English NGHIÊN CỨU ĐỘT BIẾN VÙNG KHỞI ĐỘNG GEN hTERT VÀ BIỂU HIỂN hTERT mRNA TRONG MÁU NGOẠI VI Ở BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN CÓ HBsAg DƯƠNG TÍNH

NGHIÊN CỨU ĐỘT BIẾN VÙNG KHỞI ĐỘNG GEN hTERT VÀ BIỂU HIỂN hTERT mRNA TRONG MÁU NGOẠI VI Ở BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN CÓ HBsAg DƯƠNG TÍNH NGHIÊN CỨU ĐỘT BIẾN VÙNG KHỞI ĐỘNG GEN hTERT VÀ BIỂU HIỂN hTERT mRNA TRONG MÁU NGOẠI VI Ở BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN CÓ HBsAg DƯƠNG TÍNH NGHIÊN CỨU ĐỘT BIẾN VÙNG KHỞI ĐỘNG GEN hTERT VÀ BIỂU HIỂN hTERT mRNA TRONG MÁU NGOẠI VI Ở BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN CÓ HBsAg DƯƠNG TÍNH NGHIÊN CỨU ĐỘT BIẾN VÙNG KHỞI ĐỘNG GEN hTERT VÀ BIỂU HIỂN hTERT mRNA TRONG MÁU NGOẠI VI Ở BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN CÓ HBsAg DƯƠNG TÍNH MINISTRY OFEDUCATION AND TRAINING MINISTRY OFNATIONAL DEFENCE VIETNAM MILITARY MEDICAL UNIVERSITY === * === PHAM CHAU STUDY ON hTERT PROMOTER MUTATIONS AND hTERT mRNA EXPRESSION IN PERIP

MINISTRY OF

EDUCATION AND TRAINING

MINISTRY OFNATIONAL DEFENCE

VIETNAM MILITARY MEDICAL UNIVERSITY

=== * ===

PHAM CHAU

STUDY ON hTERT PROMOTER MUTATIONS AND hTERT

mRNA EXPRESSION IN PERIPHERAL BLOOD OF HEPATOCELLULAR CARCINOMA PATIENTS WITH

THIS DISSERTATION WAS COMPLETED AT

VIETNAM MILITARY MEDICAL UNIVERSITY

Scientific Supervisors:

1 Dr Ngo Tat Trung

2 Dr Duong Quang Huy

Reviewer 1: Associate Professor, Dr Vu Van Khien

Reviewer 2: Prof Dr Nguyen Linh Toan

Reviewer 3: Associate Professor, Dr Nguyen Dang Ton

The dissertation was defended at the School-Level DissertationDefense Council at the Vietnam Military Medical University at

The dissertation can be accessed at:

1 National Library of Vietnam

2 Library of the Vietnam Military Medical University

Telomeres are nucleoprotein structures composed of repetitiveDNA sequences located at the ends of chromosomes, serving to protectchromosome integrity The stable maintenance of telomere length isclosely associated with the processes of cancer cell formation andprogression Cancer cells can preserve telomere length throughtelomerase reactivation Telomerase activation occurs via variousmechanisms during oncogenesis, with telomerase re-expressionobserved in 90% of hepatocellular carcinoma (HCC) cases

The primary cause of increased telomerase activity specific tocancer is mutations in the promoter region of the telomerase reverse

transcriptase gene (hTERT) The most common mutations in the hTERT promoter region are C228T and C250T, with the C228T

mutation being the predominant alteration This mutation is detectedearly, from low-grade dysplastic nodules to high-grade dysplasticnodules in cirrhotic patients, ultimately leading to HCC development.Telomerase activity has also been shown to correlate positively with h

TERT mRNA expression both intracellularly and extracellularly Thus,

the detection of hTERT mRNA in peripheral blood serves as a potential

biomarker for cancer diagnosis and prognosis, including HCC In

Vietnam, simultaneous studies on the hTERT C228T mutation status and hTERT mRNA expression in peripheral blood for the diagnosis

and prognosis of hepatocellular carcinoma (HCC) remainunsystematic and limited Based on this reason, we conducted the

study titled: “Study on hTERT Promoter Mutations and hTERT mRNA

Expression in Peripheral Blood of Hepatocellular Carcinoma Patientswith Positive HBsAg.”

1 OBJECTIVES OF THE STUDY

1.1 To assess the value of the hTERT C228T mutation and hTERT

mRNA expression in peripheral blood for the diagnosis of

hepatocellular carcinoma with positive HBsAg.

1.2 To analyze the correlation between the hTERT C228T mutation and hTERT mRNA expression in peripheral blood with clinical, subclinical characteristics, and survival time in HCC patients with positive HBsAg.

