Oral Diseases (2015) doi:10.1111/odi.

12318
2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
All rights reserved
www.wiley.com

ORIGINAL ARTICLE

HMGB1 genetic polymorphisms in oral squamous cell

carcinoma and oral lichen planus patients
G Supic1,2, R Kozomara1,3, K Zeljic2,4, D Stanimirovic5, M Magic5, M Surbatovic1,6, N Jovic1,3,
Z Magic1,2
1

Faculty of Medicine, Military Medical Academy, University of Defense, Belgrade; 2Institute for Medical Research, Military Medical
Academy, Belgrade; 3Clinic for Maxillofacial Surgery, Military Medical Academy, Belgrade; 4Faculty of Biology, University of
Belgrade, Belgrade; 5School of Dental Medicine, Clinic of Periodontology and Oral Medicine, University of Belgrade, Belgrade;
6
Clinic for Anesthesiology, Military Medical Academy, Belgrade, Serbia

OBJECTIVES: This study examined the single nucleotide

polymorphisms (SNPs) in high-mobility group box 1
(HMGB1) gene in patients with oral squamous cell carcinoma (OSCC) and oral lichen planus (OLP).
MATERIALS AND METHODS: The study was conducted on 93 patients with OSCC, 53 patients with
OLP, and 100 controls, all Caucasians of the same ethnicity, matched by age. HMGB1 genotypes for 4 SNPs,
2262G/A (rs1045411), 1177G/C (rs3742305), 3814C/G
(rs2249825), and rs4540927, were assessed using TaqMan SNP Genotyping Assays, Applied Biosystems.
RESULTS: The HMGB1 1177GG genotype was associated with lymph-node metastasis and tumor stage in
OSCCs (P = 0.016 and P = 0.030, respectively). Genotype 1177GG resulted in poorer recurrence-free survival
(RFS), P = 0.000. The 1177G/C polymorphism was an
independent predictor of RFS compared to GG genotype, P = 0.001. The three polymorphisms were in linkage disequilibrium (LD). The AGC and GGC haplotypes
were associated with an increased oral cancer risk,
determined
over
the
haplotype
odds
ratios
(HOR = 13.316, P = 0.015, and HOR = 5.769, P = 0.029,
respectively). The AGC haplotype was related to erosive OLP progression to OSCC (HOR = 12.179,
P = 0.001).
CONCLUSIONS: HMGB1 polymorphism 1177G/C could
be associated with tumor progression and recurrencefree survival in patients with OSCC. The haplotypes of
HMGB1 gene might be associated with susceptibility to
OSCC and OLP progression to OSCC.
Oral Diseases (2015) doi: 10.1111/odi.12318

Correspondence: Gordana Supic, PhD, Assistant Professor, Institute for

Medical Research, Military Medical Academy, Crnotravska 17, 11002
Belgrade, Serbia. Tel: +381604022202, Fax: +381112662722, E-mail:
gogasupic@sezampro.rs
Received 4 December 2014; accepted 16 January 2015

Keywords: oral squamous cell carcinoma; oral lichen planus;

high-mobility group box 1; single nucleotide polymorphisms;
recurrence-free survival; haplotypes

Introduction
Oral squamous cell carcinoma (OSCC), a subgroup of head
and neck squamous cell carcinoma (HNSCC), is characterized by a high incidence of nodal metastasis and high
recurrence rate. Despite easy accessibility for regular examination, the majority of OSCC cases are diagnosed in
advanced stages that respond poorly to current cancer therapies (Scully and Bagan, 2009). The clinical history conrms the relationship between oral lichen planus (OLP), a
chronic oral inammatory disease of unknown etiology,
and oral cancer as 0.52% of patients with OLP develop
OSCC (Georgakopoulou et al, 2012). World Health Organization (WHO) classies OLP as a potentially malignant
disorder (Warnakulasuriya et al, 2007). The highest risk
of malignant transformation has been recognized for atrophic, erosive form of OLP (Lo Muzio et al, 1998, Barnard
et al, 1993). However, the reports of the risk are controversial and mechanisms involved are still unrevealed (Gonzalez-Moles et al, 2008). Recently, it has been proposed that
both HNSCC and OLP could be a good model of inammation-associated cancerogenesis (Georgakopoulou et al,
2012, Whiteside, 2005).
High-mobility group box 1 (HMGB1) is a conserved
non-histone chromatin protein involved in diverse cellular
functions, with an important role in nucleosomal stability,
transcriptional control, and proinammatory reactions. The
HMGB1 gene, located on the human chromosome 13q12,
consists of six exons and encodes a 215-amino acid polypeptide (Ferrari et al, 1996). In the intracellular milieu,
HMGB1 acts as a cotranscriptional factor, by binding to
the minor groove of linear DNA, stabilizing nucleosomes
and recruiting and interacting with numerous transcription
factors, including p53, NF-jB, VD(J) recombination

HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

activating proteins (RAG1), homeobox-containing proteins,

and steroid hormone receptors (Muller et al, 2004).
HMGB1 can be passively released into the extracellular
milieu from damaged or necrotic cells. Derived from
necrotic cells HMGB1 act as a damage-associated molecular pattern (DAMP) molecule, referred as alarmins, chemotactic necrotic markers that activate innate responses
and initiate tissue repair (Ulloa and Messmer, 2006; Campana et al, 2008). HMGB1 binds to a wide range of receptors, Toll-like receptor 4 (TLR4), TLR2, and receptor for
advanced glycation end products (RAGE) (Keyel, 2014).
Hypo-acetylated, reduced forms of HMGB1 passively
released from necrotic or dying tumor cells trigger antigen-presenting cells via TLR4, in inducing an antineoplastic immune response (Apetoh et al, 2007; ). On the other
hand, inammatory cells, including monocytes, macrophages, and dendritic cells, during apoptosis actively
secrete hyperacetylated form of HMGB1, which induces
immunological tolerance (Kazama et al, 2008).
Thus, HMGB1 has diverse effects on immunity acting
as a pro-inammatory cytokine, enhancing both innate and
adaptive immune responses that contribute to the pathogenesis of various disorders, from sepsis and autoimmunity to cancer (Ulloa and Messmer, 2006). Overexpression
of HMGB1, as well as cytoplasmic localization, has been
observed in various human carcinomas (Flohr et al, 2001;
Kuniyasu et al, 2003; Wu et al, 2008), including HNSCC
(Liu et al, 2010; Wild et al, 2012) and OSCC (Sasahira
et al, 2008; Ahn et al, 2012).
Only few studies examined HMGB1 polymorphisms and
their prognostic or predictive association in cancer. One
study examined HMGB1 polymorphisms with lung cancer
chemotherapy response (Wang et al, 2014), and another
investigated the association of HMGB1 polymorphisms with
outcome after allogeneic hematopoietic cell transplantation
(Kornblit et al, 2010). To our knowledge, this is the rst
study investigating the HMGB1 polymorphisms in HNSCC,
OSCC, or OLP. The aim of this study was to examine the
potential clinical relevance of four single nucleotide polymorphisms, SNPs, within the HMGB1 gene in patients with
OSCC, as well as the association between HMGB1 polymorphisms and their haplotypes with OSCC and OLP.

Materials and methods

Subjects
This study was performed at the Military Medical Academy, Belgrade,
following approval by the Ethics Committee of Military Medical Academy, Belgrade, Serbia. Studied groups consisted of 93 cases of histologically conrmed OSCC, 53 patients with erosive form of OLP, and 100
controls, without previous cancer history. All the participants in the study
were Caucasians with the same ethnicity, matched by age. Informed consent was obtained from patients and that the study was performed in
accordance with the Declaration of Helsinki. The patients with OSCC
were staged according to the TNM classication system for oral and oropharyngeal tumors (Warnakulasuriya et al, 2007). All patients received
primary surgery followed by radiotherapy, while 24 patients with OSCC
(25.8%) received neoadjuvant cisplatin/5-uorouracil chemotherapy.

HMGB1 genotyping
Genomic DNA was extracted from primary tumors using a TRIZOL
reagent (Invitrogen, Karlsruhe, Germany) and from the blood of controls
using a Blood Prep Isolation Kit (Qiagen, Hilden, Germany). Genotyping

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of the four SNPs within the HMGB1 gene 2262G/A (rs1045411), 1177G/
C (rs3742305), 3814C/G (rs2249825), and rs4540927 was carried out by
TaqMan SNPs Genotyping Assays (Applied Biosystems, Warrington,
UK), according to the manufacturers protocols. The SNP genotyping
was performed blinded to patient status, and a random 10% of the samples were repeated to validate genotyping procedures.

Statistical analysis
Statistical analysis was performed with the SPSS 20.0 software (IBM
Corp., Armonk, NY, USA). The association between categorical variables
was estimated by the chi-square test or Fishers exact test. All reported P
values were two-sided, and all associations were considered signicant
when P values were less than 0.05.
The overall survival (OS) was calculated from the time of surgery
until death from any cause or last follow-up. Recurrence-free survival
(RFS) was calculated from the time of surgery until the rst observation
of any recurrence (local, regional, or distant) or death due to any cause.
If a patient had no recurrences or died, RFS was censored at the time of
the last follow-up. The association between these polymorphisms with
the OS and RFS was analyzed using KaplanMeier survival curves and
compared to the logrank test. Hazard ratios (HR) for median RFS and
OS between groups were estimated from Cox proportional hazard regression analysis, with 95% condence interval (95% CI). Variables found
signicant in the univariate analysis, including those with signicance
level below 20%, were subsequently analyzed together in multivariate
analysis using Coxs regression. The multivariate analysis also included
adjustments for other variables such as age (<58 vs. 58 years) and sex.
The Cox model was performed using the forward stepwise method,
which removed variables with P < 0.100.
To detect departure from the HardyWeinberg equilibrium, haplotype
frequencies and pairwise linkage disequilibrium (LD) were estimated
using the Haploview 4.2 software, based on the expectation maximization
algorithm (Barrett et al, 2005), with LD = 1 indicating complete linkage
disequilibrium, and LD = 0 indicating absent LD. The Thesias software
v.3.1 was used for testing the association of different haplotypes with
clinicopathological data and for haplotype-based survival analysis, based
on Cox proportional hazards survival regression (Tregouet and Garelle,
2007). The most frequent haplotype was taken as the reference haplotype.
TRANSFAC database search (Tsunoda and Takagi, 1999) was utilized to
analyze whether analyzed polymorphisms created a putative transcriptional factor binding sites.

