Determining the frequency of genotype and allele of TNF-α – 308 and TGF-β1-509 in HCC patients with HBsAg (+). Analysing the association of TNF-α-308, TGF-β1-509 polymorphism with risk of HCC and some clinical and subclinical characteristics of HCC patients.
MINISTRY OF EDUCATION MINISTRY OF DEFENCE AND TRAINING VIETNAM MILITARY MEDICAL UNIVERSITY PHAN THI HIEN LUONG STUDYING POLYMORPHISM OF TNFα 308 G>A AND TGFβ 1509C>T IN HEPATOCELLULAR CARCINOMA PATIENTS WITH HBsAg POSITIVE Specialty: INTERNAL MEDICINE Code: 9 72 01 07 THE SUMMARY OF THE MEDICAL DOTORAL THESIS HANOI – 2020 THIS DOCTORAL THESIS WAS COMPLETED AT VIETNAM MILITARY MEDICAL UNIVERSITY Scientific Instructors: A/PROF. Nguyen Ba Vuong A/PROF. Tran Viet Tu 1st Contradictor: A/PROF. Vu Van Khien 2nd Contradictor: PROF. Nong Van Hai 3rd Contradictor:A/PROF. Hoang Thanh Tuyen The doctoral thesis will be reported to The Grading and Examinations Committee hold at Vietnam Military Medical University at Searching for the dissertation at: National Library Vietname Military Medical University’s library INTRODUCTION Hepatocellular carcinoma (HCC) is one of the common malignant tumors globally with the main cause is chronic hepatitis B virus (HBV) infection This cancer has a poor prognosis with a short survival time, due to limited early diagnosis. The host's single nucleotide polymorphism (SNP) plays an important role in determining the body's immune response to HBV infection, affecting HCC formation Research on gene polymorphism helps stratify risk subjects, improve early detection and treatment results. Recently, many studies in the world have shown the influence of TNFα308 G> A and TGF – β1509C>T is associated with an increased HCC risk. The carriers of A allele of TNF α 308 G>A, T alleles of TGF – β1509 C>T increase the risk of HCC higher than the other carriers of alleles The polymorphisms influence HCC progression in controlling the concentration of the corresponding cytokines. In the microbiological environment, these cytokines interact, support and enhance the effects of each other Vietnam is a country, located in the epidemic zone of hepatitis B virus, having a high incidence of HCC with extremely serious consequences Therefore, the thesis "Studying polymorphism of TNFα308 G>A and TGFβ1509C>T in hepatocellular carcinoma patients with HBsAg positive" was conducted with two objectives Determining the frequency of genotype and allele of TNFα – 308 and TGFβ1509 in HCC patients with HBsAg (+) Analysing the association of TNFα308, TGFβ1509 polymorphism with risk of HCC and some clinical and subclinical characteristics of HCC patients. * New contributions of this doctoral thesis This is the first scientific study in Vietnam researching on frequency of genotype, allele of TNFα308 G>A, TGF β1509 C>T in healthy people, chronic hepatitis B and HCC with HBsAg (+) * TNFα – 308 G>A + The AA genotype only appeared in HCC group, which was 0.98%, the frequency of alleles of A TNF α 308 G>A was low in healthy people, 5.4%, which was similar to Asian population + The frequency of allele A in the healthy group, chronic hepatitis B and HCC was 5.4%, 5.83% and 13.2%, respectively. There was differences in the frequency of genotypes GG, GA and A allele among HCC groups compared to healthy, chronic hepatitis B groups * TGF–β1 509 C>T + The frequency of T allele in the healthy group, HBV and HCC was 52.9%; 61.67% and 63.24%, respectively There was difference in the frequency of T allele in HCC and healthy groups The study showed that A allele, genotype containing allele A of TNFα 308 increased the risk of HCC compared with G allele and genotype containing allele G. Alen T, genotype containing allele T of TGFβ1509 increased the risk of HCC compared with C allele and genotype contain C allele Two polymorphisms interacted synergistically with each other increasing the risk of HCC * The doctoral thesis arrangement: This thesis contains 113 pages (without references and appendixes): Introduction: 02 pages, Chapter 1 Overview: 31 