Introduction
Head and neck cancers, including cancers of the
pharynx, larynx, nasopharynx, oropharynx, salivary glands, oral
cavity, nasal cavity and external auricle, rank as the seventh most
common group of cancers worldwide (1). Although tobacco and alcohol use are
well-known risk factors, Epstein-Barr virus (EBV) infection has
also been implicated in numerous cases (1-3).
Among these cancers, oral cavity cancer is the most
prevalent, with 377,713 new cases reported globally in 2020. The
global age-standardized incidence rate (ASR) for oral cavity cancer
was 6.0 for men and 2.3 for women (4). In the same year, 133,354 cases of
nasopharynx cancer, 98,412 cases of oropharynx cancer, 84,254 cases
of hypopharynx cancer, 184,615 cases of larynx cancer and 53,583
cases of salivary gland cancer were reported globally (4). In Thailand, oral cavity cancer has
consistently ranked among the most common malignancies in men, as
demonstrated by population-based cancer registry data, multicenter
studies, and national epidemiological analyses spanning from the
1990s to recent years (5-11).
Globally, the incidence of early-onset cancer in
young adults increased by 79.1% from 1990 to 2019, with a projected
increase of 31% by 2030 (12,13). A
similar trend was observed for oral cavity and pharyngeal cancers
among young adult women (12).
Adolescents and young adults are defined as individuals aged 15-49
years (12-14).
The causes behind the rising cancer rates in younger
populations, whether genetic or environmental/lifestyle-related,
remain unclear. Proposed hypotheses include chronic inflammation,
smaller family sizes, gut microbiome alterations, processed food
consumption, microplastic exposure, sedentary lifestyles, family
history of cancer, early screenings, germline mutations and other
genetic alterations (15,16). Understanding potential risk factors,
including EBV reactivation, may support early prevention strategies
for head and neck cancer (3). EBV
may also participate in infection events with other viruses,
including human papillomavirus, BK polyomavirus, human
cytomegalovirus and herpes simplex virus (3). Currently, there is no screening
biomarker for head and neck cancer in Thailand. Thus, head and neck
cancer is only detected after the patient exhibits signs and
symptoms. Furthermore, cases of oncogenic EBV infections in head
and neck cancer are limited in Thailand (5-11).
Previous reports have suggested that EBV infection
may promote the progression of oral squamous cell carcinoma (OSCC)
(17,18). A meta-analysis of 13 case-control
studies (686 patients with OSCC and 433 controls) confirmed a
statistically significant association between EBV infection and
increased OSCC risk (19).
Furthermore, a study involving 315 Thai participants detected EBV
infection in 27.2% of normal oral exfoliated cells and 72% of oral
cancer cases (20). EBV is
transmitted through saliva and genital secretions (21). Mekmullica et al (22) reported that EBV antibody-positive
status in the Thai population was associated with low family income
(≤10,000 baht/month) and age >1 year. Pongpakdeesakul et
al (23) identified alcohol
consumption, second-hand smoke and using tap water for brushing
teeth as risk factors for EBV DNA reactivation in blood
samples.
EBV is separated into the α, β and γ subfamilies.
Nearly 95% of the population is infected with EBV throughout life
(24). EBV comprises linear
double-stranded DNA, ~172 kb in length. EBV contains at least 11
genes spanning EBV-encoded RNAs (EBER-1 and EBER-2), EBV nuclear
antigens (EBNA-1-6) and latent membrane proteins (LMP-1, and LMP-2A
and -2B). The latent phase of EBV infection is associated with
numerous types of cancer, such as lymphoma, Hodgkin lymphoma and
nasopharyngeal carcinoma, due to the expression of EBERs, EBNA-1
and the three LMPs, among others (25,26).
PCR could be used to detect EBV reactivation in blood or saliva.
DNA-positive results indicate that the virus is actively
replicating (27-29).
EBV and TNF-α interact in a complex,
context-dependent manner, with TNF-α functioning as both a tumor
promoter and a cancer inhibitor depending on EBV activity. TNF-α
can inhibit the lytic replication of EBV by reducing the expression
of viral proteins such as BamHI Z Epstein-Barr virus
replication activator and R transactivator. TNF-α affects the
glutathione peroxidase 4 protein and can inhibit EBV reactivation.
Furthermore, EBV-infected T cells exhibit increased secretion of
TNF-α, potentially promoting cancer development (30-32).
BamHI Z fragment leftward open reading frame 1 (BZLF-1) also
suppresses TNF-α production to facilitate viral propagation, while
LMP-1 downregulates TNF-α receptor 1, thereby preventing apoptosis
and promoting proliferation (30-32).
Currently, updated data on EBV reactivation risk
factors in the Thai population are limited and mostly focus on
antibody detection (5-11),
and evidence linking genetic mutations to EBV infection is also
limited. Therefore, the present study aimed to detect EBV
reactivation using quantitative PCR (qPCR) and to investigate
associated risk factors in normal oral buccal cells using logistic
regression.
Materials and methods
Specimens
A total of 982 human oral buccal DNA samples were
collected from donors across Thailand (samples collected from the
village population of Ubon Ratchathani and Phayao province) between
December 2016 and March 2022, as reported in previous studies
(33,34), and analyzed using qPCR as described
subsequently. Participant age ranged between 3 and 90 years.
Individuals who were easy to contact or reach were included.