Results
Clinicopathological characteristics
The characteristics of the OSCC cases, OLP cases, and
controls are listed in Table 1. While patients with OSCC
were predominantly male, OLP cases were predominantly
female. The median patient age was 58 years in both
groups, range 3680 years in OSCCs, and range 37
84 years in OLP group (Table 1). Of the 93 tumors, 19
were WHO stage II tumors, and 74 were stage III tumors.
The recurrence rate in patients with OSCC was high, 58%
(54/93). On an individual patient basis, 96% of patients
with disease recurrence had their recurrence within 2 years
after surgery. A total of 37 patient deaths (78.7%) are preceded by a tumor recurrence (P = 0.000). We observed
signicant differences in smoking habits between OSCCs
and controls, as well as OSCCs and OLP cases
(P = 0.000) (Table 1).
Genotyping
In analyzing the polymorphism rs4540927, only one genotype variant was identied in all of the studied subjects,
and therefore classied as mutation. We observed no association in the incidence of SNP 2262G/A, 1177G/C, and
3814C/G between OSCC and controls (Table 1). In all,

HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

Table 1 Demographic characteristics and polymorphisms prevalence in

OSCC, OLP, and controls
Variables

OSCC

OLP

Controls

Pa,b,c

Sex
Male
Female

69
24

14
39

64
36

NSa
0.000b
0.000c

Age (median)
58<
58
Smoking
Never

Table 2 Association of HMGB1 gene polymorphisms with clinicopathological variables of OSCC patients

Variables

44
49

23
30

48
52

NSa,b,c

26

34

65

Ever
Alcohol drinking
No

67

19

35

0.000a
NSb
0.000c

64

42

80

Yes
HMGB1 rs3742305
GG (wt)
GA (het)
AA (mut)
HMGB1 rs2249825
CC (wt)
CA (het)
AA (mut)
HMGB1 rs1045411
GG (wt)
GC (het)
CC (mut)

29

11

20

50
37
6

38
13
2

64
30
6

NSa,b,c

63
27
3

38
12
3

66
26
8

NSa,b,c

48
40
5

36
15
2

64
30
6

NSa,b,c

0.074a
NSb,c

wt/ht/mut, number of wild-type, heterozygote and variant (mutant) genotypes.

a
P OSCC vs. Controls.
b
P OLP vs. Controls.
c
P OSCC vs. OLP.

analysis of 1177G/C polymorphism of HMGB1 gene

revealed that a total of 50 patients with OSCC (53.8%) had
a GG genotype, whereas 37 patients (39.8%) had GC genotype and six patients (6.4%) had CC genotype. Patients
with OSCC with the 1177GG genotype had a higher prevalence of advance tumor stage III (P = 0.016), and compared with combined GC+CC genotypes, this difference
was even more prominent (P = 0.010, Table 2). Study of
3814C/G HMGB1 polymorphism revealed that 63 patients
(67.8%) had a CC genotype, 27 patients (29%) had a CG
genotype, and three patients (3.2%) had a GG genotype.
Patients with OSCC with wt CC genotype of 3814 C/G
polymorphism had a higher prevalence of nodal metastasis
and advance tumor stage III (P = 0.019), compared with
combined CG+GG genotypes (variant allele G carriers)
(Table 2). No signicant associations were observed
between the 2262G/A HMGB1 gene polymorphism and
clinicopathological characteristics in the OSCC cases
(Table 2). No signicant differences were found according
to HMGB1 genotype for OLP patients characteristics,
including oral hygiene, Candida albicans infection,
amalgam dental llings, or dental implants (data not
shown).
Survival outcomes in OSCCs
Analyses of individual SNPs associations with survival
outcomes revealed that the 1177G/C polymorphism
signicantly impacted recurrence-free survival (RFS)

Histological grade
1
28
2/3
65
p/pa
Nucleus grade
1
17
2/3
76
p/pb
Nodal status
N0
23
N+
70
p/pb
Tumor size
T1/2
69
T3/4
24
p/pb
Stage
II
19
III
74
p/pb

HMGB1
2262G/A
wt/ht/mut

HMGB1
1177G/C
wt/ht/mut

HMGB1
3814C/G
wt/ht/mut

14/14/0
34/26/5
NS

17/9/2
33/28/4
NS

19/8/1
44/19/2
NS

8/7/2
40/33/3
NS

12/2/3
38/35/3
0.010b

13/2/2
50/25/1
NS

10/11/2
38/29/3
NS

7/15/1
43/22/5
0.016/0.010b

11/11/1
52/16/2
0.060/0.019

38/27/4
10/13/1
NS

36/28/5
14/9/1
NS

45/21/3
18/6/0
NS

10/10/2
38/30/2
NS

7/14/1
43/23/5
0.030/0.018b

11/10/1
52/17/2
0.124/0.042b

N total number of patients.

a
Age according to median value of 58 years (range 3980).
b
P values for wild-type vs. combined heterozygote and variant homozygote genotype.
Values given in bold are signicant (P < 0.05).
NS, nonsignicant; statistical analysis performed by Fishers exact test or
chi-square test, where appropriate.

