0%) 19 (63.3%) 0.001** 0.001** 0.9
Splenomegaly 18 (90%) 24 (40.0%) 25 (83.3%) 0.09 0.09 0.9
Low grade fever 15 (75%) 18 (30.0%) 6 (20.0%) 0.001** 0.001** 0.3
L.L edema 14 (70%) 24 (40.3%) 14 (46.7%) 0.08 0.02* 0.003**
Hepatic Encephalopathy 14 (70%) 18 (30.0%) 8 (26.7%) 0.03* 0.001** 0.004**
History of Jaundice 14 (70%) 24 (40.0%) 22 (73.3%) 0.06 0.08 0.6
History of Ascites 14 (70%) 0 0 0.06 0.001** 0.001**
Radiological finding
Ultrasound
20 (100.0%) 59 (98.34%) 30 (100.0%) 0.07 0.9 0.9
-Liver Cirrhosis
Splenomegaly 18 (90%) 56 (93.34%) 29 (97.0%) 0.09 0.03* 0.1
-Ascites 14 (70%) 0 0
-PVT 0 (0.0%) 20 (33.3%) 0 (0.0%) 0.002** - 0.002**
-Focal lesion 0(0.0%) 60(100.0%) - - - -
CT (Focal lesion) 0(0.0%) 60(100.0%) 0% - - -
All parameters are represented as frequency and percent.
P. value bearing (a) initial is significantly different between G2 and G3A groups.
P. value bearing (b) initial is significantly different between G2 and G3B groups.
P. value bearing (c) initial is significantly different between G3A and G3B groups.
*P value ≤ 0.05 significant while **P value ≤ 0.01highly significant.

Table (2): Child-Pugh classification in patients of the studied groups.

G2 G3 P. value
(N=20) A (N=60) B (N=30) A B C
A 7 (35.0%) 12 (20.0%) 12 (40.0%)
Child B 9 (45.0%) 30 (50.0%) 18 (60.0%) 0.001** 0.001** 0.001**
C 4 (20.0%) 18 (30.0%) 0 (0.0%)
*P value ≤ 0.05 significant while **P value ≤ 0.01highly significant.

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Table (3):Comparison between all studied groups as regard serum clusterin (ng/ml) and heat
shock protein 70 (ng/ml)
Serum clustrin assay range: 2 ng/ml – 140 ng/ml)
P. Value
G1(N=10) G2(N=20) G3A(N=60) G3B (N=30
A B c
10.9±0.5 11.2±0.6 457.2±24.0 115.5±2.9
0.01* 0.001** 0.001**
(2 - 22) (6 – 41) (205 – 880) (95 – 136)
Serum HSP70 assay range: 0.8 ng/ml – 40 ng/ml
3.5±0.3 6.6±2.2 87.8±3.7 33.8±0.5
0.04* 0.03* 0.001**
(1 – 8.6) (3 – 15.2) (53 – 166) (20 – 36.5)
*P value ≤ 0.05 significant while **P value ≤ 0.01highly significant.

Regarding serum level of AFP There was highly significant increase in G3a (240.1ng/mL)
compared to other groups G1 (2.1 ng/mL) (P value= 0.001), G2 (10 ng/mL) and G3B (80
ng/mL) (P value= 0.01,).
The comparison between healthy control group (G1) and cirrhotic (G2) patients revealed that
CLU cutoff was 11.1 ng/ml with a sensitivity of 57%, specificity of 77 % and accuracy of 65 %.
Moreover, HSP 70 cutoff was 3.5 ng/ml with sensitivity of 76%, specificity of 69 % and
accuracy of 74 %, on the other hand, AFP cut off 4.4 ng/ml with sensitivity of 95 %, specificity
of 92 % and accuracy of 94 %.(Fig1)

Fig. (1): Receiver operating characteristic (ROC) curve of S.CLU and S.HSP 70 as a diagnostic
marker for HCC with normal or high Alpha Fetoprotein level. (G1 X G2)
The comparison between healthy control group (G1) and HCC patients without any intervention
(G3A) revealed that serum CLU cutoff was 132.2 ng/ml with sensitivity of 96%, specificity of
97 % and accuracy of 95 %, furthermore, serum HSP 70 cut off was 38.0 ng/ml with sensitivity
of 94%, specificity of 95 % and accuracy of 98 %. While AFP cut off was 114.4 ng/ml with
sensitivity of 90 %, specificity of 92 % and accuracy of 91 %.(Fig 2)

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Fig. (2): Receiver operating characteristic (ROC) curve of S.CLU and S.HSP 70 as a diagnostic
marker for HCC with normal or high Alpha Fetoprotein level. (G1 X G3A)