Individuals living in Thailand were included, and there were no
other inclusion criteria. Individuals who could not perform oral
swirling with PBS and patients with cancer were excluded. Among the
samples, 599 included information on sex, health status and life
history (such as presence of mouth ulcers, congenital diseases,
family history of cancer, soft drink consumption, alcohol use and
smoking). This information was used for risk factor assessment via
regression analysis. Percentages based on demographic data (599
samples) were used to generate a surface chart (contour), with
patients divided into various age groups.
All variable factors (such as presence of mouth
ulcers, congenital diseases, family history of cancer, soft drink
consumption, alcohol use and smoking) were used for univariate
regression model analysis, and only significant variables
(P<0.05) were used for the multivariate regression model
analysis.
The samples used in these experiments were collected
between 2016 and 2022, and leftover samples were used. Therefore,
some DNA samples were depleted or unavailable. The number of lost
samples was ~30 samples.
The required sample size was calculated using the
following formula: N=Z21-a
P(1-P)/d2 (35), where
EBV prevalence (P) ranged between 3.8 and 33.75%, Z=1.96 for a 95%
confidence level, and d=0.01 or 0.05, resulting in a sample size
range of 56-1,404. The study was approved by the Human Ethics
Committee Ubon Ratchathani University (Ubon Ratchathani, Thailand;
approval no. UBU-REC-68/2567; valid from March 20, 2024, to March
19, 2026). All procedures followed The Declaration of Helsinki
(36), Belmont Report (37), Council for International
Organizations of Medical Sciences guidelines (38) and the International Conference on
Harmonization in Good Clinical Practice standards (39). Written informed consent was obtained
from all participants or their legal guardians (for patients <18
years old).
DNA extraction
Oral buccal cells were collected 1 h after tooth
brushing using 10 ml sterile 1X PBS. Mouthwash samples were
centrifuged at 8,000 x g for 5 min at room temperature and
resuspended in 500 µl lysis buffer (10 mM Tris-HCl, pH 7.8;
5 mM EDTA; 0.5% SDS) with 25 µl proteinase K (20 mg/ml
stock) (40). After protein
precipitation using 400 µl of 5 M potassium acetate, the
supernatant was mixed with an equal volume of isopropanol. DNA was
pelleted by centrifugation at 10,000 x g for 10 min at 4˚C,
and washed with 70% ethanol. The supernatant was removed, and the
DNA was resuspended in Tris-EDTA buffer (10 mM Tris, pH 7.8; 1 mM
EDTA), and then stored at -20˚C (34).
EBV DNA detection via qPCR
EBV DNA (targeting EBNA-1 and LMP-1
genes) was detected using qPCR with specific primer sets (17,41).
For EBNA-1, the following primers were used: Forward (primer
name, QP3), 5'-CCACAATGTCGTCTTACACC-3' and reverse (primer name,
QP4), 5'-ATAACAGACAATGGACTCCCT-3' (41). For LMP-1, the following
primers were used: Forward, 5'-CAGTCAGGCAAGCCTATG-3' and reverse,
5'-CTGCTTCCGGTGGAGATG-3'. The expected PCR product sizes were 99 bp
for EBNA-1 and 106 bp for LMP-1 (17). The B95 cell line was used as a
positive control for EBV DNA detection. The B95-8 cell line
sequence was identified according to the V01555.2 EBV genome from
the National Center for Biotechnology Information (NCBI), and
authenticated prior to experimentation. B95 cells were cultured in
RPMI medium (Lonza Group, Ltd.) supplemented with 10% FBS (HyClone;
Cytiva), 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate and
2 mM glutamine at 37˚C with 5% CO2 (42).
EBV genome, strain B95-8 profiling was performed by
Professor Jaap M. Middeldorp (Department of Pathology, VU
University Medical Center, Amsterdam, The Netherlands), and the
profile matched the reference profile available from NCBI (43).
The EBNA-1 and LMP-1 primers were
validated in previous studies by Stevens et al (41) and Heawchaiyaphum et al
(17). EBNA-1 and
LMP-1 primers were detected in duplicate.
Reactions were prepared using 5X FiREPOL Eva Green
qPCR Mix Plus (Solis BioDyne), with a final composition consisting
of 1X FiREPOL master mix, 0.4 pM of each primer (forward and
reverse), 2 µl DNA template and distilled water (DW) to a final
volume of 20 µl. The qPCR conditions were as follows: Initial
activation at 95˚C for 12 min, 40 cycles of denaturation at 95˚C
for 15 sec and annealing/elongation at 60˚C for 30 sec, and melting
at 65-95˚C (0.5˚C/5 sec/step).
Conventional PCR
Conventional PCR was performed as described
previously (44). Detection of the
TNF-α promoter (rs1799964; -1031 T>C) was performed using
allele-specific PCR. For the T allele, the following primers were
used: Forward, 5'-AAGGCTCTGAAAGCCAGCTG-3' and reverse,
5'-CCAGACCCTGACTTTTCCTTCA-3'. For the C allele, the following
primers were used: Forward, 5'-GAAGCAAAGGAGAAGCTGAGAAGAC-3' and
reverse, 5'-CTTCCATAGCCCTGGACATTCT-3'.
The expected PCR product sizes were 444 and 316 bp
for the TT genotype, 444, 316 and 174 bp for the TC genotype, and
444 and 174 bp for the CC genotype (44).
