Nghiên cứu tỷ lệ biểu lộ và đột biến gen LMP1 của virus epstein barr và HLA trên bệnh nhân ung thư vòm mũi họng tại thành phố cần thơ tt tieng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Specialization: Biotechnology Code: 62 42 02 01 TRINH THI HONG CUA STUDY ON THE RATE OF EXPRESSION AND MUTATION OF LMP1 GENE OF EPSTEINBARR VIRUS AND HLA GENOTYPING ON NASOPHAGYNGEAL CARCINOMA PATIENTS AT CAN THO CITY Can Tho, 2020 THE STUDY WAS COMPLETED AT CAN THO UNIVERSITY Instructor 1: Assoc Prof Dr Tran Ngoc Dung Instructor 2: Prof Dr Phan Thi Phi Phi The dissertation was defended at the university examination committee At……………………………, Can Tho University At………………………………………………… Reviewer 1: Reviewer 2: The dissertation is available in Libraries: Central Library of Can Tho University National Library of Vietnam LIST OF PUBLICATION RELATED TO THE THESIS Trinh Thi Hong Cua, Tran Ngoc Dung, Hoang Đuc Trinh, Duong Thi Loan 2017 Clinical and histopathological characteristics of nasopharyngeal carcinoma patients in Can Tho Oncology Hospital Journal of practical medicine - JPM 12 (1064) 2017, ISSN: 1859-1663, pp.42-43 Trinh Thi Hong Cua, Tran Ngoc Dung, Tran Van Be Nam, Phan Thi Phi Phi 2018 The ratio of EBV LMP1 gene in fresh samples of nasopharyngeal carcinoma patients at Can Tho Oncology Hospital Vietnam Medical Journal, ISSN: 18591868, August - No 1&2, 2018, pp.141-142 Trinh Thi Hong Cua, Tran Ngoc Dung, Ta Van To, Phan Thi Phi Phi 2018 Frequency and mutation of LMP1 gene of Epstein-Barr Virus in nasopharynx biopsy samples of nasopharyngeal carcinoma patients at Can Tho Oncology Hospital Can Tho University Journal of Science, ISSN: 18592333, Special issue on Biotechnology (2019) (1), pp 66-71 DOI: 10.22144/ctu.jsi.2019.008 Chapter INTRODUCTION 1.1 Background Nasopharyngeal carcinoma (NPC) is a malignant tumour originating primarily from epithelial cell lining the nasopharynx (Bui Dieu, 2012) Vietnam was the country with the average incidence (5.4 people/100.000 people/year) (Bui Dieu, 2011) The disease was often diagnosed late, resulting in poorer treatment outcomes and increased mortality Therefore, the urgent need now is to find a solution to help diagnose the disease in the early stage of NPC The loss 30bp mutation of Latent Membrance Protein (LMP1) of Epstein-Barr Virus (EBV), which is considered to be a decisive factor in the pathogenesis of NPC and Human Leukocyte Antigen (HLA) - terrain gene factor, which are sensitive to NPC, contribute to explanation of the incidence of disease differences across geographic areas Therefore, can the loss 30bp mutation of LMP1 gene be considered as a decisive factor in early diagnosis? Are the two factors interrelated? Starting from the above analysis, the thesis “Study on the rate of expression and mutation LMP1 gene of Epstein-Barr Virus and HLA genotyping on nasopharyngeal carcinoma patients at Can Tho city” was implemented 1.2 Objectives (1) Determinate the rate of the presence and mutation of LMP1 gene of Epstein-Barr Virus on NPC patients (2) Determinate the frequency of common HLA genotype in nasopharyngeal carcinoma patients (3) Find out the value of the LMP1 gene mutation and common HLA genotype to diagnose of nasopharyngeal carcinoma patients 1.3 Scientific signifficance of the these 1.3.1 Scientific and education values - Providing scientific data on the rate of the presence, mutation and types of gene mutations (loss 30bp mutation) of EBV’s LMP1 gene and the frequency of common HLA alleles genotype of nasopharyngeal carcinoma patients in the Mekong Delta, southern Vietnam - Providing a scientific document and a prerequisite for further research 1.3.2 Practical values The research has contributed to providing information on the pathogenesis of nasopharyngeal