LMP1-EBV Gene Deletion Mutations and HLA Genotypes of Nasopharyngeal Cancer Patients in Vietnam

Nasopharyngeal carcinoma (NPC) is the most common cancer among head and neck cancers in Vietnam. We aimed to identify the rate of a 30 bp deletion mutation of the LMP1-EBV gene in nasopharyngeal biopsy tissue samples, the HLA genotypes of NPC patients, and the relationship between these two targets. Patients with NPC at Can Tho Oncology Hospital from September 2014 to December 2018 were selected. A length of 30 bp of the del-LMP1-EBV gene was analyzed using a PCR technique, and the HLA genotypes in patients’ blood samples were analyzed with PCR-SSO technology. HLA-B*15 gene carriers had the highest risk of 30 bp LMP1-EBV gene deletion mutation, which was found in 51 out of 70 patients (72.9%). Carriers of the HLA-B*15 allele had a 4.6-fold increased risk of a 30 bp del-LMP1-EBV gene compared with non-carriers of this allele. The initial identification of NPC was related to the 30 bp del-LMP1-EBV gene and high frequencies of the -A*02, -B*15, -DRB1*12, -DQB1*03, and -DQA1*01 HLA alleles. Our study results suggest an association of the 30 bp del-LMP1-EBV gene and the HLA-B*15 allele with NPC susceptibility.


Introduction
Malignant tumors called nasopharyngeal carcinomas mostly develop from the layer of differentiated epithelial cells that covers the nasopharynx. They are one of Vietnam's ten most common malignancies among head and neck cancers [1,2]. The human leukocyte antigen (HLA) genetic component of a patient's vulnerability to the disease, Epstein-Barr virus (EBV) infection, and the effect of dietary components are the three categories of pathogenic variables that are associated with the pathophysiological features of NPC. The latent membrane protein 1 gene of EBV (LMP1-EBV) is one of the EBV genes discovered in most nasopharyngeal biopsies of patients with NPC [1,[3][4][5][6]. It is a gene encoding the membrane protein LMP1, a unique EBV gene product capable of causing transformation, inhibiting epithelial cell differentiation, and acting as an initiating protein for the malignant transformation of cells [4,[6][7][8]. In addition, the evasion of the EBV immune response by mutations in the LMP1-EBV gene, especially the 30 bp deletion mutation of the LMP1-EBV gene, produces non-immunogenic LMP1 products; it is also the way that EBV initiates tumorigenesis and growth [9,10]. Many studies have also mentioned a second factor related to the biological pathogenesis of NPC: the genetic factor of the HLA gene in susceptible patients with NPC. This factor explains the prevalence of NPC in certain geographical Pathophysiology 2023, 30 2 areas [11][12][13]. However, the HLA molecule-a product of the HLA gene-has many functions, including the antigen presentation process of recognizing a host's immune cells. Therefore, another problem is the relationship between HLA gene sensitivity and the mutation status of the LMP1-EBV gene, especially the 30 bp gene deletion mutation of the LMP1-EBV gene, which is a common mutation in nasopharyngeal cancer that previous studies have recognized.
Our study's objectives are to ascertain the frequency of HLA genotypes in nasopharyngeal cancer patients treated at a Vietnam oncology hospital, the rate of 30 bp gene deletion mutations in the LMP1-EBV gene in nasopharyngeal biopsy samples, and the relationship between these two variables.

Materials
Nasopharyngeal biopsy tissue samples of NPC patients were obtained from Can Tho Oncology Hospital in the Mekong Delta region (from September 2014 to December 2018), as well as blood samples from NPC patients.

Selection Criteria
Patients with NPC who were treated at Can Tho Oncology Hospital were included, with no age or gender restrictions. For the nasopharyngeal biopsy tissue samples, fresh, unprocessed biopsy tissue was collected in an amount of 0.5-4 mg. Nasopharyngeal biopsy tissue samples embedded in paraffin blocks were used in cases when fresh biopsy tissue samples did not reach the standard amount. Collected tissue samples were embedded in paraffin blocks. The histopathological results for the same patient were embedded in a paraffin block for the duration of tissue sample collection, and reading of the results did not exceed 4 weeks after collection (about 10 slices, with a thickness of 5 µm). Blood samples of NPC patients showed the presence of the LMP1-EBV gene in the nasopharyngeal biopsy tissue sample.

Exclusion Criteria
The exclusion criteria included patients with recurring nasopharyngeal cancer, cancer revisiting periodically during treatment, those residing in the study region for less than one year, those not living in the provinces of the Mekong Delta, or those who did not agree to participate in the study. Figure 1 depicts a cross-sectional description of the study and analysis.