To assess the long region of the TNF-α
promoter, 20-25 randomly selected EBV-positive and EBV-negative
samples from a previous study were used (23). The primers, which were previously
designed and original in-house primers for amplification of the
long promoter region, were as follows: Forward,
5'-AGCTGTGGGGAGAACAAAAGG-3' and reverse,
5'-GAGGGCGGGGAAAGAATCAT-3'. The PCR product size was 1,102 bp.
Reactions were prepared using a 5X FiREPOL
Ready-to-Load Master Mix (Solis BioDyne) with the following
components: 1X FiREPOL master mix, 0.4 pM of each primer (forward
and reverse), 3 µl DNA template and DW to a final volume of 25 µl.
The PCR conditions were as follows: Initial activation at 95˚C for
5 min, 40 cycles of denaturation at 95˚C for 1 min, annealing at
58˚C for 1 min and elongation at 72˚C for 1 min, followed by a
final elongation step at 72˚C for 5 min. PCR products were analyzed
by 2% agarose gel electrophoresis in 1X Tris-acetate-EDTA buffer at
100 V for 40 min.
DNA sequencing
Sanger sequencing was performed to analyze the
1,102-bp region of the TNF-α promoter (long region),
covering genomic region NC_000006.12 (positions 31574417-31575499).
The aforementioned 20-25 randomly selected samples were sequenced
to verify both EBV-positive and EBV-negative cases. Sequence data
were analyzed using BioEdit (version 7.2; https://bioedit.software.informer.com/7.2/), a
biological sequence alignment editor, and compared against the
GenBank reference sequence (region, NC_000006.12; positions,
31574417-31575499; https://www.ncbi.nlm.nih.gov/nuccore/NC_000006.12).
Statistical analysis
Statistical analysis was conducted using IBM SPSS
software version 16 (SPSS, Inc.). Data are presented as n (%).
Pearson's χ2 test was applied to compare categorical
variables between groups. For qPCR, each sample was analyzed in
duplicate (technical replicates). Both univariate and multivariate
logistic regression analyses were performed to evaluate
associations [P-value, odds ratio (OR) and 95% CI]. P≤0.05 was
considered to indicate a statistically significant difference.
Results
EBV DNA detection via qPCR and risk
factors
Oral buccal cells were collected from 982 donors in
Thailand, including 301 male (30.7%) and 681 female (69.3%)
patients aged 3-90 years (mean age, 45.39±14.99 years). qPCR
detected EBV-positivity in 350 out of 974 individuals (36%) based
on the EBNA-1 gene, and in 458 out of 885 individuals (52%)
based on the LMP-1 gene. Co-positivity for both
EBNA-1 and LMP-1 was observed in 196 out of 981
individuals (20%) (Table I). DNA
from the B95-8 cell line was used as a positive control.
 | Table IEpstein-Barr virus-status by age, sex
and TNF-α genotype (rs1799964; -1031 T>C). |
Table I
Epstein-Barr virus-status by age, sex
and TNF-α genotype (rs1799964; -1031 T>C).
| | EBNA-1 | LMP-1 | Both genes
positive |
|---|
| Variable | Positive, n
(%) | Negative, n
(%) | Total, n | Positive, n
(%) | Negative, n
(%) | Total, n | Positive, n
(%) | Negative, n
(%) | Total, n |
|---|
| Age, years | | | | | | | | | |
|
3-10 | 56(56) | 44(44) | 100 | 32(32) | 68(68) | 100 | 12(12) | 88(88) | 100 |
|
11-20 | 118(36) | 209(64) | 327 | 190(68) | 89(32) | 279 | 87(27) | 240(73) | 327 |
|
21-30 | 58(31) | 128(69) | 186 | 93(63) | 55(37) | 148 | 41(21) | 151(79) | 192 |
|
31-40 | 43(43) | 56(57) | 99 | 38(39) | 59(61) | 97 | 20(20) | 80(80) | 100 |
|
41-50 | 14(16) | 73(84) | 87 | 31(36) | 56(64) | 87 | 8(9) | 79(91) | 87 |
|
51-90 | 61(35) | 114(65) | 175 | 74(43) | 100(57) | 174 | 28(16) | 147(84) | 175 |
|
Total | 350(36) | 624(64) | 974 | 458(52) | 427(48) | 885 | 196(20) | 785(80) | 981 |
|
P-value | <0.001 | <0.001 | <0.001 | | | | | | |
| Sex | | | | | | | | | |
|
Female | 233(35) | 442(65) | 675 | 339(55) | 276(45) | 615 | 138(20) | 543(80) | 681 |
|
Male | 117(39) | 184(61) | 301 | 119(44) | 153(56) | 272 | 58(19) | 243(81) | 301 |
|
Total | 350(36) | 626(64) | 976 | 458(52) | 429(48) | 887 | 196(20) | 786(80) | 982 |
|
P-value | 0.190 | | | 0.002 | | | 0.719 | | |
|
Odds ratio
(female vs. male; 95% CI) | 0.829
(0.626-1.098) | | 1.579
(1.185-2.105) | | 1.065
(0.756-1.499) | |
| TNF-α
genotype | | | | | | | | | |
|
TC | 134(40) | 205(60) | 339 | 141(42) | 197(58) | 338 | 59(17) | 282(83) | 341 |
|
CC | 71(37) | 121(63) | 192 | 59(31) | 132(69) | 191 | 18(9) | 175(91) | 193 |
|
Total | 205(39) | 326(61) | 531 | 200(38) | 329(62) | 529 | 77(14) | 457(86) | 534 |
|
P-value | 0.562 | | | 0.014 | | | 0.012 | | |
|
Odds ratio
(TC vs. CC; 95% CI) | 1.114
(0.773-1.605) | | 1.601
(1.100-2.331) | | 2.034
(1.161-3.563) | |
There was a significant association between
EBNA-1 positivity and age. In particular, individuals aged
3-10 years had a higher prevalence of EBNA-1 positivity
(P<0.001) than other groups, while those aged 11-20 years showed
a higher prevalence of LMP-1 and dual gene positivity
(P<0.001) than other groups (Table
I).