(Figure 1a), but not overall survival (OS) (Figure 1b).

Recurrence-free survival proved statistically signicant
differences among these genotypes with the median values
of 7 months for GG genotype, 12 months for GC, and
7 months for CC (P = 0.000, Figure 1a). Median RFS
was improved for minor allele C carriers, for patients
showing the GC/CC genotype (12 vs. 7 months;
P = 0.000, Figure 1a). Although the OS of OSCC patients
with a GG genotype of 1177G/C polymorphism tended
toward worse survival compared with GC/CC genotype
(median OS 36 vs. 60 months), differences in did not
reach statistical signicance (P = 0.104, Figure 1b). Other
individual SNPs under study did not show association
with RFS nor OS (data not shown).
In univariate analysis, relevant prognostic factors were
tested as predictors for OS and RFS for patients with
OSCC (Table 1). Variables found statistically signicant
in the univariate analysis, including the variables with signicance level below the 20%, were subsequently analyzed together in multivariate analysis. Univariate analysis
revealed that alcohol use, tumor size (T1/2 vs. T3/4), stage
(stage III vs. II), nodal status (lymph node positive, N+ vs.
lymph node negative, N0), and recurrences were signicant prognostic indicators for overall survival (Table 3).
In the nal multivariate model, alcohol, tumor size, nodal
status, and recurrences persisted as signicant adverse factors for overall survival in patients with OSCC, while the
contribution of other variables was lost (Table 4).
Signicant prognostic indicators for recurrences-free
survival were nucleus grade, stage, lymph-node status, and
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HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

RECURRENCE-FREE SURVIVAL

(a)

RECURRENCE-FREE SURVIVAL
P = 0.000

Cum survival

Cum survival

P = 0.000

1177 GC+CC

1177 GC

1177 GG

1177 GG

1177 CC

Time (months)

Time (months)

OVERALL SURVIVAL

OVERALL SURVIVAL

(b)

1177 CC

1177 GG

1177 GC

Time (months)

P = 0.104

Cum survival

Cum survival

P = 0.222

1177 GG

1177 GC+CC

Time (months)

Figure 1 (a) Recurrence-free survival (RFS) and (b) overall survival (OS) analysis of 1177G/C polymorphism of HMGB1 gene (KaplanMeier curves,
compared by the logrank test)

1177G/C polymorphism in HMGB1 gene (Table 3). In the

nal multivariate model, nodal metastasis and 1177G/C
polymorphism are independent adverse risk factors for
recurrences-free survival (Table 4). Multivariate analysis
showed that minor allele C carrier patients (heterozygous
or homozygous) had the signicantly decreased hazard
risk (HR = 0.317, [0.1620.622] 95% CI, P = 0.001)
compared with homozygous patients for the HMGB1
1177 G major allele, as a reference group. Based on the
comparison of individual genotypes, heterozygous subjects
for 1177G/C polymorphism have a reduced risk
(HR = 0.232 [0.1170.463], P = 0.000). Otherwise, when
the minor allele is set as the reference allele instead, major
allele is an independent prognostic factor for poor recurrence-free survival in OSCCs, HR = 3.153 [1.6086.183]
95% CI, P = 0.001.
Haplotype analysis and linkage disequilibrium
Although there were no differences in the distribution of
HMGB1 genotypes between OSCC and controls, we
hypothesized that haplotypes, the combined effects of
HMGB1 polymorphisms, could be more prominent than
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the effect of individual polymorphisms in the susceptibility to oral cancer or oral lichen planus. The haplotype frequency estimates were obtained by Haploview v.4.2 and
Thesias software v.3.121, and the results from the two
software packages were consistent with each other. Haploview 4.2 software was used to identify the linkage disequilibrium structure and to ensure that the markers were
appropriate for inclusion in the haplotype estimates. The
three polymorphisms we examined are in linkage disequilibrium (Figure 2), and ve haplotypes had a frequency
>1.0% (Table 5). Permutation tests were used to assess
the signicance of the individual and prole haplotype frequency differences. The haplotype frequency differences
between the OSCC and controls for SNP 2262G/A,
1177G/C, and 3814C/G combinations indicated that the
AGC and GCC haplotypes had a higher incidence in
OSCC cases than in controls (10% vs. 1.5%, and 9% vs.
2.2%, respectively) and could be associated with an
increased oral cancer risk, determined over the haplotype
odds ratios (HOR = 13.316 [1.656107.051], P = 0.015;
and HOR = 5.769 [1.19627.824], P = 0.029, respectively; Table 5). Differences in haplotype distribution

HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

(a)

OLP experienced progression to OSCC at the time of the

last follow-up.
Thesias software was also utilized to evaluate the combined effect of the three HMGB1 polymorphisms in LD
on OSCC and OLP patients clinicopathological characteristics and/or survival, by comparison with the most
frequent haplotype GGC. No signicant associations of
the HMGB1 haplotypes with the clinicopathological data
were observed (data not shown).