2 NOVEL CONTRIBUTIONS OF THE DISSERTATION

This is the first study in Vietnam to simultaneously identify the

hTERT C228T mutation and hTERT mRNA expression in peripheral

blood in HCC patients with positive HBsAg, comparing it withcorresponding parameters in cirrhosis patients with positive HBsAgand chronic hepatitis B The results are as follows:

- The rate of the hTERT C228T mutation in the peripheral blood of

HCC patients with positive HBsAg was 25.2%, while this mutationwas not detected in patients with HBV-related cirrhosis or chronichepatitis B (p < 0.001)

- The hTERT C228T mutation has a diagnostic value for HCC with

a specificity of 100% Combining the hTERT C228T mutation with

plasma AFP for diagnosing HCC compared to the cirrhosis, chronichepatitis B, and non-cancer groups showed good AUC values (0.81,0.88, and 0.85, respectively), which were higher than when AFP wasused alone (AUC values of 0.76, 0.84, and 0.81, respectively)

- The rate of hTERT mRNA positivity in the peripheral blood of

HCC patients with positive HBsAg was 16.4%, which was higher thanthat in the cirrhosis group (8.1%) and the chronic hepatitis B group(3.1%), with a statistically significant difference (p < 0.001) Positive h

TERT mRNA was associated with a 6.19-fold (95% CI: 1.82 - 21.05)

and 3.71-fold (95% CI: 1.63 - 8.48) higher risk of HCC compared to

negative hTERT mRNA (chronic hepatitis B group and non-cancer

group, respectively), p = 0.001, but not significant when comparedwith the cirrhosis group

The rate of the hTERT C228T mutation increased progressively

with liver failure severity according to the Child-Pugh score, and it wasassociated with abdominal lymph node metastasis (p = 0.01), portalvein thrombosis (p = 0.04), and disease stage

HCC patients with the hTERT C228T mutation had a significantly

shorter survival time than those without the mutation (21.3 ± 2.7 vs.30.7 ± 1.5 months, respectively, p = 0.005)

3 STRUCTURE OF THE DISSERTATION

The dissertation consists of 138 pages, including: Introduction: 2 pages,Literature Review: 36 pages, Study Subjects and Methods: 25 pages,Research Results: 35 pages, Discussion: 37 pages, Conclusion: 2 pages,Recommendations: 1 page The dissertation includes 44 tables, 16 charts,and 8 figures It references 138 sources (11 Vietnamese sources, 127English sources)

CHAPTER 1: LITERATURE REVIEW

1.1 hTERT Promoter region mutations in HCC

Mutations in the hTERT promoter region are the most frequent

somatic mutations observed in various types of cancer Two commonmutation types, C228T and C250T, involve the substitution of cytidinewith thymidine (C>T) at positions 1,295,228 and 1,295,250 onchromosome 5, respectively (also known as -124 bp and -146 bp fromthe transcription start site, referred to as 124C>T and 146C>T) Studieshave also shown that the C228T mutation occurs more frequently than

the C250T mutation in most cancers Mutations in the hTERT promoter

region affect gene expression and contribute to the malignanttransformation of hepatocytes, as evidenced by several studies.Telomerase activity is crucial for maintaining telomere length,enabling cancer cells to acquire unlimited replicative potential

In recent years, hTERT has been considered to have a direct role in

the pathogenesis of HCC, beyond its function in telomerase

reactivation to maintain telomere length Several studies have shown

that hTERT functions as a transcriptional activator, stimulating the

transcription of target genes involved in the Wnt signaling pathwayand NF-κB, thereby promoting cell migration, resisting apoptosis, and

renewing stem cells In HCC, higher expression levels of hTERT have been observed in most cases with mutations in the hTERT promoter region compared to those without this mutation, and increased hTERT

expression is strongly correlated with HCC progression Based on the

significant correlation between the hTERT promoter region and the CTNNB1 mutation, as well as the interaction between hTERT and the

Wnt/β-catenin signaling pathway, it is suggested that mutations in the

hTERT promoter region, along with activation of the Wnt/β-catenin

signaling pathway, contribute to the malignant transformation of liver

cells Additionally, the integration of HBV DNA into the hTERT promoter region may initiate hTERT transcription, providing another

mechanism for hepatocellular carcinogenesis The integration of HBVDNA into the human hepatocyte genome in HBV-related HCC hasbeen confirmed, and the integration sites are not random but

predominantly occur near or within the hTERT gene.