Using LMP-1 gene detection, EBV positivity
was significantly more prevalent in female patients (55%) compared
with male patients (43%) (P=0.002; OR, 1.579; 95% CI, 1.185-2.105).
In the LMP-1 positive group, the TNF-α genotype
TC (42%) was more common than the TNF-α genotype
CC (31%) (P=0.014; OR, 1.601; 95% CI, 1.100-2.331).
Additionally, individuals positive for both EBNA-1 and
LMP-1 had higher frequencies of the TC genotype (17%)
than the CC genotype (9%) (P=0.012; OR, 2.034; 95% CI,
1.161-3.563) (Table I; Fig. 1). EBV status by age, sex and
TNF-α status is shown in Table
I. The association between TNF-α (-1031 TC) and dual
gene positivity (EBNA-1- and LMP-1-positive) is shown
in Fig. 1.
Results for the 599 buccal samples are presented in
Table II. The proportion of
samples positive for both EBNA-1 and LMP-1 was
13%.
| Table IIAssociation between lifestyle factors
and Epstein-Barr virus status. |
Table II
Association between lifestyle factors
and Epstein-Barr virus status.
| | EBNA-1 | LMP-1 | Both genes
positive |
|---|
| Variable | Positive, n
(%) | Negative, n
(%) | Total, n | Positive, n
(%) | Negative, n
(%) | Total, n | Positive, n
(%) | Negative, n
(%) | Total, n |
|---|
| Sex | | | | | | | | | |
|
Male | 99(33) | 200(67) | 299 |
113(45)a | 136(55) | 249 | 42(14)a | 257(86) | 299 |
|
Female |
108(36)a | 191(64) | 299 | 84(31) | 188(69) | 274 | 34(11) | 265(89) | 299 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Age, years | | | | | | | | | |
|
3-10 | 56(56)a | 44(44) | 100 | 32(32) | 68(68) | 100 | 12(12) | 88(88) | 100 |
|
11-20 | 44(35) | 83(65) | 127 | 36(30) | 83(70) | 119 | 13(10) | 114(90) | 127 |
|
21-30 | 14(19) | 58(81) | 72 | 27(73)a | 10(27) | 37 | 9(12) | 63(88) | 72 |
|
31-40 | 19(19) | 81(81) | 100 | 31(35) | 57(65) | 88 | 8(8) | 92(92) | 100 |
|
41-50 | 43(43) | 56(57) | 99 | 34(38) | 56(62) | 90 | 20(20)a | 79(80) | 99 |
|
51-90 | 31(31) | 69(69) | 100 | 37(43) | 50(57) | 87 | 14(14) | 86(86) | 100 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Children, n | | | | | | | | | |
|
1 | 21(37)a | 36(63) | 57 | 19(37) | 32(63) | 51 | 10(18)a | 47(82) | 57 |
|
2 | 30(34) | 58(66) | 88 | 33(42)a | 45(58) | 78 | 13(15) | 75(85) | 88 |
|
3 | 5(31) | 11(69) | 16 | 3(25) | 9(75) | 12 | 2(12) | 14(88) | 16 |
|
4 | 0 (0) | 3(100) | 3 | 0 (0) | 3(100) | 3 | 0 (0) | 3(100) | 3 |
|
No children
(0) | 120(36) | 214(64) | 334 | 105(36) | 185(64) | 290 | 37(11) | 297(89) | 334 |
|
Total | 176(35) | 322(65) | 498 | 160(37) | 274(63) | 434 | 62(12) | 436(88) | 498 |
| Sexual partners,
n | | | | | | | | | |
|
1 | 52(33) | 106(67) | 158 | 52(38) | 86(62) | 138 | 24(15)a | 134(85) | 158 |
|
2 | 3(50)a | 3(50) | 6 | 1(17) | 5(83) | 6 | 0 (0) | 6(100) | 6 |
|
3 | 0 (0) | 2(100) | 2 | 1(50)a | 1(50) | 2 | 0 (0) | 2(100) | 2 |
|
No sexual
partners (0) | 121(36) | 211(64) | 332 | 106(37) | 182(63) | 288 | 38(11) | 294(89) | 332 |
|
Total | 176(35) | 322(65) | 498 | 160(37) | 274(63) | 434 | 62(12) | 436(88) | 498 |
| Sexual
activity | | | | | | | | | |
|
Yes | 101(31) | 223(69) | 324 |
121(44)a | 154(56) | 275 | 47(15)a | 277(85) | 324 |
|
No |
106(39)a | 168(61) | 274 | 76(31) | 170(69) | 246 | 29(10) | 245(90) | 274 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Congenital
disease | | | | | | | | | |
|
Yes | 20(30) | 46(70) | 66 | 21(36) | 37(64) | 58 | 8(12) | 58(88) | 66 |
|
No |
186(35)a | 343(65) | 529 |
175(38)a | 285(62) | 460 | 68(13)a | 461(87) | 529 |
|
Total | 206(35) | 389(65) | 595 |
196(38)a | 322(62) | 518 | 76(13) | 519(87) | 595 |
| Family history of
cancer | | | | | | | | | |
|
Yes | 8(26) | 23(74) | 31 | 9(31) | 20(69) | 29 | 3(10) | 28(90) | 31 |
|
No |
199(35)a | 368(65) | 567 |
188(38)a | 304(62) | 492 | 73(13)a | 494(87) | 567 |
|