carcinoma, supporting the more accurate diagnosis and prognosis of nasopharyngeal carcinoma patients, thereby contributing to more practical treatment effects 1.4 New contributions of the thesis - This is the first research work on pathogenesis mechanisms of nasopharyngeal carcinoma in the Mekong Delta - The LMP1 - PCR method can be applied to identify the loss 30bp mutation LMP1 gene at health facilities in Can Tho city or generally Mekong Delta Chapter OVERVIEW MATERIALS 2.1 Nasopharyngeal carcinoma 2.1.1 Clinical characteristic of nasopharyngeal carcinoma: Depending on the location of the tumor, the direction and degree of invasion (ears, nose, eyes, neck lymph nodes, etc.) 2.1.2 Histopathology of nasopharyngeal carcinoma: according to the World Health Organization 2005 2.1.3 Classification of T, N, M and state of nasopharyngeal carcinoma: according to the Union for Internation Cancer Control (UICC) 2010 2.2 Epstein-Barr Virus - Epstein-Barr Virus belongs to group I (dsDNA virus), order Herpesvirales, family Herpesviridae, subfamily gammaherpesviridae, genus Lymphocryptovirus, species Human Herpesviridae - The LMP1 gene is about 2.6 kb of side, located from 166483-169088, encoding the LMP1 protein consisted of 386 amino acids, dividing into active intracellular signal pathways and this is the key to develop and proliferate for malignant cells of disease (Kang and Kieff, 2015) - The presence and mutation of LMP1 gene (the lost segment 30bp mutation type) in biopsy tissue samples of NPC patients was confirmed in the world and in Vietnam: 81% (34/42) LMP1 (+) 56% (19/34) Del 30 bp (Hui et al., 2008); 85.7% (18/21) Del 30 bp (Tang et al., 2008); 100% (20/20) LMP1 (+) 90% (18/20) Del 30 bp (Pham Thi Nguyet Hang, Phan Thi Phi Phi ctv, 2003); 60% Del 30 bp (Nguyen-Van D et al., 2008) - The role of mutant losing segment of 30bp LMP1 gene can help EBV survived and escaped the supervision of the immune system (Tang et al., 2008) 2.3 HLA – A terrain factors 2.3.1 HLA gene structure - Cluster of HLA gene is a region containing many polymorphic genes arranged relatively close to each other and located on the short wings of chromosome (paragraph 6p21.3), particularly, genes coding for β-microglobuline (constituent components create HLA I class) at the 15th chromosome (15q21-q22.2) (Phan Thi Phi Phi, 2007), in which: + HLA I class: including HLA-A, HLA-B, HLA-C genes encoding for corresponding HLA antigens + HLA II class: including HLA-DR, -DQ, -DP genes encoding for corresponding HLA antigens - Haplotype is the allele combination in each locus on a single chromosome Fig 2.12: HLA gene position on 6th chromosome Source: https://ghr.nlm.nih.gov/art/large/hla.jpeg, retrieved July 12, 2014 2.3.2 Role of HLA genotype in the pathogenesis of nasopharyngeal carcinoma The pathogenesis of nasopharyngeal carcinoma is related to the EBV antigen presentation role of HLA moleculars of nasopharyngeal carcinoma cells (peptid segment of LMP1) and the role of Tc cells involved in the immune response of EBV infection (cited by Tran Ngoc Dung, 2000; Su et al., 2013) 2.3.3 HLA susceptibility genotype to nasopharyngeal carcinoma Studies show that the frequency of an HLA antigen can be high (susceptible gene) or low (protective gene) in some diseases, called susceptibility gene of diseases The same, in NPC, some HLA alleles were founded with high frequency: HLA-A*02,-A*11, -A*24, -A*33; -B*07, -B*15, -B*46; DRB1*04,-DRB1*09, -DRB1*12; -DQB1*03, -DQB1*05, DQB1*06 (Tran Ngoc Dung, 2000; Yu et al., 2009; Su et al., 2013; Wang and Wang., 2014) 2.4 Molecular biology techniques applied in the diagnosis of LMP1 EBV and HLA genotyping Currently, to study the LMP1 EBV gene, there are two widely used molecular biology techniques: polymerase chain reaction