The above results indicate that serum CLU level and serum HSP 70 level are better diagnostic
markers for HCC detection than serum AFP level.
There was a positive association among AFP and CLU, AFP and HSP 70 (r: +0.425, +0.274
respectively) and There was a positive association among CLU and AFP, CLU and HSP 70 (r:
+0.425, +0.778 respectively). Also, there was a positive correlation between HSP 70 and AFP,
HSP 70 and CLU (r: +0.274, +0.778 respectively).
There was a positive association among AFP and both AST and ALT (r: +0.241, +0.258
respectively). Also, WBC count showed positive correlation with AFP (r: +0.400). In addition,
there was a positive correlation between CLU and both AST (r: +0.366) and WBC count (r:
+0.228). Finally, there was a positive correlation between HSP 70 and AST (r: +0.382).

DISCUSSION

HCC accounts for more than 80% of the world's primary liver cancers. HCC has a heavy disease
burden and is a major cause of cancer-related mortality in several regions of the world and is
reported to be the world's most common cause of cancer-related mortality.16 For men, the risk is
higher than for women, and it rises as they get older.17
It is believed that hepatocellular carcinoma advances through a multi-step process in which
dysplastic nodules form in the cirrhotic liver, advancing with the accumulation of genetic
mutations to HCC. The development of neoplasia involves many components including,
insensitivity to growth-inhibitory signals, inhibition of apoptosis, unlimited replicative potential,
tissue invasion through metastasis and persistent angiogenesis.18
HCC surveillance is one of the preventive approaches to reduce the burden of HCC via early
tumor identification and effective early management. In patients with liver cirrhosis, HCC
monitoring is suggested.16
The standard HCC monitoring test approved by the American Association for the Study of Liver
Diseases is liver ultrasonography (AASLD).16 Detection of high serum AFP levels is widely
employed as an adjunct to liver ultrasonography in blood-based surveillance studies. The normal
serum AFP level range is 10-20 ng/ml and the diagnostic value is commonly considered to be
>400 ng/ml. Nevertheless, two-thirds of HCC sufferers with a nodule smaller than 4 cm of serum

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AFP >200 ng/ml do not have AFP in up to 20 % of HCC sufferers. However, the lack of AFP
specificity and sensitivity has clarified the need for a new tumor marker to distinguish HCC from
benign liver disorders. 18
Recent studies have identified HSP70 gene as the most abundantly up regulated gene in early
HCC. Similarly, CLU has been suggested as a viable biomarker for the early detection of HCC. 19
Secretory CLU provided a potential molecular target for HCC therapy.20
Precision medicine with novel adjuvant therapy using HSP70 mRNA transfected dendritic cell
(DC) improved prognosis of HSP70-expressing HCC cases.21HCC patients with elevated CLU
expression also showed poor oxaliplatin response.22
The aim of this research was to examine the diagnostic value of CLU and HSP70 in HCC
patients in order to improve the outcome of HCC patients through early diagnosis.
The demographic data retrieved from this study revealed a significant older age difference
between HCC group and other studied groups in agreement with the report from Velazquez et
al.23who observed that patients over 54 years of age were four times at risk of developing HCC
than younger ones. The data also revealed that 83.3 % of HCC patients are males andthis is
compatible with the study conducted by El-Zayadi et al., 24 who noticed that HCC was more
prevalent in males than females. The authors stated that hepato-carcinogenesis could be
modulated by estrogens and androgens and attributed the higher occurrence of HCC in males due
to differences in exposure to risk factors.
In the current research, there was a significant association among HCC and smoking. These
results were in agreement with Siegel et al. 25 who recorded that 50.2% of patients with HCC had
a smoking history. Heavy smokers have an increasing risk about 50% higher than nonsmokers,
this may be due to presence of cytochrome P450 system which is highly inducible by smoking
mainly cytochrome 1A1 that is a type of cytochrome p450 that is present in the lung and is
important for metabolizing polycyclic aromatic hydrocarbons inhaled by smoking converting
these procarcinogen into active carcinogen by hydroxylation reactions.26
Viral markers were analyzed in all subjects enrolled into the current study. The data showed that
almost all HCC patients (96.7%) were HCV infected. These results are consistent with data from
Atti 27. While HBV is regarded worldwide as a significant risk of liver cirrhosis and HCC, over
the past 2 decades, the incidence of HBV infection in Egypt has decreased, whereas HCV has
risen. Egypt may have the highest prevalence of HCV globally. 28
The Liver function tests of patients enrolled in this study showed a highly significant difference
between the liver cirrhosis and HCC without radiological intervention groups regarding AST.
Wang et al. 29 evaluated the impact of hepatitis using AST and ALT as indices for injury to
hepatocytes. The significant elevation of both AST and ALT with high level of AFP may be due
to their release into the blood from damaged hepatocytes, their activities have been broadly
known as useful tools for detecting liver diseases like HCC. 30Also, cases of HCC were
associated with low platelet counts. This finding is in agreement with Velazquez et al., 31
Furthermore; it reflects a greater hepatic dysfunction and highlights the consequences of chronic
disease.
This study revealed a highly significant difference in AFP levels between HCC and control
group. In agreement with Wang et al.29who found increased AFP level in HCC
patientscompared to control.
The current study revealed that serum CLU levels in the HCC group were higher compared to
the control and liver cirrhosis groups, suggesting its function in carcinogenesis. Similarly, Nafee
et al.32recorded a substantial increase in serum CLU in HCV-related HCC sufferers.