Total | 209(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Betel nut
chewing | | | | | | | | | |
|
Yes | 23(33) | 46(67) | 69 | 15(23) | 51(77) | 66 | 5(7) | 64(93) | 69 |
|
No |
184(35)a | 345(65) | 529 |
182(40)a | 273(60) | 455 | 71(14)a | 454(86) | 525 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 518(87) | 594 |
| Mouth ulcer | | | | | | | | | |
|
Yes | 69(29) | 171(71) | 240 | 85(41)a | 120(59) | 205 | 30(12) | 210(88) | 240 |
|
No |
138(39)a | 220(61) | 358 | 112(35) | 204(65) | 316 | 46(13)a | 312(87) | 358 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Alcohol
consumption | | | | | | | | | |
|
Yes | 89(32) | 190(68) | 279 |
116(51)a | 111(49) | 227 | 45(16)a | 234(84) | 279 |
|
No |
118(37)a | 201(63) | 319 | 81(28) | 213(72) | 294 | 31(10) | 288(90) | 319 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Smoking | | | | | | | | | |
|
Yes | 29(36)a | 51(64) | 80 | 34(49)a | 36(51) | 70 | 19(24)a | 61(76) | 80 |
|
No | 178(34) | 340(66) | 518 | 163(36) | 288(64) | 451 | 57(11) | 461(89) | 518 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Hot tea
consumption | | | | | | | | | |
|
Yes | 48(31) | 107(69) | 155 | 62(48)a | 67(52) | 129 | 26(17)a | 129(83) | 155 |
|
No |
159(36)a | 284(64) | 443 | 135(34) | 257(66) | 392 | 50(11) | 393(89) | 443 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Coffee
consumption | | | | | | | | | |
|
Yes | 36(35)a | 68(65) | 104 | 38(40)a | 58(60) | 96 | 16(15)a | 88(85) | 104 |
|
No |
171(35)a | 323(65) | 494 | 159(37) | 266(63) | 425 | 60(12) | 434(88) | 494 |
|
Total |
207(35)a | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(13) | 522(87) | 598 |
| Soft drink
consumption | | | | | | | | | |
|
Yes | 91(47)a | 101(53) | 192 | 61(32) | 127(68) | 188 | 24(17)a | 116(83) | 140 |
|
No | 116(29) | 290(71) | 406 |
136(41)a | 197(59) | 333 | 52(13) | 354(87) | 406 |
|
Total | 207(35) | 391(65) | 598 | 197(38) | 324(62) | 521 | 76(14) | 470(86) | 546 |
| Herbal water
consumption | | | | | | | | | |
|
Yes | 5(26) | 14(74) | 19 | 7(64)a | 4(36) | 11 | 3(16)a | 16(84) | 19 |
|
No |
202(35)a | 377(65) | 579 | 190(61) | 123(39) | 313 | 73(13) | 506(87) | 579 |
|
Total |
207(35)a | 391(65) | 598 | 197(61) | 127(39) | 324 | 76(13) | 522(87) | 598a |
For the 599 buccal samples with available
demographic and lifestyle data (including sex, age, number of
children, number of sexual partners, sexual activity, congenital
disease, family history of cancer, betel nut chewing, mouth ulcers,
alcohol use, smoking and beverage consumption), univariate and
multivariate regression analyses were conducted.
Univariate analysis revealed that mouth ulcers, sex,
hot tea consumption and sexual activity were significantly
associated with EBV positivity (Table
SI).
Univariate analysis revealed significant
associations between EBNA-1 positivity and several factors,
including mouth ulcers, soft drink consumption, and the age groups
of 3-10, 11-20, 21-30 and 41-50 years (P<0.05; Table SI). Being in the 11-20-year-old
age group, alcohol consumption, smoking status, sexual activity and
hot tea consumption were significantly associated with LMP-1
positivity (P<0.05; Table SI).
Notably, both alcohol consumption and smoking status were
significantly associated with positivity for both EBV genes
(P<0.05). Mouth ulcers were significantly associated with
EBNA-1 gene positivity based on univariate analysis
(P=0.014; OR, 0.952) (Table
SI).
Multivariate analysis indicated that EBNA-1
positivity was significantly associated with alcohol and soft drink
consumption, being in the 11-20-year-old age group, being in the
21-30 year-old age group, and having four children. LMP-1
positivity was associated with sex, while smoking status was
significantly associated with dual gene positivity (Fig. 2).