Discussion

(b)

(c)

Figure 2 D pairwise linkage disequilibrium (LD) plots of analyzed

2262G/A, 1177G/C, and 3814C/G polymorphisms in the HMGB1 gene.
(a) LD plots for OSCC cohort; (b) LD plots for OLP cohort; (c) LD
plots for control group. The red shaded boxes correspond to the paired
D values (showed in percentages) between the single nucleotide polymorphisms (SNPs). Shades of pink: LOD > 2, D < 1; red: LOD > 2,
D = 1; white: LOD < 2, D < 1. LODlogarithm of odds. The gray
shaded boxes correspond to the paired r2 between the SNPs. White:
r2 = 0; shades of gray: 0 < r2 < 1; black: r2 = 1

between OLPs and controls were not signicant, while

AGC haplotype had a higher incidence in OSCC cases
compared to OLPs (10% vs. 1.8%), determined over the
haplotype odds ratios (HOR = 12.179 [2.61956.647],
P = 0.001; Table 5). However, none of the patients with

HMGB1 protein has diverse cellular functions, with an

important role in chromatin architecture, transcriptional
regulation, DNA repair, and proinammatory reactions.
HMGB1, acting as a pro-inammatory cytokine, plays an
important role in both innate and adaptive immune
responses, which contribute to the pathogenesis of various
disorders, from sepsis and autoimmunity to cancer development and metastasis (Ulloa and Messmer, 2006; Kornblit et al, 2008; Zeng et al, 2012). All of these features
have placed HMGB1 in the center of a number of recent
cancer biology studies. Overexpression of HMGB1, as
well as its cytoplasmic localization, has been observed in
a variety of human solid carcinomas, including the colon
(Kuniyasu et al, 2003), breast (Flohr et al, 2001), nasopharyngeal (Wu et al, 2008), HNSCC (Liu et al, 2010;
Wild et al, 2012), and OSCC (Sasahira et al, 2008; Ahn
et al, 2012). A number of studies have demonstrated that
genetic variation in genes involved in tumor immunity,
immunotolerance, and inammation could be involved in
host protection against carcinomas (Apetoh et al, 2007;
Campana et al, 2008). To our knowledge, our study is the
rst one investigating the HMGB1 polymorphisms in
OSCC and OLP, and the rst study demonstrating that the
variation in HMGB1 could be associated with tumor progression and recurrence-free survival.
Our analysis showed that homozygote genotype for the
1177G/C major allele was signicantly associated with the
tumor progression and the decreased recurrence-free survival in patients with OSCC. Polymorphism 1177G/C was
associated with lymph-node metastasis occurrence and
advanced tumor stage in patients with OSCC (P = 0.016
and P = 0.030, respectively). In OSCC cases, genotype
1177GG resulted in poorer recurrence-free survival (RFS)
(P = 0.000), but failed to demonstrate an overall survival
(OS) benet (P = 0.104). Multivariate analysis revealed
that HMGB1 1177G/C polymorphism is an independent
prognostic factor for recurrence-free survival in our OSCC
cohort. Most likely salvage chemotherapy might have
played a role in the non-superior outcome of the OS.
However, this discordance could also be related to the relatively low number of patients or the duration of followup.
Our ndings that HMGB1 polymorphism was an independent predictor of recurrence-free survival in OSCC and
associated with tumor progression supported previous
studies, which reported HMGB1 overexpression in a variety of human cancers, including OSCC and HNSCC (Sasahira et al, 2008; Liu et al, 2010). Immunohistochemical
staining revealed that expression levels of HMGB1 were
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HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

Table 3 Univariate analysis of different prognostic factors in relation to recurrence-free survival (RFS) and overall survival (OS), according to Cox proportional hazards regression analysis
Overall survival
Univariate analysis

HR [95% CI]

Sex
Age
Smoking
Alcohol
Nucl. grade
Hyst. grade
Stage
T
N
Recurrences
Chemotherapy
rs22498258a
rs3742305a
rs1045411a

0.760
1.482
1.751
2.427
1.179
1.452
4.205
2.019
3.561
4.764
0.712
0.805
0.653
0.769

Recurrence-free survival
P

[0.3771.532]
[0.8252.661]
[0.8463.624]
[1.4074.185]
[0.8041.729]
[0.77510.061]
[1.50711.734]
[1.1233.631]
[1.4069.017]
[9.6872.343]
[0.4041.255]
[0.4611.406]
[0.3971.073]
[0.4691.261]

0.443
0.188
0.131
0.001
0.399
0.712
0.006
0.019
0.007
0.000
0.240
0.445
0.093
0.298

HR [95% CI]
1.140
1.295
1.090
1.123
0.748
0.905
2.074
0.959
2.972

0.898
0.600
0.302
0.645

[0.5832.227]
[0.7312.296]
[0.5592.124]
[0.6831.848]
[0.4861.152]
[0.6851.194]
[1.0284.186]
[0.5881.565]
[1.3986.321]
[0.4861.659]
[0.3471.037]
[0.1610.567]
[0.3821.090]

P
0.702
0.376
0.801
0.647
0.188
0.480
0.042
0.867
0.005

0.731
0.067
0.000
0.102

HR indicates a hazard ratio; CI, condence interval.