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Furthermore, Kang et al.33 used tissue microarray to reveal immunohistochemical

overexpression of CLU in surgically resected HCCs. Additionally, Hsieh et al. 34indicated that in
human hepatoma, dysregulation of the CLU gene was most probably attributed to cellular
responses to external stresses or micro-environmental shifts that may have a significant impact
on gene or protein expression.
The current study indicated that serum HSP 70 was higher in the HCC patients compared to both
liver cirrhosis and control groups. This finding denoted the role of HSP70 in hepato-
carcinogenesis. Also, Cervello et al. 35and Gehrmann et al. 12noticed that serum HSP70 levels
in HCC patients were substantially higher relative to healthy people and hepatitis patients. The
authors reported that HSP70 serum may be a beneficial biomarker for the differentiation between
HCC and other hepatic diseases.
Several studies have investigated the diagnostic efficiency of biomarkers under study in HCC. In
the current research, the comparison between the control group (G1) versus HCC patients
without any intervention (G3A) revealed that serum CLU was more sensitive, specific and
accurate than AFP. This indicated that CLU was superior to AFP as a diagnostic marker in HCC
patients.
Similarly, Wang et al.29 demonstrated that serum CLU with a 50 ng/ml cutoff level
outperformed serum AFP with a 15 ng/ml cutoff level in diagnostic sensitivity (91% versus
67%), specificity (83% versus 76%), predictive values that are positive and negative (93% versus
88% and 77% versus 47% respectively).
Additionally, the study by Kimura et al. 36performed three-step proteome analyses on serum
samples of HCC patients and 83 up-regulated proteins were found, of which CLU was the most
substantially over-expressed.Using another group of HCC specimens, the overexpression of CLU
was verified by ELISA and the authors indicated that CLU was a possible novel serum indicator
for HCC. Nafee et al., 2012 also confirmed the same finding.32
On the contrary, Ramadan et al. 37concluded that serum AFP with a 137 ng/ml cutoff level
outperformed serum CLU with a 135 ng/ml cutoff level in diagnostic sensitivity (77.3% versus
70.5%), specificity (100% versus 90%), predictive values that are positive and negative (100%
versus 86.1% and 83.3% versus 77.6%, respectively). In addition, the authors used combined
parallel method to improve diagnostic sensitivity (95.5 %) and negative predictive value (95.7
%) over the single use of AFP, but this led to a decrease in specificity of 88 % and a positive
predictive value of 87.5%. Therefore, combined parallel approach improved the sensitivity at the
expense of the specificity.
The present study revealed a highly significant difference regarding the serum level of HSP 70 in
HCC patients before treatment and HCC patients post treatment. This finding is attributed to the
ability of HSP70 to directly inhibit apoptosis as it is an effective inhibitor of apoptosis which is
constitutively over expressed in tumors. The anti-apoptotic potential of HSP70 should draw
attention towards the attractive role it could play in targeted therapy for cancer 38&11.
A positive association among AFP and both CLU and HSP70 was found in this study. Also,
there was a positive association among CLU and HSP70. Furthermore, there was a positive
correlation between both CLU, HSP70 and AST. These results were in agreement with
Ramadan et al.37and Mostafa et al. 39.

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CONCLUSION

Serum CLU and HSP70 levels in HCC patients were significantly higher than in non-HCC
patients. CLU and HSP70 are promising and potentially complementary candidate biomarkers
for effective detection of HCC at early stage. AFP shouldn’t be used as the sole diagnostic
biomarker for HCC. The combined assay of serum AFP, CLU and HSP70 would provide a
sensitive, rapid and easily accessible method for improving the diagnostic performance of
laboratory screening for HCC. Further studies with large sample size are required to investigate
and validate CLU and HSP70 as reliable sensitive and specific serum biomarkers for early
HCC detection.

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 3, 2021, Pages. 98 - 108
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