Overall, significant risk factors for EBV positivity
included smoking, sex, soft drink consumption and age of 21-30
years.
Individuals aged 21-30 years showed high
LMP-1 gene positivity. Individuals aged 31-40 had a high
level of alcohol consumption (79%), and individuals aged 31-40,
41-50 and 51-90 had a high level of sexual activity (90, 87 and
95%, respectively) (Fig. 3;
Tables II and SII).
 | Figure 3Exposure to risk factors of oral
cancer across age groups. Colors represent the range percentage of
exposure to environmental risk factors: 0-20%, green; 21-40%, blue;
41-60%, orange; 61-80%, red; 81-100%, yellow. Lighter colors and
darker colors indicate lower and higher percentages, respectively
(Table SII). Factors included
Epstein-Barr virus status, sex, congenital disease, mouth ulcers,
family history of cancer, smoking and sexual activity.
EBNA-1, Epstein-Barr nuclear antigen-1; LMP-1, latent
membrane protein-1. |
DNA sequencing
TNF-α promoter mutations were most commonly
identified at the following SNPs in EBV-positive vs. EBV-negative
individuals: rs1452146766, TTTT>TTTTT (→T), 66.7 vs. 9.1%;
rs1799964, T>C (-1031 promoter), 58.3 vs. 33.3%; rs1554283139,
CCCCCCC>CCCCCAC, 25 vs. 0.0%; rs924800313, C>A, 11.1 vs.
0.0%; rs1799724, C>T, 8.3 vs. 0.0%; and rs1771099055,
CCCCC/CCCCCC, 7.7 vs. 0.0% (Table
III; Figs. 4 and Fig. S1, Fig.
S2, Fig. S3, Fig. S4, Fig.
S5, Fig. S6, Fig. S7, Fig.
S8, Fig. S9, Fig. S10, Fig. S11 and Fig. S12). Detection of the TNF-α
promoter (size, 1,102 bp) is shown in Fig. S13.
| Table IIIPrevalence of TNF-α promoter
SNPs by EBV status. |
Table III
Prevalence of TNF-α promoter
SNPs by EBV status.
| | | EBV | |
|---|
| RS no. | SNP | Positive, n
(%) | Negative, n
(%) | Sequence from NCBI
(5'-3') |
|---|
| rs1800629 | G>A (-308) | 0/3 (0.0) | 2/8 (25.0) | GGGGCATG [G/A]
GGACGGGGTT |
| rs1452146766 | TTTT>TTTTT
(->T) | 2/3 (66.7) | 1/11 (9.1) | GG [-/T]
GAGGGGCATGGGG |
| rs1799964 | T>C (-1031) | 14/24 (58.3) | 5/15 (33.3) | GAAGA [T/C]
GAAGGAAAA |
| rs1799724 | C>T | 2/24 (8.3) | 0/15 (0.0) | CCCCCCCTTAA [C/T]
GAAGACAGGG |
| rs1554283139 |
CCCCCCC>CCCCCAC | 6/24 (25.0) | 0/15 (0.0) | CCCCC [-/A]
CTTAACGAAG |
| rs4248158 | C>T | 1/24 (4.2) | 0/15 (0.0) | CCTCCAGGAC [C/T]
TCCAGGTATGG |
| rs943806159 | G>C (found
G>T) | 1/24 (4.2) | 0/15 (0.0) | ATACAG [G/C]
GGACGTTTAAGAA |
| rs1277698900 | C>T | 1/24 (4.2) | 0/15 (0.0) | GGGGA [C/T]
GTTTAAGAA |
| rs1466575171 | A>T | 1/24 (4.2) | 0/1 (0.0) | GGCCAC [A/G]
CACTGGGGCCC |
| rs2533238182 | G/- (found
G>T) | 1/24 (4.2) | 0/15 (0.0) | GGGGCCCTGA [G/-]
AAGTG |
| rs899519990 | G>T | 1/24 (4.2) | 0/15 (0.0) | GAAGTGAGA [G/T]
CTTCATGAAAAAAAT |
| rs1771099055 | CCCCC/CCCCCC | 1/13 (7.7) | 0/14 (0.0) | CAAG [-/CC]
AGCTCCTTCTCCCC |
| rs924800313 | C>A | 1/9 (11.1) | 0/15 (0.0) | TTTTCCCTCCAAC
[C/A/G] CCGTTTT |
Conventional PCR
A significant association was also observed between
the TNF-α mutation (TC) and mouth ulcers in individuals aged
3-50 years. The TC genotype was more common in individuals with
mouth ulcers (42.4%) compared with the CC genotype (27.6%)
(P=0.043; OR, 1.933; 95% CI, 1.016-3.678; Table IV). The expected PCR product sizes
were 444 and 316 bp for the TT genotype, 444, 316 and 174 bp for
the TC genotype, and 444 and 174 bp for the CC genotype (38) (Figs.