Signicant values, P < 0.05, are bolded.
a
HR was analyzed in minor allele C carrier patients (heterozygous or homozygous) compared to homozygous patients for the HMGB1 1177 G major
allele, as a reference group.

Table 4 Multivariate Cox proportional hazards regression analysis of survival in OSCC patients
Multivariate analysis
Overall survival
Alcohol
T
N
Relapse
Recurrence-free survival
N
rs3742305 (C carriers vs. wt GG)
rs3742305 (GC vs. wt GG)
rs3742305 (CC vs. wt GG)

HR [95% CI]

1.876
1.449
3.142
4.923

[1.0673.299]
[1.0352.028]
[1.1968.250]
[2.36710.242]

0.029
0.031
0.020
0.000

2.290 [1.0185.153]
0.317a [0.1620.622]
0.320b [0.1600.638]
0.296b [0.0651.348]

0.045
0.001
0.001
0.115

Signicant values, P < 0.05, are bolded.

HR indicates a hazard ratio; CI, condence interval.
a
HR was analyzed in minor allele C carrier patients (heterozygous or
homozygous) compared to homozygous patients for the HMGB1 1177G
major allele, as a reference group.
b
HR was analyzed for each of the genotypes, heterozygous GC or homozygous CC compared to homozygous patients for the HMGB1 1177G
major allele, as a reference group.

signicantly higher in a metastatic OSCC cell line (HSC3)

compared with non-metastatic OSCC cell line (HSC4)
(Sasahira et al, 2008). HMGB1 overexpression was significantly associated with tumor progression and shorter disease-free and overall survival in patients with HNSCC
(Liu et al, 2010). Additionally, overexpression of HMGB1
enhanced the invasiveness and metastasis of hepatocellular
carcinoma via activation of caspase-1 (Yan et al, 2012).
Previously, polymorphisms in HMGB1 and HMGB2 genes
were signicantly associated with lung cancer platinumbased chemotherapy response (Wang et al, 2014). High
levels of serum HMGB1, detected prior to and 24 h after
radioembolization therapy, were associated with poor outcome in colorectal cancer patients with liver metastasis
(Fahmueller et al, 2013). A variant allele of TLR4 affects
Oral Diseases

the binding of HMGB1 to TLR4, and predicts early

relapse after anthracycline-based chemotherapy in breast
cancer patients (Apetoh et al, 2007). Thus, HMGB1,
through its interaction with TLR4, could contribute to
antitumor immunity inuencing the extent of processing
and the presentation of tumor-associated antigens, which
could be inuenced by chemotherapy and radiotherapy.
There were no differences in the distribution of HMGB1
genotypes between OSCC and controls, as well as
between OLP and controls, indicating that the individual
HMGB1 polymorphisms may not have a role in the susceptibility to oral cancer or oral lichen planus. However,
haplotype effect might be more signicant than a single
polymorphism in susceptibility to OSCC or OLP. The
haplotype frequency differences between the OSCC and
controls for SNP 2262G/A, 1177G/C, and 3814C/G combinations indicated that AGC and GCC haplotype could
be associated with an increased oral cancer risk, while
AGC haplotype could be related to erosive OLP progression to OSCC. However, these haplotypes are rather rare
(<10% of OSCC and OLP cases, and controls); therefore,
these ndings must be interpreted with caution. Additionally, as a result of moderate to strong LD and haplotype
effects, genotyped polymorphisms could also represent
surrogate markers for other distant or novel polymorphisms that could inuence the gene expression of
HMGB1.
None of the analyzed SNPs are located in the region
with enhancer elements in intron 1, which enhances the
HMGB1 transcriptional activity up to threefold (Lum and
Lee, 2001; Kornblit et al, 2007, 2010), or within silencer
element, which reduces HMGB1 transcription to one-sixth
(Zeng et al, 2012). Our TRANSFAC database search only
conrmed previous ndings of several putative transcriptional factors binding sites within the region of intron 1
(Sp1, AP1, AP4, and USF), while only the 3814 C/G
polymorphism created a binding site for a transcription

HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

Table 5 Haplotype odds ratio (HOR) analysis of HMGB1 polymorphisms 2262G/A (rs1045411), 1177G/C (rs3742305), and 3814C/G (rs2249825), in
OSCC, OLP, and controls, compared to the most common GGC haplotype

Haplotype Frequencies

Haplotypes

OSCC,
N (%)

OLP,
N (%)

Controls,
N (%)

Haplotypic Odds Ratio

HORa,b,c[95% CI], P

GGC
AGC

57 (62)
9 (10)

41 (78)
9 (1.8)

75 (75)
15 (1.5)

GCC

8 (9)

15 (2.8)

2 (2.2)

GGG
AGC

1 (1.7)
15 (17)

1 (1)
7 (13)

2 (2)
20 (19.5)

Referent
13.316a [1.656107.051], P = 0.015
NSb
c
12.179 [2.61956.647], P = 0.001
5.769a [1.19627.824], P = 0.029
1.382b [0.6392.987], P = 0.410
NSc
NSa,b,c
NSa
0.146 b [0.0151.434], P = 0.099
1.767c [0.9043.454], P = 0.096