pages, Chapter 2: Subjects and methods: 17 pages, Chapter 3 Results: 30 pages, Chapter 4 Discussion: 30 pages, Conclusion: 02 pages, Recommendations: 01 page It includes 46 tables, 23 figures, and 125 references (14 Vietnamese references and 111 English references) CHAPTER 1: OVERVIEW 1.1 Overview of hepatocelular carcinoma Hepatocelular carcinoma is the sixth most common cancer among the 10 common cancers worldwide Located in Southeast Asia, high epidemic area, Vietnam has a high incidence of HCC. Corresponding to the high prevalence and mortality rates of HCC, the status of HBV infection is also worrisome. According to Robert G et al (2011), Vietnam is one of the countries which has the highest HBV infection rate in the world with nearly 12% of people having HBsAg (+), about 10 million people living with chronic HBV. In 2018, GLOBOCAN had notified the ASRI (age specific standard index) of HCC in Vietnam for both sexes is 23,2, ranked fourth globally. HCC is closely related to HBV infection. However, the reality is that not all patients infected with HBV will develop HCC. Therefore, both HBV and patients play important role, they interact with each other to cause the final disease 1.2 Single nucleotide polymorphism (SNP) The human genome, largely preserved during evolution, at least 99.5% of genes between any two individuals will be identical, so the difference is only 0.1% 0.5% This is the key to creating each individual is unique due to different characteristics (shape, inherited disease ) Many factors in the genome contribute to 0.1% of the difference, of which SNP (singlepolymorphism) plays an important role. SNP is defined as a single base change on a DNA sequence, this change accounts for a proportion of ≥ 1% in the large community and continue to be inherited for the next generation. SNP can be located in different regions of the gene, in the promoter region, it plays an important role in controlling the start and level of transcriptional activity of the gene TNFα – 308 G>A gene TNFα gene (Tumor necrosis factor alpha) is located in locus 21.3 of the short arm chromosome 6 (6p21.3). TNFα gene has many polymorphism, especially the polymorphism at 308 in the promoter region plays an important role. This SNP has 2 types of alleles: the allele G, the other rarer type, the allele A The frequency of genotypes and alleles of TNFα 308G>A polymorphisms varied among different populations TGFβ1 509C>T gene TGFβ1 gene (Transforming Growth Factor, Beta 1) is located on the long arm of chromosome 19 (19q13.113.3) Many single polymorphisms have been identified in the TGFβ1 gene, of which SNP at the C509T site at the promoter region has important role. It is related to gene transcription capability, producing TGF β plasma The frequency of allele and genotype of TGFβ1509 is quite diverse in different populations 1.3. TNFα 308G>A, TGFβ1509C>T and HCC risk Recently, many studies in the world have shown the influence of polymorphism of TNFα 308 G> A, TGFβ1509 C>T with the risk of chronic hepatitis B and HCC due to HBV. The researchers suggest that these polymorphisms affect the risk of HCC through controlling the concentration of TNFα, TGFβ plasma TNFα 308 G/A gene Teixeira A.C. et al (2013) studied the association of TNFα 308 G/A polymorphism with HCC risk in Brazil in 120 HCC patients and 202 healthy people The result had shown there was an increased HCC risk in people carriers A allele, GA gene of TNF–α308 compared to people carriers G allele, GG gene with (OR = 1,82; 95%CI = 1,073,08; p = 0.0351), and (OR = 2,51; 95%CI = 1,39 4,51; p = 0,0031). Tsai J. F. et al (2017) studied the effect of TNF α 308 G>A on HCC risk related to HBV in 200 HCC infected with HBV patients and 200 cirrhosis with HBV infected patients, concluded that GA gene carriers versus GG carriers have an increased risk of HCC with OR = 2,34; 95% CI (1,29 – 4,25); p = 0,004 TGFβ1509C>T gene Ma J. (2015) studied 393 patients with hepatitis C found that the risk of HCC was higher in patients with TT gene than CC