Summary thesis’ doctor of medicine: Research of hepatitis C virus genotypes in hepatocellular carcinoma patients

This research aiming to: Describing clinical and characteristics laboratory features and identifying HCV genotype on patients of HCC. Evaluating relationship between HCV genotypes and some clinical characteristics laboratory features,on patients of HCC.

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE

108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

-

NGUYEN THI THANH THUY

RESEARCH OF HEPATITIS C VIRUS

GENOTYPES IN HEPATOCELLULAR

CARCINOMA PATIENTS

Speciality: Gastroenterology Code: 62.72.01.43

SUMMARY THESIS’ DOCTOR OF MEDICINE

HANOI – 2018

THE THESIS WAS DONE IN: 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

Supervisor:

1 Prof PhD Mai Hong Bang

2 Ass Prof PhD Cao Minh Nga

Day Month Year

The thesis can be found at:

1 National Library of Vietnam

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

3 Central Institute for Medical Science Infomation and Tecnology

1 Introduction

Hepato cellular carcinoma (HCC) is a popular cancer ranking the sixth in the world, in which ranking fifth in male and eighth in female with many changes over time This is the third reason causing cancer-death in Asia-Pacific region

Beside hepatitis B, hepatitis C is an important causal factor related to hepato cellular carcinoma (HCC) Hepatitis C virus (HCV) causes hepato cellular carcinoma (HCC) by promoting the inflammation and fibrosis process on liver, eventually causing cirrhosis and hepato cellular carcinoma (HCC) Although there are lots of debates about the mechanism causing HCC of HCV, recent researches had mentioned the role of HCV genotype In Vietnam, there have been researches on HCV genotypes in patients with acute

or chronic hepatitis C, but not having adequate researches about HCV genotypes in HCC patients on foundation of hepatitis C virus From the above-mentioned reasons, we conduct this research aiming to:

1.1 Describing clinical and characteristics laboratory features and identifying HCV genotype on patients of HCC

1.2 Evaluating relationship between HCV genotypes and some clinical characteristics laboratory features,on patients of HCC

2 Necessity

Six (06) HCV genotypes have been identified currently In Vietnam, researches on chronic hepatitis C patients are most commonly found in genetype 6, followed by genotype 1 then genotype 2 Some authors referred to the high risk of causing hepato cellular carcinoma on patients that infect HCV with genotype 1 (1b); but other researches referred to the high risk on patients that infect HCV with genotype 3 Therefore, researching the role of HCV genotypes on patients of hepato cellular carcinoma is essential work, contributing to clarify disease’s mechanism

3 New contributions of thesis

Thesis identified genotype and subtype of hepatitis C virus

on 68 patients with HCC and 63 patients with HCV chronic hepatitis Thesis concurrently showed the risk of hepato cellular carcinoma in patients with HCV genotype 1b were 4.92 times higher than patients

of HCV non-1b (p = 0.008; OR = 4.92; 95% CI: 1.52 – 15.96)

Thesis includes 44 tables, 14 graphs, and 146 reference documents in which 29 Vietnamese documents and 117 English documents

CHAPTER 1 OVERVIEW 1.1 Epidemiology of hepato cellular carcinoma (HCC):

Hepato cellular carcinoma is a popular cancer ranking sixth in the world In Vietnam, author Vuong Anh Duong (2010) certified liver cancer ranking the third among other cancer diseases in male According to statistic of author Nguyen Dinh Song Huy (2015) from

2010 to 2014, patients with HCC increased year by year mostly in male patients The main reason was hepatitis B virus and hepatitis C

virus

1.2 Biological characteristics of hepatitis C virus

1.2.1 Characteristics of form and structure: Hepatitis C virus

(HCV) is a virus in the Flaviviridae family with structure of RNA

single sequence

1.2.2 Genotype characteristics

During replication period, HCV must use RNA poly merase enzyme, which is not capable of correcting in RNA synthetic process that diversifies HCV gene set, so it is classified HCV in different types

Genotype identification and classification are based on nucleotide order If discrepant order of nucleotide > 20% we have different genotypes If discrepant order of nucleotide ≤ 20% we have different subgroup of same genotype

It is identified currently six (06) HCV genotypes causing disease Genotype 1 and 3 are globally distributed, in which genotype 1 is the most common (46%), followed by genotype 3 (22%), genotype 2 (13%), and genotype 4 (13%)

Most of techniques previously focused on 5’UTR region (5’ untranslated region or also called 5’NC: 5’ non-coding) in identifying quality and quantity of HCV, because of its high conservation among different genotypes of HCV However it only based on 5’NC region, it is not sufficient to distinguish familiar subtypes in the same HCV type So, genotype identification nowadays are based on coding regions such as core region, NS5B region, NS4 or E1 region