Reference haplotype is GGC as the most frequent corresponding to the intercept of the logistic model.
HOR indicates haplotype odds ratio; 95% CI, 95% condence interval.
P-value, probability from the chi-square/Fishers exact test comparing the haplotype distribution for controls and cases. Values given in bold are signicant (P < 0.05). Haplotype distribution between: aOSCC vs. controls, bOLP vs. controls, and cOSCC vs. OLP.

factor v-myb (Kornblit et al, 2007). The GG genotype of

this polymorphism has previously been associated with
the LPS-induced HMGB1 production by peripheral blood
leukocytes in patients with major trauma (Zeng et al,
2012). Nevertheless, an inuence of the other effects of
polymorphisms on gene expression cannot be overlooked.
The polymorphism 1177G/C is located in intron 4, near
the fourth exonintron boundary. Polymorphisms in the
exonintron boundaries can alter the mRNA splicing and
stability, or inducing the enhancer functions. Deng and
coauthors reported that 1176G/C polymorphism in intron
4 near the exonintron boundary contained sequences
characteristic of an enhancer that could affect the expression of HMGB1 (Deng et al, 2013). Additionally, potential regulation of HMGB1 expression at the level of
mRNA translation or mRNA stability is suggested by the
existence of long conserved 30 untranslated regions (30
UTR) (Bustin, 1999). Within the 30 UTR of HMGB1
gene, there is a potential binding site for the transcriptional repressor cut homeodomain protein, CCAAT displacement protein. Tumor suppressors p53 and p73a have
a role in the transcriptional regulation of the HMGB1 gene
interacting with CCAAT-binding transcription factor 2
(CTF2) (Nagatani et al, 2001). Whereas p53 downregulates the activity of the HMGB1 promoter, p73a upregulates it (Uramoto et al, 2003). Thus, polymorphism within
this region could affect the interaction with p53 or p73a,
inuencing the activity of the HMGB1 promoter. Additionally, the 2262G/A polymorphism located in the 30
UTR could interfere with binding of microRNAs, consequently changing the mRNA stability.
There is an increasing need for understanding the
molecular basis of oral carcinogenesis and to identify
potential molecular markers to justify a more aggressive
adjuvant approach. Increasing evidence suggests that
inammation and tumor-induced immunosuppression,
involved in host protection against tumors, are among the
key hallmarks of cancer. Chronic inammation could
cause dysregulation of cell cycle control and oxidative

stress, which can provoke DNA damage resulting in

malignant transformation. Our ndings suggest that
HMGB1 1177G/C polymorphism could be considered as a
marker of tumor progression and a marker for poor recurrence-free survival in patients with OSCC. Additionally,
HMGB1 haplotypes, rather than individual polymorphisms, could be associated with an increased risk of
susceptibility to oral cancer. Further studies on the functional relevance of these polymorphisms are warranted to
conrm the important role of HMGB1 in cancer and to
evaluate HMGB1 as a potential target for the development
of novel anticancer therapies.
Acknowledgements
This research was supported by a Military Medical Academy,
Belgrade, Serbia, institutional grant 06/10/A1.

Author contributions
G. Supic designed study, performed experiments, analyzed data,
and wrote the manuscript, R. Kozomara collected and analyzed
data, drafted paper, K. Zeljic performed experiments, analyzed
data, drafted paper, D. Stanimirovic collected and analyzed data,
drafted paper, M. Surbatovic collected and analyzed data, drafted
paper, M. Magic collected and analyzed data, drafted paper, N.
Jovic collected and analyzed data, drafted paper, Z. Magic
designed study, analyzed data, and drafted and revised the manuscript.

References
Ahn MY, Kwon SM, Cheong HH et al (2012). Toll-like receptor
7 agonist, imiquimod, inhibits oral squamous carcinoma cells
through apoptosis and necrosis. J Oral Pathol Med 41: 540
546.
Apetoh L, Ghiringhelli F, Tesniere A et al (2007). Toll-like
receptor 4-dependent contribution of the immune system to
anticancer chemotherapy and radiotherapy. Nat Med 13: 1050
1059.
Oral Diseases