gene with OR = 1,820, p = 0.03. Patients carrier T allele compared with C allele increased the risk of HCC with OR= 1,383, p = 0.028 Combination TNFα308G>A, TGFβ1509C>T HCC, complex disease, is affected by many factors, of which the role of a gene is very important, but in fact, no single polymorphism can fully explain the complex mechanisms of disease, each SNP plays only a very small role in the development of disease. Therefore, the new trend is studying the combination of genotypes and alleles of different SNPs with the risk of disease, help stratify This helps increase the value of early detection and improve the effectiveness of disease treatment Bei C.H (2014) evaluated the effect of 6 SNP include IL2, IFNγ, IL1β, IL6 and IL10 genes with risk of HCC in China in 720 HCC patients and 784 healthy people, had concluded that although not a single SNP increased the risk of HCC, but combination of 6 SNP had increased HCC risk with OR = 1,821; 95%CI = 1,078 3,075. The authors said that interactions between SNPs increase the risk of HCC. El Din N.G. (2017) assessed the effects of TNFα 308 and TGF –β1509 on cirrhosis progression due to HCV showed that combination of TNFα 308 AA and TGFβ1509 TT compared with TNF (GG) + TGF (CC) increases risk of cirrhosis with OR = 6,4; 95%CI = 1,490 – 27,641; p = 0,013. The results showed that these was a synergistic effect of 2 genes because TNFα alone increased the risk of cirrhosis by 2.8 times, TGFβ1 increased the risk of cirrhosis by 2.9 times 1.4. Studies TNFα 308G>A and TGFβ 1509C>T in Vietnam Recently, the role of polymorphisms for HCC has begun to be studied in Vietnam. However, as far as we know, there have been no studies on TNF α 308 and TGFβ 509 in HCC patients. In 2012, Dunstan studied in 2350 healthy Vietnamese people, genetic analysis was conducted in England. The result showed that the percentage of A allele TNFα 308 was 7%. Tran Dinh Tri (2017) studied the gene TNFα 308 G>A in patients with stomach cancer showed that the percentage of allele A was 17.1%. Such research results have been noted that the percentage of A allele of TNFα 308 in the Vietnamese population is low, similar to other Asian countries The polymorphism TGFβ1 509 C>T has been extensively studied in the world with certain roles in many diseases. However, in the Vietnamese population, although we have tried to search, no studies on this polymorphism have been published CHAPTER 2: SUBJECTS AND METHODS 2.1. Subjects The study included 102 HCC infected with HBV patients, 60 chronic hepatitis B patients, 102 healthy people 2.1.1. Study group Inclusion criteria: Diagnosis of HCC was base on the guidance of the Ministry of Health of Vietnam in 2012, has 1 of the following 2 criteria + Anatomical or histopathological evidence of histology + Typical image on contrast CT scan or contrast MRI with αFP ≥ 400 ng/ml Patients with HBsAg (+) The patient was first diagnosed with HCC Exclusion criteria Patients with HIV infection Patients with HCV infection Patients with alcoholic liver disease The liver is damaged by drugs or chemicals Metastatic liver cancer from other organs Patients were need liver biopsy for diagnosis but having contraindication to liver biopsy (platelets A polymorphism and clinical, subclinical, and stage of HCC Tables 3.16, 3.17, 3.18, 3.19 and 3.20 showed that there was no difference between TNFα308G>A polymorrphism with some clinical and subclinical characteristics such as age, tumor characteristics, platelets, αFP, HBV DNA, portal venous thrombosis, metastasis, HCC stage 4.3. TGFβ 1509 C>T polymorphism and HCC risk 4.3.1. Frequency of genotypes, alleles of TGFβ1509 C>T CT genotype predominates in all groups T allele in HCC patients (63,24%), chronic hepatitis B (61,67%), healthy people (52,94%) There was a significant difference in the frequency of alleles in HCC group compared to healthy group, with p = 0.035 4.3.2. TGFβ 1509 C>T polymorphism and HCC 4.3.2.1. TGFβ 1509 C>T polymorphism and HCC risk The control group is healthy people Table 