Beside epidemiological sense, clinical practice identifies HCV genotype for predicting treatment response, treatment duration Although existing conflict and debate, both domestic and international researches have recognized role of genotypes 1 and 3 affecting hepato cellular carcinoma |In Vietnam, genotype 6 is most common, followed by genotype 1 and 2, genotype 3 has not been reported in general population, except some special addicted drug users that users HCV infected drugs The role of genotype initially in hepato cellular carcinoma has been researched but sporadically and unsystematically It is a reason we carry out this topic

1.2.3 Process of hepatitis C replication: It occurs in cell’s

cytoplasm due to synthetic process via RNA

1.3 Natural progression of hepatitis C virus infection

1.3.1 Acute hepatitis C: Most have no symptoms

1.3.2 Chronic hepatitis C: 75-85% of HCV infection will progress

chronic hepatitis, which is a risk leading to cirrhosis and hepato cellular carcinoma

1.4 Mechanism of causing hepato cellular carcinoma due to HCV infection

Chronic HCV infection causes hepato cellular carcinoma mainly by direct way Oppositely, patients of chronic hepatitis C often develop into hepato cellular carcinoma (HCC) on cirrhosis In addition, indirect way via cirrhosis HCV causes hepato cellular

carcinoma via HCV protein such as core, NS3, NS4B and NS5A

CHAPTER 2 OBJECT AND METHOD OF RESEARCH

2.1 Object of research

Researching group: 68 patients were diagnosed HCC by surgery, they treated at Hepatoma Department of Cho Ray Hospital from October 2012 to December 2015

Controlling group: 63 patients were diagnosed chronic hepatitis C and had no abnormal tumor in liver They treated at Hepatitis Department of Cho Ray Hospital from October 2012 to December 2015

2.1.1 Criteria of reasrching group (HCC groupe)

2.1.1.1 Selecting criteria:

Patients were diagnosed primarily HCC to base on surgery evidences with Anti HCV (+) and HBsAg (-), and identified HCV genotype at Medicine Faculty of University of Medicine and Pharmacy, Ho Chi Minh City

2.1.1.2 Exclusive criteria:

Patients did not meet selecting criteria Those had cancer in other organs, dilating cholangioma, metastatic tumors, and secondary liver cancer diagnosis Patients that is alcoholics, addicted to

cigarettes, and accompanied with acute and chronic diseases

2.1.2 Criteria of controlling group ( HCV groupe)

2.1.2.1 Selecting criteria:

Patients were excluded abnormal tumors in liver by

ultrasound with Anti HCV (+) and HBsAg (-) tested to identify HCV

genotype at Medicine Faculty of University of Medicine and

Pharmacy, Ho Chi Minh City

2.1.1.2 Exclusive criteria

- Did not meet selecting criteria

- To have abnormal tumors in liver

- Patients had infection, glomerular diseases, alcoholics, and other chronic diseases

2.2 Method of research

2.2.1.Research design: This is cross-sectional research with

controlling comparison

2.2.2 Sample size: Selecting sample that are convenient for both

groups, researching group of 68 HCC patients with and controlling group of 63 HCV patients

2.2.3 Research facilities: Machine system Cobas 6000 Analyzer

Series of Hitachi Kit automatic detaching and extracting DNA/RNA

of Roche Set kit of quantitative and HCV genotype identification

“Accupid HCV Genotyping Kit” of Khoa Thuong Company; and some other machines, equipment and facilities

2.2.4 Research content and criteria

2.2.4.1 Clinical research: To record symptoms HCC

2.2.4.2 Subclinical research

2.2.4.2.1 Tests of peripheral blood cells

2.2.4.2.2 Tests of blood chemistry

2.2.4.2.3 Immunoassay

2.2.4.2.4 Tests of HCV quantitative and identifying HCV genotype

- Time taking blood: when hospitalizing

- Tests were carried out at Microbiology Faculty of |University

of Medicine and Pharmacy, Ho Chi Minh City

Process consists of five (5) steps: sample receiving – processing, RNA extraction, reverse transcription of RNA to cDNA, DNA replication by real time PCR Both primer and probe were designed basing on sequence of core genes in HCV by Khoa Thuong Company After identifying HCV genotype by realtime PCR,we continued identify HCV subtypes by sequencing

2.2.4.3 Evaluating tumors condition in ultrasound, computed tomography or magnetic resonance imaging

2.2.4.4 Liver biopsy: Sending tumors’ samples for histiology

2.2.4.5 Evaluating liver function according to Child-Pugh

2.2.4.6 Evaluating relationship between HCV genotype and some subclinical, clinical characteristics

2.3 Data collecting and processing:

- Collecting date: Collecting tests and clinical information according to the medical reports

- Processing data according to the method of medical statistics, using SPSS 13 software

2.4 Ethical issue in research: To ensure medical ethics in research

CHAPTER 3 RESEARCH RESULTS 3.1 General characteristics of researching group

Table 3.1 Age characteristics of researching group

Age

HCC group (n=68)