HMGB1 polymorphisms in oral carcinomas and oral lichen planus

G Supic et al

Barnard NA, Scully C, Eveson JW, Cunningham S, Porter SR

(1993). Oral cancer development in patients with oral lichen
planus. J Oral Pathol Med 22: 421424.
Barrett JC, Fry B, Maller J, Daly MJ (2005). Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21: 263265.
Bustin M (1999). Regulation of DNA-dependent activities by the
functional motifs of the high-mobility-group chromosomal proteins. Mol Cell Biol 19: 52375246.
Campana L, Bosurgi L, Rovere-Querini P (2008). HMGB1: a
two-headed signal regulating tumor progression and immunity.
Curr Opin Immunol 20: 518523.
Deng C, Deng G, Wang Y (2013). HMGB1 gene polymorphisms
in patients with chronic hepatitis B virus infection. World J
Gastroenterol 19: 51445149.
Fahmueller Y, Nagel D, Hoffmann R et al (2013). Immunogenic
cell death biomarkers HMGB1, RAGE, and DNAse indicate
response to radioembolization therapy and prognosis in colorectal cancer patients. Int J Cancer 132: 23492358.
Ferrari S, Finelli P, Rocchi M, Bianchi M (1996). The active
gene that encodes human high mobility group 1 protein
(HMG1) contains introns and maps to chromosome 13. Genomics 35: 367371.
Flohr AM, Rogalla P, Meiboom M et al (2001). Variation of
HMGB1 expression in breast cancer. Anticancer Res 21:
38813885.
Georgakopoulou EA, Achtari MD, Achtaris M, Foukas PG, Kotsinas A (2012). Oral lichen planus as a preneoplastic inammatory model. J Biomed Biotechnol 2012: 759626.
Gonzalez-Moles MA, Scully C, Gil-Montoya JA (2008). Oral
lichen planus: controversies surrounding malignant transformation. Oral Dis. 14: 229243.
Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA (2008). Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of highmobility group box-1 protein. Immunity 29: 2132.
Keyel P (2014). How is inammation initiated? Individual inuences of IL-1, IL-18 and HMGB1. Cytokine 69: 136145.
Kornblit B, Munthe-Fog L, Petersen SL, Madsen HO, Vindelv
L, Garred P (2007). The genetic variation of the human
HMGB1 gene. Tissue Antigens 70: 151156.
Kornblit B, Munthe-Fog L, Madsen HO, Strm J, Vindelv L,
Garred P (2008). Association of HMGB1 polymorphisms with
outcome in patients with systemic inammatory response. Crit
Care 12: R83.
Kornblit B, Masmas T, Petersen SL et al (2010). Association of
HMGB1 polymorphisms with outcome after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant
16: 239252.
Kuniyasu H, Chihara Y, Takahashi T (2003). Co-expression of
receptor for advanced glycation end products and the ligand
amphoterin associates closely with metastasis of colorectal
cancer. Oncol Rep 10: 445448.
Liu Y, Xie C, Zhang X et al (2010). Elevated expression of
HMGB1 in squamous-cell carcinoma of the head and neck and
its clinical signicance. Eur J Cancer 46: 30073015.

Oral Diseases

Lo Muzio L, Mignogna MD, Favia G, Procaccini M, Testa NF,

Bucci E (1998). The possible association between oral lichen
planus and oral squamous cell carcinoma: a clinical evaluation
on 14 cases and a review of the literature. Oral Oncol 34:
239246.
Lum HK, Lee K-LD (2001). The human HMGB1 promoter is
modulated by a silencer and an enhancer-containing intron.
Biochim Biophys Acta 1520: 7984.
Muller S, Ronfani L, Bianchi M (2004). Regulated expression
and subcellular localization of HMGB1, a chromatin protein
with a cytokine function. J Intern Med 255: 332343.
Nagatani G, Nomoto M, Takano H et al (2001). Transcriptional
activation of the human HMGB1 gene in cisplatin-resistant
human cancer cells. Cancer Res 61: 15921597.
Sasahira T, Kirita T, Oue N et al (2008). High mobility group
box-1-inducible melanoma inhibitory activity is associated
with nodal metastasis and lymphangiogenesis in oral squamous
cell carcinoma. Cancer Sci 99: 18061812.
Scully C, Bagan JV (2009). Oral squamous cell carcinoma: overview of current understanding of aetiopathogenesis and clinical
implications. Oral Dis 15: 388399.
Tregouet DA, Garelle V (2007). A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics 23: 10381039.
Tsunoda T, Takagi T (1999). Estimating transcription factor bindability on DNA. Bioinformatics 15: 622630.
Ulloa L, Messmer D (2006). High-mobility group box 1
(HMGB1) protein: friend and foe. Cytokine Growth Factor
Rev 17: 189201.
Uramoto H, Izumi H, Nagatani G et al (2003). Physical interaction of tumour suppressor p53/p73 with CCAAT-binding transcription factor 2 (CTF2) and differential regulation of human
high-mobility group 1 (HMG1) gene expression. Biochem J
371: 301310.
Wang Y, Li XP, Yin JY et al (2014). Association of HMGB1
and HMGB2 genetic polymorphisms with lung cancer chemotherapy response. Clin Exp Pharmacol Physiol 41: 408415.
Warnakulasuriya S, Johnson NW, van der Waal I (2007).
Nomenclature and classication of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 36: 575580.
Whiteside TL (2005). Immunobiology of head and neck cancer.
Cancer Metastasis Rev 24: 95105.
Wild C, Brandau S, Lot R et al (2012). HMGB1 is overexpressed
in tumor cells and promotes activity of regulatory T cells in
patients with head and neck cancer. Oral Oncol 48: 409416.
Wu D, Ding Y, Wang S, Zhang Q, Liu L (2008). Increased
expression of high mobility group box 1 (HMGB1) is associated with progression and poor prognosis in human nasopharyngeal carcinoma. J Pathol 216: 167175.
Yan W, Chang Y, Liang X et al (2012). High-mobility group
box 1 activates caspase-1 and promotes hepatocellular carcinoma invasiveness and metastasis. Hepatology 55: 18631875.
Zeng L, Zhang A, Gu W et al (2012). Clinical relevance of single nucleotide polymorphisms of the high mobility group box
1 protein gene in patients with major trauma in southwest
China. Surgery 151: 427436.