S14 and S15).
| Table IVTNF-α (-1031; TC) mutation and
presence of mouth ulcers. |
Table IV
TNF-α (-1031; TC) mutation and
presence of mouth ulcers.
| TNF-α
(-1031) | Mouth ulcers, n
(%) | No mouth ulcers, n
(%) | Total, n | P-value (odds
ratio; 95% CI) |
|---|
| TC | 81 (42.4) | 110 (57.6) | 191 | 0.043 (1.933;
1.016-3.678) |
| CC | 16 (27.6) | 42 (72.4) | 58 | |
| Total | 97 | 152 | 249 | |
Discussion
The present study demonstrated that EBV reactivation
varied across age groups, with a high EBV prevalence observed in
individuals aged 11-20 and 21-30 years compared with individuals in
other age groups. A previous report has shown that EBV variants,
such as the 30-bp deletion LMP-1 variant, are associated
with malignant transformation (20). According to the World Health
Organization, the key genes used for investigating EBV infection
include Bacillus amyloliquefaciens (strain H) W repeat type
II restriction enzyme, EBNA-1 and EBER (45,46).
The present study identified several risk factors
for EBV reactivation in oral buccal cells, including age, tobacco
use and alcohol consumption; factors that are also linked to oral
cancer risk in Thailand (47).
Tobacco smoking and alcohol consumption were more prevalent among
men, whereas betel nut chewing was more commonly observed among
women in the present study, which was consistent with a previous
study (48). In a previous study,
significant associations were observed between oral cancer and
tobacco smoking (OR, 4.47; 95% CI, 2.00-9.99), alcohol consumption
in women (OR, 4.16; 95% CI, 1.70-10.69) and betel nut chewing (OR,
9.01; 95% CI, 3.83-21.22) (49),
all of which exhibited dose-response effects. Smoking is relatively
uncommon among Thai women; however, betel nut chewing remains
widespread, especially among older women (49). The univariate analysis revealed an
association between LMP-1 status and sexual activity. These
findings suggested that changes in traditional oral habits, such as
reduced betel nut chewing and use of traditional cigars, may have
contributed to the decline in oral cancer rates among both men and
women in Thailand (49).
The findings of the present study regarding the
association between TNF-α mutation and EBV status also
reinforced findings from previous meta-analyses that highlighted
the impact of the TNF-α gene on OSCC and oral potentially
malignant disorders. TNF-α position -308 mutation has been
associated with increased oral cancer risk (50,51).
TNF-α levels in both saliva and serum are being explored as
potential biomarkers for early OSCC detection, tumor staging,
differentiation and prognosis. However, TNF-α levels are also
influenced by general inflammation and common oral diseases, thus
complicating their interpretation (51). In the oral cavity, TNF-α is
modulated by both the oral microbiome and periodontal diseases
(51). One study found that TNF-α
levels in patients with OSCC (28.9±14.6 pg/ml) were significantly
higher than those in patients with oral premalignant lesions
(10.5±7.4 pg/ml) and healthy controls (3.0±1.0 pg/ml) (P<0.01)
(52). Another study reported
salivary TNF-α levels of 27.75±30.94 pg/ml in patients with OSCC,
compared with 8.6±7.27 pg/ml in controls (53).
The TNF-α (-1031 T/C) SNP has been associated
with severe adult periodontitis in the Japanese population
(54). Although the C allele is
linked to higher TNF-α cytokine levels than the T allele, the
difference is not statistically significant due to concurrent
mutations in other regions (55).
In the present study, EBV reactivation was associated with the
TNF-α (rs1799964; -1031 T/C) mutation. Consistent with
earlier findings (44), the present
study also demonstrated that this mutation was linked to the
presence of mouth ulcers in oral samples from the Thai population.
In other populations, such as in Iran, the same SNP has been
associated with both the risk and severity of oral lichen planus
(44). Future research should
examine the annual frequency of mouth ulcers and their potential
link to oral cancer. The complex relationship among EBV, the
TNF-α (rs1799964; -1031 T/C) mutation and mouth ulcers
warrants further investigation. These factors (such as mouth
ulcers, alcohol consumption or smoking), along with EBV DNA and
TNF-α cytokine levels, may ultimately be useful for oral cancer
screening and head and neck cancer diagnostics. TNF-α (-1031 T/C)
variants and mouth ulcers appeared to be associated in individuals
aged 3-50 years. This indicates that the findings of the present
study are exploratory, and thus, require further validation.
In a previous study, oral cancer was one of the most
common forms of head and neck cancer, ranking as the sixth most
common cancer among Thai men and being among the leading cancers in
Thailand based on the mean annual ASR of the 2019-2021 period
(5-11).
Trends indicate an increasing incidence of oral cavity cancer in
men (5-11).
In another study, the youngest male patient with
oral cancer was 15 years old, while the youngest female patient was
18 years old (44,47). The median age among younger patients
was 33.5 years (interquartile range, 42.5-24.5) (44,47).
Nasopharyngeal cancer has been reported in male patients as young
as 10-19 years old, with the incidence markedly increasing after
the age of 50 years. Oropharyngeal and hypopharyngeal cancers in
female patients have also been found, starting in the
40-44-year-old age group. Nasopharyngeal cancer in male patients
often appears earlier, between the ages of 10 and 19 years, while
oropharyngeal and hypopharyngeal cancers are more commonly observed
in the 30-39-year-old age group (56). The cause of rising cancer rates
among younger individuals remains unclear, and it continues to be
debated whether this stems from genetic or environmental factors
(15,16). The high prevalence of EBV in
individuals aged 11-30 years should be closely examined as a
potential risk factor.
A review of previous studies on EBV status in head
and neck cancers in the Thai population published between 1991 and
2025 was conducted by including studies employing PCR/qPCR methods
targeting the BamHI N leftward frame 1, LMP-1 and
EBNA-1 genes, as well as those involving the serological
detection of anti-EBV IgG (20,23,57-76).
