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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: Prof PhD Mai Hong Bang Ass Prof PhD Cao Minh Nga Reviewer: This thesis will be presented at Institute Council at: 108 Institute of Clinical Medical and Pharmaceutical Sciences Day Month Year The thesis can be found at: National Library of Vietnam Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences 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 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 then genotype Some authors referred to the high risk of causing hepato cellular carcinoma on patients that infect HCV with genotype (1b); but other researches referred to the high risk on patients that infect HCV with genotype Therefore, researching the role of HCV genotypes on patients of hepato cellular carcinoma is essential work, contributing to clarify disease’s mechanism 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) Research had also identified linear relationship between genotype 1b and HCV load in patients with hepato cellular carcinoma (HCC) Thesis outline: Thesis was presented in 128 pages including introduction pages, overview 38 pages, object and method of research 20 pages, research results 32 pages, discussion 33 pages, conclusion pages and petition pages Thesis includes 44 tables, 14 graphs, and 146 reference documents in which 29 Vietnamese documents and 117 English documents CHAPTER 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 and are globally distributed, in which genotype is the most common (46%), followed by genotype (22%), genotype (13%), and genotype (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 and affecting hepato cellular carcinoma |In Vietnam, genotype is most common, followed by genotype and 2, genotype 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 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 RESEARCH RESULTS 3.1 General characteristics of researching group Table 3.1 Age characteristics of researching group HCC group HCV group Age (n=68) (n=63) p n Rate % n Rate % ≤ 40 0,0 12.7 41 – 60 26 38.2 40 63.5 < 0,001 > 60 42 61.8 15 23.8 Total 68 100.0 63 100.0 Average age 64.81 ± 8.82 52.13 ± 9.3 < 0,001 Maximum age 85 71 Minimum age 47 32 HCC group mainly over 60 age, majority of male Table 3.2 Genders’ characteristics of researching group HCC group HCV group Gender (n=68) (n=63 p n Rate % n Rate % Female 13 19.1 35 55.6 < 0.001 Male 55 80.9 28 44.4 Total 68 100.00 63 100.00 Comment: Male patients accounts 80.9%; the ratio of male/female = 4.24 There is gender discrepancy between researching and controlling group 3.2 Clinical symptoms and laboratory tests of HCC and HCV groups 3.2.1 Clinical symptoms Table 3.3 Some symptoms of mechanical energy HCC group HCV group (n=68) (n=63) p n Rate n Rate % % No 19 27.9 55 87.3 < Fatigue 0.001 Yes 49 72.1 12.7 No 20 29.4 57 90.5 Pain in right < hypochondrium Yes 0.001 48 70.6 9.5 No 35 51.5 60 95.2 < Weight loss 0.001 Yes 33 48.5 4.8 Digestive No 36 52.9 48 7.2 0.006 disorder Yes 32 47.1 15 23.8 No 47 69.1 53 84.1 Anorexia 0.04 Yes 21 30.9 10 15.9 Fatigue, weight loss and pain in right hypochondrium are common symptoms in the HCC group Symptoms of mechanical energy Table 3.4 Some signs HCC group (n=68) Entity symptoms n Rate % Liver under No 30 44.1 right Yes 38 55.9 hypochondrium No 60 88.2 Jaundice Yes 11.8 No 56 82.4 Collateral circulation Yes 12 17.6 No 59 86.8 Leg oedema Yes 13.2 HCV group (n=63) n Rate % 57 90.5 9.5 52 11 63 63 82.5 17.5 100 100 p < 0.001 0.36 < 0.001 0.003 11 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 80 60 40 73,5 10,4 20 2,9 13,2 Period Period A Period B Period C 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 1,5 5,9 29,4 63,2 Non-difference Low difference Graph 3.11 Difference levels of tumors Majority of patients with HCC were at medium differentiations accounting 63.2%; high differentiations were 29.4% 12 3.2.4 HCV-RNA load and HCVgenotype Table 3.14 Characteristics of HCV-RNA load HCC group HCV group HCV-RNA load p n Rate % n Rate % < 400000 UI/ml 49 