Additionally, a meta-analysis of 13 studies found that hTERT is the

most susceptible gene to HBV infection This integration disrupts the h

TERT gene Since the regulation of hTERT gene expression largely

depends on the promoter region, especially the core functional

segment, the integration of HBV into the hTERT promoter plays a

critical role in the formation and progression of HCC HBV integrationpromotes HCC development by creating genomic instability,enhancing the expression of adjacent genes, producing mixed virus-host transcripts, causing secondary mutations in host or viral genes,and resulting in DNA and protein copy number variations withoncogenic activity

1.2 hTERT mRNA expression in peripheral blood of HCC

hTERT mRNA is the transcription product of the hTERT gene,

which is translated into the hTERT protein, the catalytic subunit of

telomerase, a reverse transcriptase enzyme that adds repetitivetelomeric sequences to the ends of chromosomes The expression

levels of hTERT mRNA are closely correlated with telomerase

activity, both in non-cancerous tissues and in HCC tissues, indicating

that hTERT is the key determinant of this enzyme's activity Many studies have confirmed the value of hTERT mRNA as a biomarker in

tissue samples and plasma for diagnosing and prognosticating HCC

Miura N et al (2003) reported that hTERT mRNA expression was not

detected in the serum of normal individuals, and it was independentlyassociated with serum AFP levels, tumor size, and cell differentiation

grade Notably, hTERT mRNA has also been shown to be a valuable

biomarker for early-stage HCC diagnosis and a prognostic marker for

patients after intervention Therefore, the expression of hTERT mRNA

in serum may serve as an important molecular and biological indicatorfor diagnosing and predicting the biological characteristics of HCC

CHAPTER 2: RESEARCH SUBJECTS AND METHODS 2.1 Study sujects: The study included 319 patients, divided into two

groups: the study group and the control group, who were examined andtreated as inpatients at 103 Military Hospital and 108 Central MilitaryHospital

Study Group: 159 patients with HCC and HBsAg positive

Control Group: 62 patients with cirrhosis and HBsAg positive, and

98 patients with chronic hepatitis B (CHB)

Study Period: From December 2020 to August 2024

2.1.1 Inclusion criteria

- Study Group: Diagnosed according to the Ministry of Health ofVietnam’s 2020 standards and with HBsAg (+)

- Control Group:

+ Cirrhosis Group: Meets the criteria for liver dysfunctionsyndrome and portal hypertension (those who do not meet both criteriawill be confirmed for cirrhosis by Fibroscan ultrasound showingfibrosis at stage F4 combined with an APRI score > 2), and HBsAg (+) + Chronic Hepatitis B Group: HBsAg and/or HBV-DNA (+) for atleast 6 months, or HBsAg (+) and anti-HBc IgM (-), combined withliver biopsy showing chronic hepatitis or Fibroscan showing liverfibrosis from F1 to F3

2.1.2 Exclusion criteria

- Study Group: Diagnosed and treated for HCC; patients with alcoholaddiction, co-infection with HCV, HIV; secondary liver cancer; history oflong-term drug use or exposure to hepatotoxic chemicals

- Control Group: Patients with alcohol addiction, co-infection

with HCV, HIV; history of drug use causing liver damage

2.2 Study methods

2.2.1 Study design: A descriptive cross-sectional study with controls

and a longitudinal follow-up

2.2.2 Sample size and sampling method: Convenience sample size.

The sampling method was purposive, including all patients who metthe inclusion and exclusion criteria

2.2.3 Equipment and chemicals used in the study: Equipment and

chemicals for blood testing, imaging diagnosis, and detection ofhTERT C228T mutation and hTERT mRNA expression

Table 2.1 Primer sequences, peptide clamps, and probes

for detecting hTERT C228T mutation

Primer Name Sequence (5’ - 3’)

Tr-hTERT-seq-F GTCCTGCCCCTTCACCTT

Tr-pNA-hTERT NH2-CCGGAGGGGGCT-CONH2

hTERT-probe FAM-CCC+C+T+T+CCGG-Iowa Black

Table 2.2 Primer sequences, peptide clamps, and

probes for detecting hTERT mRNA

Collect blood samples from patients with HCC, liver cirrhosis withHBsAg (+), and chronic hepatitis B at the time of admission

Identify the hTERT C228T mutation and hTERT mRNA

expression in peripheral blood at the Molecular Biology Departmentand the Vietnam-Germany Medical Research Center (VGCARE) –

108 Central Military Hospital:

- Isolate and extract cfDNA/ctDNA from plasma; extract and purify total RNA from blood samples and synthesize cDNA ; synthesize cDNA using RT-PCR technique

- Identify the hTERT C228T mutation using Nested-PCR combined

with realtime PCR

- Determine hTERT mRNA expression using realtime PCR.