and sequencing techniques Particularly for HLA genotyping, Today, beside the serological techniques, there are higher resolution molecular biology techniques such as PCR-SSP, PCR-SSO, PCR-SBT Next Generation Sequencing (NGS) Chapter STUDY SUBJECTS AND METHODS 3.1 Study subjects 3.1.1 Criterial samples - Patients with the results of histopathology are nasopharyngeal carcinoma, treated at Can Tho Oncology Hospital; No restrictions on age, gender and place of residence - Nasopharyngeal biopsy samples: Fresh sample (0.5-4 mg mass; untreated) and paraffin-embedded tissues sample (used in the case that fresh samples not reach the same sample weight, at the same time for sample collection is not more than weeks; about 10 slices, thickness µm) 3.1.2 Exclusion criteria: recurrent disease; periodic examinations during treatment; histopathological results are sarcome or lymphoma; not agree to participate in the study 3.1.3 Study time: from September 2014 to December 2018 3.1.4 Study places - Collecting subjects at Can Tho Oncology Hospital - Performing of testing techniques: Histopathology tests performs at Pathology department of Can Tho Oncology Hospital; Result conformed at the pathology department of Ha Noi K Hospital; Molecular biological tests performs at Laboratory of Molecular Biology of Can Tho University of Medicine and Pharmacy; Laboratory of Molecular Biology of Institute of Biotechnology Research and Development, Can Tho University and Immunological-testing unit, Cho Ray Hospital 3.2 Materials and methods 3.2.1 Equipment, tools and chemicals: Our research used the equipment, tools, chemicals to perform DNA extraction, PCR and electrophoresis techniques; sequencing techniques on automatic sequencing system; PCR-SSO technique 3.2.2 Research methods 3.2.2.1 Study design: A cross-sectional descriptive study 3.2.2.2 Sample size and sampling method - Objective 1: + Apply the estimation formula to a ratio to calculate the sample size for objective 1: In which: α is probability of error type 1, choose α = 0.05 Z: Value from the standard distribution p: The frequency of LMP1 EBV mutation detected in nasopharyngeal biopsy samples by PCR technique is 90% (from Pham Thi Nguyet Hang, Phan Thi Phi Phi et al., 2003) d: the estimated error, choose d = 0.06 It is calculated that the sample size is 96 samples, rounded to 100 samples In fact, we studied 108 cases + Sampling method: Selected total samples that meets the sample selection criteria - From the results of Figure 4.1 and Chart 4.1, we calculated the rate of presence of LMP1 EBV gene in biopsy tissue sample of studied nasopharyngeal carcinoma patients was 64.8% (70/108) and the rate of LMP1 EBV gene not presence was 35.2% (38/108) Comparing with the results of domestic and foreign studieds with the same survey method (classical PCR technique with primer design at the location of LMP1 gene), we found that there are differences according to geographical area: our results are higher than that of author Le Thanh Ha et al., (2014) (53.1%); Hussain et al., (2015) (61.3%) but lower than Adam et al., (2011) (84.5%), Tan et al., (2003) (83%), Hui et al., (2008) (81%) This difference, in our opinion, may be due to differences in tissue sample types studied - Presence of LMP1 EBV gene in nasopharyngeal carcinoma biopsy tissue samples based on some common characteristics of the studied subjects: + Based on subjects’ biological characteristics: The results of study so that there is no difference between frequency of LMP1 EBV gene presence on biopsy tissue samples of nasopharyngeal carcinoma patients with sexes (male and female), as well as in age groups ( 0.05) 4.3 The rate of LMP1 gene mutations in nasopharynx biopsy samples of studied patients 4.3.1 By PCR and