Sample Selection
We selected 108 samples and collected all the samples that matched the sample selection criteria during the study period (objective 1), as well as randomly selecting a single sample (objective 2).

Sample Selection
We selected 108 samples and collected all the samples that matched the sample selection criteria during the study period (objective 1), as well as randomly selecting a single sample (objective 2).

DNA extraction chemicals
For chemical DNA extraction from the biopsy tissue, an Invisorb ® Spin Tissue Mini Kit (Stratec company, Birkenfeld city, Germany) was used.
For chemical DNA extraction from the whole blood samples, an Invisorb ® Spin Blood Mini Kit (Stratec company, Birkenfeld city, Germany) was used.

Chemicals Required for Automated Gene Sequencing
The following kits were used for automated gene sequencing: a BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a DNA purification GenJet Gel Extraction Kit (Thermo Scientific) using agarose gel.

Chemicals Required for Automated Gene Sequencing
The following kits were used for automated gene sequencing: a BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a DNA purification GenJet Gel Extraction Kit (Thermo Scientific) using agarose gel.

Study Content
To determine the prevalence of the HLA gene in blood samples of patients with NPC, we performed a PCR-SSO technique using a LABScan3D™ system (Luminex FLEXMAP 3D) with LABType™XR and CWD Typing Test chemicals. We analyzed the results using HLA Fusion™ LABType software ® at the immuno-assay unit of Cho Ray Hospital to identify HLA alleles from the blood samples of the study patients. The frequency of an HLA gene allele was recorded as the occurrence of a particular HLA allele at a high rate compared with the occurrence of other HLA alleles in the total HLA alleles of the survey of the HLA gene locus.

Statistical Analysis
The collected study data were entered into the EpiData 3.1 program, and statistical processing was performed using Stata 10.0 software. We used a descriptive statistics algorithm to describe the ratio values of the variables and tested the hypothesis with a chi-squared test.

Research Ethics
The study was approved by the Proposal Defense Council of the Institute of Biotechnology, Research, and Development at Can Tho University.
The study was conducted only after receiving permission from the Scientific Research Council and the Board of Directors of Can Tho Oncology Hospital.
The steps to conduct the study were approved by the dean of the department, the treating doctor at the department, and the patients' voluntary consent to participate in the study (obtained through a consent form to participate in the survey). The examination procedure was approved when a patient's condition was stable.
During the implementation of the study, the patients' rights were guaranteed: encryption for patient information was confidential, patients could refuse to participate at any time during the research process, and study participation did not increase any costs of patient treatment.

Results
During the implementation of the study, we recorded 108 NPC cases treated at Can Tho Oncology Hospital that met the sample selection criteria. From 108 participants, a total of 70 nasopharyngeal biopsy samples with the presence of the LMP1-EBV gene were found in the electrophoresis analysis (including 38 fresh biopsy tissue samples and 70 biopsy tissue samples embedded in paraffin blocks). The results for the nasopharyngeal objectives are presented as follows.

Prevalence of LMP1-EBV Gene Mutations in Nasopharyngeal Biopsy Tissue Samples of Patients with NPC
Among 70 nasopharyngeal biopsy tissue samples from patients with the presence of the LMP1-EBV gene found by electrophoresis, the study detected 72.9% (51/70) of the amplification products with 200 bp (expressing a 30 bp deletion mutation of the LMP1-EBV gene) ( Table 1 and Figure 2). treating doctor at the department, and the patients' voluntary consent to participate in the study (obtained through a consent form to participate in the survey). The examination procedure was approved when a patient's condition was stable.
During the implementation of the study, the patients' rights were guaranteed: encryption for patient information was confidential, patients could refuse to participate at any time during the research process, and study participation did not increase any costs of patient treatment.

Results
During the implementation of the study, we recorded 108 NPC cases treated at Can Tho Oncology Hospital that met the sample selection criteria. From 108 participants, a total of 70 nasopharyngeal biopsy samples with the presence of the LMP1-EBV gene were found in the electrophoresis analysis (including 38 fresh biopsy tissue samples and 70 biopsy tissue samples embedded in paraffin blocks). The results for the nasopharyngeal objectives are presented as follows.

Prevalence of LMP1-EBV Gene Mutations in Nasopharyngeal Biopsy Tissue Samples of Patients with NPC
Among 70 nasopharyngeal biopsy tissue samples from patients with the presence of the LMP1-EBV gene found by electrophoresis, the study detected 72.9% (51/70) of the amplification products with 200 bp (expressing a 30 bp deletion mutation of the LMP1-EBV gene) ( Table 1 and Figure 2). Table 1. Percentage of LMP1-EBV gene amplification products with a size of 200 bp detected in nasopharyngeal biopsy tissue samples of NPC patients studied by PCR technique and amplification product electrophoresis (n = 70).  3).