72.1 13 20.6 400000 – 800000 4.4 11.1 UI/ml < 0.001 > 800000 UI/ml 16 23.5 43 68.3 Tổng 68 100.0 63 100.0 Majority of HCC group has low HCV loads Table 3.15 HCV genotype characteristics HCV HCC group HCV group p* genotype n Rate % n Rate % Genotype 33 48.6 14 22.2 Genotype 13.2 13 20.6 0.034 Genotype 26 38.2 36 57.2 Total 68 100.0 63 100.0 * p : Adjusted according to age and gender Majority of HCC group were genotype that was higher than HCV group Table 3.16 HCV genotype of HCC patients HCV genotype Cases (n=68) Rate % 1a 11.8 Genotype 1b 25 36.8 Total 33 48.6 2a 4.4 2j 2.9 Genotype 2m 5.9 Total 13.2 6a 13.2 6b 1.5 6e 10 14.7 Genotype 6l 2.9 6s 4.4 6u 1.5 Total 26 38.2 Total 68 100 13 In HCC group,the main ratio of HCV genotype were 1b, 6e, 6a, 1a being 36.8%; 14.7%; 13.2% and 11.8% Table 3.17 The role of HCV 1b genotype on causing hepato cellular carcinoma (HCC) HCC group HCV group (n=68) (n=63) HCV OR p* genotype n Rate n Rate (95%CI) % % Non-1b 43 63.2 54 85.7 4.92 (1.52 – 1b 0.008 25 36.8 14.3 15.96) genotype Total 68 100.0 63 100.0 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 genotype Table 3.22 Relationship between 1b genotype and ALT HCV Non-1b 1b genotype Total genotype Rate Rate Rate p n n n ALT (%) (%) (%) < ULN 33 76.7 15 60 48 70.6 ≥ ULN 10 23.3 10 40 20 29.4 0.14 Total 43 100.0 25 100.0 68 100.0 Average 60.23 ± 44.4 85.61 ± 66.71 69.56 ± 54.61 0.06 40% patients with 1b genotype had ALT ≥ times of normal value, whereas in the non-1b group, the rate was only 23.3%; ALT average of 1b genotype tend to be higher than non-1b genotype, however disvrepancy was not significant 14 Bảng 3.23 Relationship between HCV - 1b genotype and AST HCV Non-1b 1b genotype Total genotype Rate Rate Rate p n n n AST (%) (%) (%) < ULN 28 65.1 28 35 51.5 ≥ ULN 15 34.9 18 72 33 48.5 0.003 Total 43 100.0 25 100.0 68 100.0 Average 76.85 ± 32.98 101.09 ± 50.5 85.76 ± 41.64 0.02 72% patients with 1b genotype increased AST ≥ times of ULN, compared with 34.9% of non-1b genotype group (p = 0.003) Average AST value of 1b genotype group that was higher than the rest (p=0.02) Table 3.24 Relationship between HCV-1b genotype and bilirubin HCV Non-1b 1b genotype Total genotype p Bilirubin Rate Rate Rate n n n TP (%) (%) (%) Normal 24 55.8 32 32 47.1 Increase 19 44.2 17 68 36 52.9 0.06 Total 43 100.0 25 100.0 68 100.0 18.48 ± Average 16.69 ± 7.31 21.56 ± 10.85 0.03 9.01 Billirubin average numeric value of 1b group was higher than the rest Table 3.27 Relationship between HCV 1b genotype and APRI index HCV Non-1b 1b genotype Total genotype p Rate Rate Rate APRI n n n (%) (%) (%) index ≤2 23 53.5 10 40 33 48.5 >2 20 46.5 15 60 35 51.5 0.28 Total 43 100.0 25 100.0 68 100.0 Average 2.42 ± 2.07 3.35 ± 2.94 2.76 ± 2.44 0.13 1b genotype group tended to increase APRI index, compared with non-1b group 15 Table 3.28 Relationship between HCV 1b genotype and thrombocyte Non-1b 1b genotype Total HCV 1b genotype p Rate Rate Rate n n n Thrombocyte (%) (%) (%) Decrease 23 53.5 12 48 35 51.5 (< 150000) 0.66 Normal 20 46.5 13 52 33 48.5 Total 43 100.0 25 100.0 68 100.0 161.7 ± Average 151.5 ± 72.2 157.97 ± 81.3 0.62 86.8 1b genotype group tended to decrease thrombocyte compared with the rest Table 3.32 Relationship between HCV 1b genotype and HCV load HCV Non-1b 1b genotype Total genotype Rate Rate Rate p HCVn n n (%) (%) (%) RNA < 35 81.4 14 56 49 72.1 400000 ≥ 0,024 18.6 11 44 19 17.9 400000 Total 43 100.0 25 100.0 68 100.0 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 < 400000 ≥ 400000 Tonnage p Genotype n Rate (%) n Rate (%) 1a 16.3 0 1b 14 28.6 11 57.9 6a 14.3 10.5 0.02 6e 10.2 26.3 Other 15 30.6 5.3 Total 49 100 19 100 16 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 differences of tumor 1b Non-1b Total genotype HCV genotype p Differences Rate Rate Rate n n n (%) (%) (%) Non-difference 100 0,0 100 Poor differences 0,0 100 100 0.05 Medium 29 67.4 14 32.6 43 100 differences High differences 13 65 35 20 100 Total 43 63.2 25 36.8 68 100 HCV 1b group tend to get HCC with poor differentiation higher tendency than the rest of the other