electrophoresis technology: In 70 samples of nasopharynx biopsy samples of studied patients with LMP1 EBV (+), by electrophoresis technology, we discovered 27.1% (19/70) of amplified products of size 230bp and 72.9% (51/70) amplified products of size 200bp So, we have detected 72.9% of cases, which may be had a missing 30bp mutation of LMP1 EBV segment This result was similar to others studies at home and abroad By the time, when comparared with previous studies at home and abroad, we have found that our results were higher than those of Nurhantari et al., (2003) (25.5%); Zhang et al., (2004) (51.51%); Hui et al., (2008) (55.9%); Boutheina et al., (2006) (66.67%), but lower than Pham Thi Nguyet Hang, Phan Thi Phi Phi et al, (2003) (90%); Zhang et al., (2002) (84%); Tan et al., (2003) (84%); Dardari et al., (2006) (84%) This difference may be due to differences in type of biopsy tissue and the histopathology, also, the presence of LMP1 EBV gene on samples studies of nasopharyngeal carcinoma patients 15 4.3.2 By sequencing technique: In total 70 samples with the presence of LMP1 EBV gene, we have randomly selected 33 samples having clear electrophoresis result, including 25 samples with size of 200bp and samples with size 230 bp (24.25%) to determine the mutation type of LMP1 EBV gene by sequencing technique The following results showed that: - The loss 30bp rate at 168266-168295 was 75.8% (25/33) 24.2% (8/33) did not detect the 30 bp deleted Fig 4.2: Results of loss 30bp mutation LMP1 EBV in the 111 sample (compared to B95-8 stratin (V01555) at position 168266-168295) - The location of the loss 30bp mutation of LMP1 EBV gene is 168266-168295, belonging to the exon gene region of LMP1 EBV (168965-168163), coding for 10 amino acids from 343352, related to the TES2 region (313-386) in carboxyl fragment of LMP1 EBV molecule This leads to the loss of epitope of LMP1 EBV molecular and this segment is important to help T 16 CD4 cells recognize antigen As a result, EBV can be avoided by the indentification of cancer anti-immune cells Fig 4.3: No loss 30bp mutation of LMP1 EBV in the 135 sample (compared to B95-8 strain (V01555) at position 168266-168295) but there is a change in nucleotide at position 168295 (T>A) - We have also founded the changes of some more nucleotide in this location: 168295 T>A (8/8), 168225 A>T (33/33), 168308 A>G (33/33), 168320 T>C or T>G (1/33)(32/33); particularly, for the 192th of sample, there is an insert one or more nucleotide, such as: (168268 and 168269) (inserted GC → GTC), inserted more nucleotide (168276 168277, CA → CGAA), so, the total length of this segment is up to 33bp, instead of 30bp, this is the new discovery of our research 4.4 Frequency of HLA alleles genotype in studied nasopharyngeal carcinoma patients 4.4.1 Frequency of class I HLA alleles genotype - We have detected types of HLA-A alleles, of which, HLA-A*02 (40.4%), -A*11 (21.2%) and -A*24 (21.2%) are the alleles has high frequency of disease group, especially, the allele HLA-A*02 has the highest frequency in studied 17 nasopharyngeal carcinoma patients Compared to the control group, there is not statistically signigicant (p > 0.05) difference of frequency of HLA-A alleles in two group Disease group 40.4% Control group 21.2% 21.2% 9.6% Chart 4.2: Frequency of HLA-A in the disease group and the control group 25% 23.1% 9.6% 7.7% Disease group Control group Chart 4.3: Frequency of HLA-B in the disease group and the control group 18 - We have detected 16 types of HLA-B alleles, of which, the alleles HLA-B*15 (25%), -B*46 (23.1%), -B*38 (9.6%) B*07 (7.7%) have high frequency Similarly, the difference between HLA-B alleles of the disease group compared to the control group is not statistically signigicant (p > 0.05) 4.4.2 