Prevalence of HLA Gene in Blood Samples of Patients with NPC
When selecting samples for HLA gene determination in nasopharyngeal cancer patients, we randomized only 30 patients with nasopharyngeal cancer who met the nasopharyngeal cancer patient criteria, and we identified the presence of the LMP1-EBV gene in nasopharyngeal biopsy tissue samples to assess objective 1.

Prevalence of HLA Gene in Blood Samples of Patients with NPC
When selecting samples for HLA gene determination in nasopharyngeal cancer patients, we randomized only 30 patients with nasopharyngeal cancer who met the nasopharyngeal cancer patient criteria, and we identified the presence of the LMP1-EBV gene in nasopharyngeal biopsy tissue samples to assess objective 1.

Association between 30 bp Deletion Mutation Rate of LMP1-EBV Gene and Frequency of HLA Alleles
There was a statistically significant difference in the HLA-B*15 alleles of the groups with and without 30 bp deletion mutations of the LMP1-EBV gene, with OR = 4.64 and p < 0.05. This meant that patients carrying the HLA-B*15 allele had an LMP1-EBV 30 bp deletion mutation rate about 4.6 times higher than those who did not carry this allele. For the remaining alleles of -A*02, -A*11, -A*24, -B*38, -B*46, -DRB1*12, -DRB1*09, -DQB1*03, and -DQA1*01, there were no statistical differences between the two groups (Table 5).