HCC patients will undergo different treatment interventionsaccording to the Ministry of Health of Vietnam’s 2020recommendations

2.2.5 Research indicators

- Age and gender distribution across the study groups

- Clinical and paraclinical characteristics, liver failure severity,tumor burden, disease stages (BCLC and TNM) in the HCC group, andtreatment methods based on disease stages Follow-up of HCC patients

to record the time of death and survival duration at the end of the study

- hTERT C228T mutation: Positive/negative hTERT mRNA

expression: Positive/negative

- Diagnostic value of hTERT C228T mutation and hTERT mRNA

expression combined with AFP in comparison to the cirrhosis, chronichepatitis B, and non-cancer groups

- Correlation of hTERT C228T mutation and hTERT mRNA

expression with various factors (age, gender, clinical symptoms, liverenzyme activities AST/ALT, liver function, peripheral blood plateletcount, plasma AFP levels, tumor burden, disease stage, and survivalduration) in the study group

2.3 Data Analysis and Processing: Data were stored and managed

using Excel Office 365 Statistical analysis was performed using SPSSversion 26

2.4 Research ethics: The research proposal was approved by the

Ethics Council of the Military Medical University, Decision No.22/2021/CNChT-HĐĐĐ All patients voluntarily participated in thestudy, and the procedures were carried out in strict compliance with theregulations of the Ministry of Health Genetic mutation and expressiontests did not affect the treatment process or impose any financialburden on the patients

Evaluate the diagnostic value of

hTERT C228T mutation and hTERT

mRNA expression in HCC with

HBsAg (+)

Clinical, subclinical characteristics, and survival time

Rate of hTERT C228T mutation and hTERT mRNA expression in peripheral

blood

Control Group (n=160): Cirrhosis with HBsAg (+) (62), Chronic Hepatitis B (98)

HCC with HBsAg (+) Group

(n=159)

Study Subjects (n=319)

Chapter 3 RESEARCH RESULTS 3.1 Characteristics of the study HCC group

3.1.1 Age and gender characteristics

The mean age of the HCC patient group was 58.30 ± 11.39years 91.2% of the patients were male, with a male-to-female ratio of10.36:1

3.1.2 Clinical and paraclinical characteristics of HCC

Common subjective symptoms: right upper quadrant/hepaticregion pain (46.5%) The main objective physical finding wassplenomegaly

(12.6%)

Research design diagram

Median values of some laboratory tests: AST 51.5 (U/L); ALT 48.3(U/L); Total Bilirubin 14.9 (µmol/L); Prothrombin Time 85.0% Theproportion of patients with AFP ≥ 20 ng/mL was 63.5% (median102.93 ng/mL).

The majority of HCC patients had tumors located in the right lobe(70.4%), a single tumor (69.8%), a tumor size < 5cm (50.9%), and amean tumor size of 6.8 ± 3.9cm 15.1% of patients presented withtumor invasion forming a portal vein thrombosis (PVT), and 11.3%had abdominal lymph node metastasis

The majority of HCC patients had Child-Pugh class A cirrhosis(80.5%), tumors at stage 0/A (48.4%) according to the BCLC(Barcelona Clinic Liver Cancer) staging system, and stage II (44.0%)according to the TNM (Tumor, Node, Metastasis) staging system

3.2 Value of hTERT C228T mutation and hTERT mRNA

expression in the diagnosis of HCC with HBsAg (+)

Table 3.1 Rate of hTERT C228T mutation and hTERT mRNA

expression in HCC patients (n = 159) Characteristics Number (n) Rate (%)

The percentage of hepatocellular carcinoma patients with the

hTERT C228T mutation was 25.2%, and the percentage with hTERT

mRNA expression in peripheral blood was 16.4%

Table 3.2 Comparison of the hTERT C228T mutation rate

between the HCC Group and the Control Groups

Study Subjects hTERT C228T Mutation

p Positive n(%) Negative n(%)

< 0.001

The hTERT C228T mutation was observed only in the

hepatocellular carcinoma (HCC) group and was not detected in theHBV-induced cirrhosis and chronic hepatitis B groups, with astatistically significant difference (p < 0.001)

Table 3.3 Comparison of hTERT mRNA expression between the

HCC Group and the Control Groups Study

Negative n (%)

-0.001Chronic

Hepatitis

3 (3.1) 95 (96.9)HCC 26 (16.4) 133 (83.6) 3.7

1

1.63 8.48

-0.001Non-

cancerous

8 (5.0) 152 (95

.0)

The positive rate of hTERT mRNA in hepatocellular carcinoma

(HCC) patients was significantly higher compared to the chronichepatitis B group (16.4% vs 3.1%, respectively, p = 0.001) and the

non-cancerous group Positive hTERT mRNA was associated with a

higher risk of HCC, with an odds ratio of 6.19 (95% CI: 1.82 - 21.05) inthe chronic hepatitis group and 3.71 (95% CI: 1.63 - 8.48) in the non-

cancerous group, compared to negative hTERT mRNA.