Frequency of class II HLA alleles genotype - We have detected 12 types of HLA-DRB1 and of wich, HLA-DRB1*12 (17.3%), -DRB1*09 (13.8%), -DRB1*04 (12.1%), -DRB1*08 (12.1%), -DRB1*15 (12.1%) are the alleles with high frequency in the disease group Particularly, when compared with control group, the HLA-DRB1*08 was an allele that can risk of nasopharyngeal carcinoma (with OR = 8.098, p=0.025) and vice versa, the allele HLA -DRB1*12 is the allele that can reduce the risk (with OR = 0.335, p = 0.011) 17.3% 13.8% 12.1% 12.1% 12.1% Disease group Control group Chart 4.4: Frequency of HLA-DRB1 in the disease group and the control group 19 - We have detected types of HLA-DQB1 and types of HLA-DQA1, of which, the allele have high frequency were HLA-DQB1*03 (44.7%), -DQB1*05 (21.4%) -DQB1*06 (17.9%); HLA-DQA1*01 (35.7%), -DQA1*03 (28.6%) and DQA1*06 (21.4%) Similarly, when compared with control group, we have founded that the allele DQB1*03 is also an allele that can reduce the risk of nasopharyngeal carcinoma with OR = 0.367, p = 0.014 4.4.4 Frequency of HLA haplotypes appearing in nasopharyngeal carcinoma patients - In the results, there are some HLA haplotypes have appeared, of which the hallotype A*02-B*15, A*24-B*46 A*11-B*46; DRB1*08-DQB1*03, DRB1*15-DQB1*05 have appeared higher in the disease group than the control group In contrast, the haplotypes A*02-B*46 A*11-B*15; DRB1*09DQB1*03, DRB1*12-DQB1*03 have appeared higher in the control group than the disease group, but these differences were not statistically significant (p>0.05) 4.5 The value of LMP1 EBV gene mutation and the frequency of HLA alleles genotype to diagnose nasopharyngeal carcinoma To find out the value of the mutation of LMP1 EBV gene and HLA genotype in the diagnosis of nasopharyngeal carcinoma, we use the chi-square test and analyzed on the relationship between two NPC patient groups, with and without the loss 30bp of LMP1 EBV gene Similarly, for the relationship between the two NPC patient groups, with and without the presence of HLA genotype alleles, that were recorded as being susceptible to nasopharyngeal carcinoma 20 4.5.1 Relationship between the loss 30bp mutation of LMP1 EBV gene with stage of disease, histopathological type and high frequency of HLA alleles in studied NPC patients The results showed that: - No difference was found between loss 30bp mutation LMP1 EBV gene and stage of disease of studied patients (p > 0.05) - The rate of loss 30bp mutation of LMP1 EBV gene in undifferentiated carcinoma of nasopharyngeal type (85.7%) was higher than the rate of nonkeratinzing squamous cell carcinoma type (64.3%) and this difference is statistically significant, with p = 0.048 - Among all HLA alleles genotype, only HLA-B*15 allele was an allele that can risk of loss 30bp mutation LMP1 EBV gene (with OR = 4.640, p = 0.018) Others have not seen any difference 4.5.2 Relationship between frequency of HLA alleles genotype with stage of disease, histopathological type in studied patients - Among all of HLA alleles genotype, only HLA-A*02 allele has a statistically significant difference between the two histopathological type group (undifferentiated carcinoma of nasopharyngeal type and nonkeratinizing squamous cell carcinoma type, with p = 0.034) The others alleles have not difference - Similarlly, among all of HLA alleles genotype, only two HLA-B*15 and HLA-DQA1*03 alleles reduced the risk of late stage of disease by 12.2% and 17.8%, there are no difference for other alleles 21 4.5.3 The sensitivity, specificity of some HLA haplotypes to diagnose nasopharyngeal carcinoma: We have found that the HLA haplotypes with high specificity to diagnose nasopharyngeal carcinoma are A*11-B*46, A*24-B*46, DRB1*08-DQB1*03, DRB1*15-DQB1*05 and A*02-B*46 (all p