HCC group mainly over 60 age, majority of male

Table 3.2 Genders’ characteristics of researching group

Gender

HCC group (n=68)

3.2 Clinical symptoms and laboratory tests of HCC and HCV groups

HCV group (n=63)

HCV group (n=63)

right

hypochondrium

< 0.001

Liver under right hypochondrium is common entity symptom in

HCC group; discrepancy is significant compared with HCC group

3.2.2 Blood chemistry characteristics

Table 3.5 Characteristics of liver enzyme

Researching group

Enzyme

HCC group (n=68)

HCV group (n=63)

Table 3.6 Characteristics of APRI Index

Table 3.7 Thrombocyte characteristics

HCC group with incresing rate of thrombocyte < 100.000 G/L is higher than HCV group, average value of thrombocyte is lower than HCV group

Table 3.10 Characteristics of AFP test

n Rate % n Rate %

< 0.001

Most of HCC group has normal amount of AFP

3.2.3.Characteristics of tumor images

Table 3.11 Some characteristics of tumor images on abdominal ultrasound

On ultrasound, liver size of researching group is normal with even liver-edge, not thrombosis of portal vein; but majority of livers are poor reverberation

3.2.4 Dividing periods in HCC group base on BCLC

Graph 3.10 Dividing periods in HCC group base on BCLC Most of patients in researching group were in early and very early periods (account 86.7%)

3.2.5 Characteristics of tumor differentiation

Graph 3.11 Difference levels of tumors

Majority of patients with HCC were at medium differentiations accounting 63.2%; high differentiations were 29.4%

Non-difference Low difference

3.2.4 HCV-RNA load and HCVgenotype

Table 3.14 Characteristics of HCV-RNA load

Majority of HCC group has low HCV loads

Table 3.15 HCV genotype characteristics

p * : Adjusted according to age and gender

Majority of HCC group were genotype 1 that was higher than HCV group

Table 3.16 HCV genotype of HCC patients

HCV group (n=63)

1b

p * : Adjusted according to age and gender

The ratio of 1b genotype in researching group was higher than controlling group, discrepancy was significant The risk of getting hepato cellular carcinoma (HCC) of 1b HCV group was 4.92 times higher than non-1b HCV group

3.3 Relationship between some clinical symtoms , laboratory test of HCC and HCV genotype

3.3.1 Relationship between some clinical symptoms and HCV

genotype: There was no relationship

3.3.2 Relationship between some laboratory test and HCV

Table 3.24 Relationship between HCV-1b genotype and bilirubin HCV

Rate (%)

Table 3.27 Relationship between HCV 1b genotype and APRI index HCV

Table 3.28 Relationship between HCV 1b genotype and

thrombocyte HCV 1b

Table 3.32 Relationship between HCV 1b genotype and HCV load HCV

44% HCC patients with HCV 1b genotype had virus load ≥

400000 UI/ml, whereas non-1b group was only 18.6% Discrepancy was significant with p=0.024

Table 3.33 Relationship between HCV genotypes and HCV load

HCV 1b genotype group has higher HCV load than the rest

of genotypes, significant discrepancy

Table 3.38 Relationship between HCV 1b genotype and the

Rate (%)

CHAPTER 4 DISCUSSION

4.1 General characteristics of HCC patients’

4.2 Clinical HCC, characteristics, laboratory features and HCV genotype on patients’

4.2.1 Clinical symtoms

Common symptoms include fatigue (72.1%), pain in right hypochondrium(70.6%), weight loss (48.5%), digestive disorder (47.1%) and anorexia (30.9%) Most of symptoms found in HCC group were higher than controlling group (p < 0.05) Fatigue is symptom non-specific, depending on patients’ subjective feeling and disease period Pain in right hypochondrium is a symptom which is specific relatively and most common in HCC Author Dao Van Long (2007) found that 73.7% of HCC patients having pain in right hypochondrium Author Doan Thai Ky (2015) recorded the rate of pain in right hypochondrium is 79% In addition to fatigue and pain

in right hypochondrium, research group recorded symptoms such as weight loss, digestive disorder and anorexia were lesser frequency, familiar with some domestic researches such as Duong Minh Thang (2007) and Thai Thi Phuong Lien (2011) These are, however, not

typical symptoms that may be present in various diseases

Physical signs: According to medical literature, hepatomegaly is a common sign of HCC Symptom of hepatomegaly

at diagnosed period was found out about 90% of HCC patients in Africa and Asia, compared 50 75% in America and Europe Our HCC group recorded 55.9% hepatomegaly Other less common symtoms include collateral circulation (17.6%), jaundice (11.8%), and leg oedema (13.2%)

4.2.2 Characteristics of transaminase enzyme: In clinic, AST and