3.21. TGFβ 1509 C> T polymorphism and HCC risk (Control is healthy people) Genotype and allele CC HCC n= 102 14 Healthy n=102 Genotype (n) 23 OR (95%CI) Ref p 14 CT TT CC TT + CT CT + CC TT Alen C Alen T 47 41 14 88 61 41 75 129 50 29 23 79 73 29 Allele (2n) 96 108 1,544 (0,712 – 3,351) 2,323 (1,026 – 5,258) Ref 1,830 (0,881 – 3,799) Ref 1,692 (0,943 – 3,036) Ref 1,529 (1,029 – 2,271) 0,270 0,041 0,102 0,077 0,035 The TT genotype compared to the CC genotype, increases the risk of HCC, with OR = 2,323; 95% CI (1,026 – 5,258); p = 0,041 T allele was significantly associated with increased risk HCC, compared to C allele, OR = 1,529; 95% CI (1,029 – 2,271); p = 0,035 Tables 3.22 and 3.23 genotypes and alleles of TGFβ1509 did not affect the risk of HCC, when using control groups were without HCC, chronic hepatitis B 4.3.2.2 TGFβ 1509 C>T polymorphism and clinical, subclinical, and stage of HCC Tables 3.24, 3.25, 3.26, 3.27 and 3.28 showed that there was no difference between TGFβ1509C>T polymorphism with some clinical and subclinical characteristics such as age, tumor characteristics, platelets, αFP, HBV DNA, portal venous thrombosis, metastasis, HCC stage 4.4 Combination of TNFα 308 G>A, TGFβ 1509 C>T polymorphisms We see GG genotype of TNFα308 G>A and CC genotype of TGFβ1509 C>T appeared significantly more in the without HCC group than the HCC group The genotype containing allele A of TNFα 308 and genotype containing allele T of TGFβ1509 increased the risk of HCC compared to the remaining genotypes. Therefore, we consider GG to be a good genotype compared to AA and CC as a good genotype compared to TT, we divide the appearance of two genes as follows: 15 Good combination: having 2 genes GG and CC are homozygous Moderate combination: having 1 gene GG or CC in homozygous Bad combination: neither GG gene nor CC gene in homozygous 4.4.1. TNFα –308G>A, TGFβ 1509C>T polymorphism and HCC risk The control group is healthy people Table 3.32. TNFα – 308 G>A, TGFβ 1509 C> T polymorphism and HCC risk (Control is healthy people) Genotype and allele Good combination Moderate combination Bad combination HCC n= 102 72 21 Healthy n=102 23 68 11 OR (95%CI) p Ref 2,706 (1,169 – 6,261) 4,879 (1,688 – 14,098) 0,017 0,003 The control group is healthy people Moderate combination, bad combination increases the HCC risk compared to good combination, OR=2,706; 95%CI (1,169 – 6,261);p=0,017 and OR=4,879; 95%CI (1,688 – 14,098); p= 0,003 The control group is chronic hepatits B + healthy people Table 3.33. TNFα – 308 G>A, TGFβ 1509 C> T polymorphism and HCC risk (Control is healthy people + healthy people) Genotype and allele HCC n= 102 Good combination Moderate combination Bad combination 72 21 Without OR (95%CI) HCC n = 162 32 Ref 112 2,286 (1,031 – 5,070) 18 4,148 (1,571– 10,956) p 0,038 0,003 The moderate, bad combination increases HCC risk compared to good combination, with OR = 2,286; 95% CI (1,031 – 5,070); p = 0,038 and OR = 4,148; 95% CI (1,571 – 10,956); p = 0,003 When using control group was chronic hepatitis B patient, we did not see an association for HCC risk with polymorphisms 4.4.2. The relation between combination of polymorphisms and some clinical, subclinical characteristics of HCC patients 16 According to tables 3.35, 3.36, 3.37, 3.38, and 3.39, there was no effect of combining polymorphisms with some clinical, subclinical characteristics: age, tumor characteristics, platelets, αFP, HBV DNA, thrombosis portal vein, metastases, HCC stage CHAPTER 4: DISCUSSION 4.1. Clinical and subclinical characteristics of HCC patients HCC patients had an average age of 57.4 ± 9.7. The youngest is 37 years old, the oldest is 80 years old. The majority of patients aged 5170 years old were 67.2%. Male / female ratio: 11.8 /1 At the time of diagnosis, the percentage of patient without symptoms was 13,7%. The average size of total tumor: 10,80 ± 12,02 cm, size tumor 10 cm was high percentage (37.3%), portal vein thrombosis (22.5%), other metastases (19.6%). Subclinical indicators fluctuate, αFP