Discussion
By using the PCR technique with an LMP1 primer pair (168,373-168,174), we detected 70/108 (64.81%) nasopharyngeal biopsy tissue samples of the patients with the presence of the LMP1-EBV gene (230 bp and 200 bp). Amplification products of 200 bp in size were found in 72.9% (51/70) of the 70 samples from the LMP1-EBV gene patients ( Figure 1). As a result, the PCR method detected 72.9% of the 30 bp deletion mutations of the LMP1-EBV gene.
As we continued to compare our results with the studies of other authors worldwide, we saw differences in the rates of 30 bp deletion of the LMP1-EBV gene among studies in different geographical regions. For example, in China, the rate of 30 bp deletion mutations of the LMP1-EBV gene was 84% (36/43) (Zhang et al., 2002) [14]; Malaysia also had a similar rate of 84% (21/25) (Tan et al., 2003) [15]; Morocco (north Africa) had a rate of 84% (51/61) 30 bp deletion mutations of the LMP1-EBV gene (Dardari et al., 2006) [16]; and 6.67% (28/42) was the proportion found in a study in Tunisia (northeast Africa) (Boutheina et al., 2006) [17]. In Asia, the rate of 30 bp deletion mutations of the LMP1-EBV gene in NPC patients accounted for the highest rate (79%), followed by that in the Americas (64%) and Europe, with north Africa having the lowest rate (59%).
Using sequencing methods and the analysis results of 25 LMP1-EBV gene sequences compared with the EBV B95-8 reference strain (Genbank V01555), we randomly selected 25 samples with sizes of 200 bp and compared them with the clear electrophoresis lines in 51 samples showing the presence of the LMP1-EBV gene. There were 30 bp deletion mutations at the 168,266-168,295 position ( Figure 2). Therefore, when both PCR electrophoresis and LMP1-EBV gene sequencing were performed, the results corresponded completely, with 30 bp deletion mutations of the LMP1-EBV gene in patients with NPC.
We compared the results obtained in this study with previous results from Tran Ngoc Dung (2000) [18], who studied class-I and -II HLA alleles in 47 nasopharyngeal cancer patients from northern Vietnam using cytotoxicity based on Terasaki's technique and indirect immunofluorescence and showed that there were 11 types of HLA-A alleles with high frequencies, including -A*02 (30%), -A*11 (20%), -A*25 (17%), and -A*24 (9.4%) [18]. Compared with the results of this study, there are similarities in the frequencies of the -A*02, -A*11, and -A*24 alleles, which also accounted for the alleles with the highest proportions in the studied patients. However, there are differences in NPC patients from two different geographical regions of Vietnam. The assessment of allele frequency for locus A in Vietnamese NPC patients resulted in the -A*02, -A*11, and -A*24 alleles showing high frequency and these are the HLA alleles that often appear in individuals of Asian descent.
In the HLA-B locus, our study detected 16 alleles (Results Table). We compared this with the results of Tran Ngoc Dung (2000) [18] [21].
In short, the results for the HLA-B alleles in this study are similar to those of the above studies for some alleles, such as -B*46, -B*15, and -B*38. However, the frequency of encountering each HLA-B allele varied, which indicated the diversity of HLA-B alleles in patients with NPC.
In the pathogenesis of NPC, there are two main risk factors, namely 30 bp deletion mutations of the LMP1-EBV gene and the inherited factor of the HLA gene in NPC patients. The question continually posed in this study was whether these two risk factors in patients with NPC were associated. To answer this question, we continued to analyze the association between the 30 bp deletion mutation rate and the high frequencies of class-I HLA alleles (HLA-A and HLA-B) and class-II HLA alleles (HLA-DRB1, HLA-DQB1, and HLA-DAB1) in patients with NPC.
From Table 5, we could see no relationship between the 30 bp gene deletion mutation of LMP1-EBV and the high frequency of HLA-A alleles in the study sample (p > 0.05). Although the alleles HLA-A*02, HLA-A*11, HLA-A*24, and HLA-A*33 had high frequencies of occurrence in this study, these frequency results are similar to those of previous studies, both in Vietnam and abroad. According to the results of several previous studies, the HLA-A*02 allele plays an essential role in helping to present the LMP1-EBV antigen to CD8 T cells for the recognition and destruction of EBV-infected cells at the antigendetermining sites of YLLEMLWRL (125-133) and YLQQNWWTL (159-167) (Lin et al., 2004; Tang et al., 2008). A limitation in this study is that we only sequenced a short segment of the LMP1-EBV gene, the carboxyl terminus coding region, or CTAR2 (351-386), so it was not possible to make further judgments about this. However, the results of our study's genetic-sequencing analysis showed that, in addition to the 30 bp gene deletion mutation of LMP1-EBV that the study results presented, the study also recorded the occurrence of many other nucleotide changes scattered throughout the LMP1-EBV gene (locations 168,225, 168,295, and 168,320). These results suggested that there might still be point mutations at other sites, particularly in the gene region encoding the CTAR1 segment of the LMP1-EBV molecule, which could help EBV escape the immune response of host translation and might be related to the HLA-A*02 allele. This HLA-A allele is responsible for presenting EBV antigens to recognized T cells. This suggests a new study direction for the pathogenesis of nasopharyngeal cancer related to the presence of HLA-A alleles and LMP1-EBV.
In addition, from Table 5, we compared the frequencies of HLA-B*15, HLA-B*38, and HLA-B*46 alleles, as well as high-proportion HLA-B alleles, in study samples with mutation rates of 30 bp LMP1-EBV gene deletion. Carriers of the HLA-B*15 allele (4.64; 1.23-17.52; 0.018) had a 4.6-times higher risk of losing 30 bp segment mutations of the LMP1-EBV gene than those who did not carry this allele. In particular, domestic and foreign studies have not published data on this association; this can be considered a new finding about the relationship between 30 bp gene deletion mutation of LMP1-EBV and the HLA-B*15 allele in NPC patients in Vietnam that is specific this study.
This study is an important first step in the process of researching LMP1-EBV gene expression in the biopsy tissue of patients with NPC using HLA-A, -B, -DRB1, -DQB1, and -DQA1 alleles investigated using a PCR-SSO technique. The novelty of this study is that it is the first study on the biology of nasopharyngeal cancer in the Mekong Delta, Vietnam. Our results provided scientific data on the rate of mutations in the LMP1-EBV gene for NPC tissue biopsy and the frequency of HLA alleles in Vietnamese patients, as well as on the relationship between the 30 bp gene deletion mutation of LMP1-EBV and HLA-B*15 alleles in NPC patients, which has not previously been studied, either in Vietnam or the world. This scientific document can be referred to in order to update teaching methods about nasopharyngeal cancer at universities of Medicine and Pharmacy. Moreover, these results can provide a premise for further studies on the biology of nasopharyngeal cancer in Vietnam, allowing clinicians to view the pathogenesis of NPC in patients more accurately, contributing to a more practical treatment effect.

Conclusions
Through 108 nasopharyngeal biopsy tissue samples of patients with NPC in southern Vietnam, we found that 72.9% of the LMP1-EBV genes had 30 bp deletion mutations. The HLA alleles appearing most frequently in patients with NPC were -A*02, 11, and 24; -B*15 and 46; -DRB1*12, 09, and 08; -DQB1*03, 05, and 06; and -DQA1*01, 03, and 06. Compared with those who did not carry the HLA-B*15 variant, carriers were more likely to experience 30 bp deletion on the LMP1-EBV gene. Our findings indicated that the majority of Vietnam's NPC patients with EBV-associated carcinomas included the deleted version of LMP1. To further understand the LMP1 signaling system and determine the role that LMP1 sequence variation plays in the pathogenesis of EBV-associated malignancies, particularly NPC, more research using these variations is required.