genotypes (p=0.05) 17 CHAPTER DISCUSSION 4.1 General characteristics of HCC patients’ 4.1.1 Age characteristics The most common age was > 60, average age 64.81 ± 8.82 ,higher than controlling group (p < 0.001) 4.1.2 Gender characteristics Male patients account 80.9%, the ratio male/female = 4.24 Almost all researches in the world reported that male’s tendency to get HCC is higher than female (from to 10 times) 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 18 ALT enzyme are used to evaluate necrotic level of liver parenchyma and monitor the progress of liver disease A research by Tran Van Huy (2003) recorded that patients with HCC have on average value of AST = 102  43 U/L and ALT = 79  32 U/L In our research, average value of AST = 84.76  41.61 U/L and ALT = 69.56  54.61 U/L HCC group has the rate increasing liver enzyme  ULN as well as average value of liver enzyme are higher than controlling group It is proved that damage liver of HCC group is greater than controlling group 4.2.3 APRI index and thrombocyte value: Mechanism of HCV causing HCC is mainly indirect via cirrhosis Liver biopsy is considered golden standard to diagnose level of cirrhosis In our research, however, biopsy specimens were taken directly at tumor rather than liver parenchyma surrounding tumor, so ability to evaluate cirrhosis accurately was limited Thus, we conduct to compare APRI index of two groups to evaluate cirrhosis levels of these groups The results showed that 51.5% of researching group had APRI > (equivalent to F4 cirrhosis), discrepancy is significant compared with controlling group APRI average of HCC group is also higher than HCV group significantly It is proved that most of HCC group developed HCC on cirrhosis To reinforce this results, we conducted to compare thrombocyte value of HCC group and HCV group The results showed that 33.8% of HCC group had thrombocyte < 100 G/L and 51.5% with thrombocyte < 150 G/L Discrepancy the rate of decreasing thrombocyte between HCC group and HCV group showed that most of HCC group was in cirrhosis In our opinion, the reason of decreasing thrombocyte is that most of HCC patients’ gets infected by hepatitis B virus or hepatitis C virus and develop on cirrhosis Thrombocyte often decrease in cirrhosis cases, but underlying mechanism is not well understood Kajihara and partners claim that symptom of decreasing thrombocyte in this case is contributed by many factors, in which increasing speed to replace and produce thrombocyte is damaged This results is suitable with research of Mihai Olariu in 2010 found that most of patients, who was infected by HCV in long term have the decrease of thrombocyte 19 due to HCV virus repress bone marrow and reduce production of liver thrombopoietin 4.2.4 Characteristic of AFP testing: AFP is a very common cancer marker According to some authors, hepato cellular carcinoma is a high differential type; AFP did not increase in early stage and its increasing level is related to tumor’s size Research by Dao Van Long in 2007 at Bach Mai Hospital recorded 32.5% of hepato cellular carcinoma patients with AFP in normal level Similarly, Le Van Thanh (2010) and Thai Doan Ky (2015) also reported that the rate patients of HCC with AFP < 20ng/mL was 31.8% and 43.8% respectively In our research, majority of patients had tumor’s size 60 accounts 61.8%, average age 64.81 ± 8.82, in which 81.9% is male; The ratio of male/ female = 4.24 Group HCC patients had average age higher than controlling group (p < 0.001) - Clinic: symptoms include fatigue (72.1%), pain in right hypochondrium (70.6%), weight loss (48.5%), digestive disorder (47.1%), anorexia (30.9%), hepatomegaly (55.9%) Most of symptoms in HCC group are higher than controlling group (p < 0.05) - Laboratory features + Liver enzyme: 29,4% patients with ALT ≥ upper limite of normal; 48,5% with AST ≥ ULN proving liver injury in HCC group than controlling group + APRI index assess cirrhosis’s levels: 51,5% HCC patients group with APRI >2 It is shown that majority of HCC group developed on cirrhosis Difference was significant (p