The prevalence of EBV in normal tissue samples ranged from 5 to
33.7%, while in carcinoma samples, it ranged from 21 to 98% based
on PCR/qPCR results. In normal blood samples, EBV prevalence ranged
from 0 to 7.26% based on PCR/qPCR results. Using anti-EBV IgG
serology, EBV prevalence ranged from 3.1 to 97.27% in normal blood
samples and reached ≤86.5% in carcinoma cases. Examination of
existing publications from 1991 to 2025 on head and neck cancers in
the Thai population (20,23,55-76)
showed that most studies focused on individuals aged 40-67 years,
showing an ASR range of 0.1-14.68 among female patients and
0.6-15.7 among male patients. From 2001 to 2021, the prevalence and
ASR of oral cavity and oropharyngeal cancers in Thailand increased,
particularly among male patients (20,23,57-76).
The ASRs of head and neck cancers are generally
lower in younger populations (<40 years old) compared with those
in older adults. In young adults, the ASR of thyroid cancer was
14.4 per 100,000, while that of oral cavity cancer was 1-2 per
100,000 new cancer cases worldwide in 2022 (77,78).
To the best of our knowledge, there are no studies on the ASR in
this group in Thailand; only medical opinions are available, with
no research to support existing opinions.
A limitation of the present study is that qPCR was
not used to confirm cancer diagnoses. In another study, nucleic
acid sequence-based amplification or reverse transcription-PCR were
used for the detection of EBNA-1, EBNA-2, LMP-2A and
BZLF-1 (79), and
immunohistochemistry was used for the detection of EBER (pathology
confirmed), LMP-1, EBNA1, EBNA2, LMP2A and BZLF-1(77). In situ hybridization was used
for the detection of EBER-1 and EBER-2 or EBV DNA. EBNA-1,
LMP-1, LMP-2A and BZLF-1 were most commonly
used to detect EBV via qPCR (78,79).
Improvements in quantitative amplification technology are
stimulating a resurgence of interest in amplification strategies
for detecting EBV in patient samples (80).
In conclusion, the alignment of EBV reactivation,
age (11-30 years) and associated behavioral risk factors (alcohol
consumption, smoking and sexual activity) strongly mirrors the risk
profile for head and neck cancers. An association was found between
TNF-α promoter mutations [such as rs1799964 (-1031 T/C)] and
mouth ulcers or EBV. Future research should focus on integrating
EBV DNA, TNF-α gene mutations and cytokine levels into early
screening strategies for individuals with a high risk of OSCC.
Supplementary Material
TNF-α promoter mutation
(rs1799724) detected by sequencing.
TNF-α promoter mutation
(rs4248158) detected by sequencing.
TNF-α promoter mutation
(rs943806159 and rs1277698900) detected by sequencing.
TNF-α promoter mutation
(rs1771099055) detected by sequencing.
TNF-α promoter mutation
(rs1466575171) detected by sequencing.
TNF-α promoter mutation
(rs2533238182) detected by sequencing.
TNF-α promoter mutation
(rs899519990) detected by sequencing.
TNF-α promoter mutation
(rs1799964) detected by sequencing.
TNF-α promoter mutation
(rs1800629) detected by sequencing.
TNF-α promoter mutation
(rs924800313) detected by sequencing.
TNF-α promoter mutation
(rs1452146766) detected by sequencing.
TNF-α promoter mutation
(rs1554283139) detected by sequencing.
PCR detection of TNF-α promoter
(size, 1,102 bp).
Example of TNF-α promoter
(-1031; CC and TC) detected by PCR.
Example of TNF-α promoter
(-1031; TT) detected by PCR.
Univariate analysis of positivity
versus negativity of EBNA-1, LMP-1 and both
genes.
Percentage of exposure to risk factors
of oral cancer across age groups.
Acknowledgements
The B95 cell line was provided by Professor Jaap M.
Middeldorp (Department of Pathology, VU University Medical Center,
Amsterdam, The Netherlands).
Funding
Funding: The present study was funded by the Faculty of
Pharmaceutical Sciences, Ubon Ratchathani University (grant no.
Phar. UBU.0604.11-2/2568).
Availability of data and materials
The data generated in the present study are
included in the figures and/or tables of this article.
Authors' contributions
JB and SB were responsible for conceptualization
and methodology. SB was responsible for validation. SB, SP, TR, WF,
DD, FK, PL and KC performed experiments. SS, SB and JB confirm the
authenticity of all the raw data. SB and JB were responsible for
data curation. SB and JB prepared the original draft. CP, TE, SD,
JB and SB revised the manuscript. SB, JB and SD acquired funding.
All authors have read and approved the final version of the
manuscript.
Ethics approval and consent to
participate
The present study was approved by the Human Ethics
Committee Ubon Ratchathani University (Ubon Ratchathani, Thailand;
approval no. UBU-REC-68/2567). All procedures involving human
participants in the present study were performed in accordance with
the ethical standards of The Declaration of Helsinki, the Belmont
Report, the Council for International Organizations of Medical
Sciences guidelines, and the International Conference on
Harmonization in Good Clinical Practice. Written informed consent
was obtained from all participants or their legal guardians (for
patients <18 years old).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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