Oral Oncology 50 (2014) 330–338

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Etiological factors of nasopharyngeal carcinoma

Sai Wah Tsao a,⇑, Yim Ling Yip a, Chi Man Tsang a, Pei Shin Pang a, Victoria Ming Yi Lau a, Guitao Zhang a,
Kwok Wai Lo b,⇑
a
Department of Anatomy and Center for Cancer Research, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
b
Department of Anatomical and Cellular Pathology and Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, Hong Kong Special Administrative Region

a r t i c l e i n f o s u m m a r y

Article history: Nasopharyngeal carcinoma (NPC) is a common disease among southern Chinese. The major etiological
Available online 12 March 2014 factors proposed for NPC pathogenesis include genetic susceptibility, environment factors and EBV infec-
tion. In the high risk population, genetic susceptibility to NPC has been mapped to the HLA loci and adja-
Keywords: cent genes in MHC region on chromosome 6p21. Consumption of preserved food including salted fish has
EBV been implicated in its etiology in earlier studies. Its contribution to pathogenesis of NPC remains to be
Nasopharyngeal carcinoma determined. A decreasing trend of NPC incidence was observed in Hong Kong, Taiwan and Singapore
Etiology
in recent years which may be accounted by a change of dietary habits. A comprehensive epidemiological
Stromal inflammation
Immune evasion
study will help to elucidate the relative importance of various risk factors in the pathogenesis of NPC.
Despite the close association of EBV infection with NPC, the etiological role of EBV in NPC pathogenesis
remains enigmatic. EBV infection in primary nasopharyngeal epithelial cells is uncommon and difficult to
achieve. EBV does not transform primary nasopharyngeal epithelial cells into proliferative clones, which
contrasts greatly with the well-documented ability of EBV to transform and immortalize primary B cells.
Genetic alterations identified in premalignant nasopharyngeal epithelium may play crucial roles to sup-
port stable EBV infection. Subsequently, latent and lytic EBV gene products may drive clonal expansion
and transformation of premalignant nasopharyngeal epithelial cells into cancer cells. Stromal inflamma-
tion in nasopharyngeal mucosa is believed to play an important role in modulating the growth and pos-
sibly drive the malignant transformation of EBV-infected nasopharyngeal epithelial cells. Furthermore,
there are increasing evidences supporting a role of EBV infection to evade host immune surveillance.
EBV-infected cells may have selective growth advantages in vivo by acquiring a stress–resistance pheno-
type. Understanding the etiological factors and pathogenesis of NPC will contribute effectively to the pre-
vention and treatment of this disease.
Ó 2014 Elsevier Ltd. All rights reserved.

Geographical distribution of nasopharyngeal carcinoma
Nasopharyngeal carcinoma (NPC) stands out from other epithe-
lial cancers arising from the head and neck regions in its distinct
geographical distribution and close association with EBV infection
[1,2]. The anatomic site of NPC is also unique which may implicate
a contributing role of microenvironment in its pathogenesis. The
incidence rate of NPC is remarkably variable worldwide. In most
parts of the world, NPC incidence is low (<1/100,000 per year).
NPC incidence is high among ethnic southern Chinese and native
Eskimos living in Greenland and Alaska. High incidence of NPC is
⇑ Corresponding authors. Address: Laboratory Block, Department of Anatomy,
LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special
Administrative Region. Tel.: +852 28199227; fax: +852 28170857 (S.W. Tsao).
E-mail addresses: gswtsao@hku.hk (S.W. Tsao), kwlo@cuhk.edu.hk (K.W. Lo).
also seen in regions in North Africa. Globally, approximately
65,000 new cases of NPC are reported each year and more than
80% are from southern China and Southeast Asia. The highest inci-
dence rate of NPC is amongst Cantonese living in Hong Kong and
Guang-dong province of Southern China (>20 per 100,000 per year
in males) [3–5]. Relatively high incidence of NPC was also reported
in other provinces of Southern China region including GuangXi,
Fujian and Hunan. The incidence rate of NPC is low in Northern
China (1 to 5 per 100,000 persons per year). Notably, southern
Chinese migrated to non-endemic regions still retains a much
higher incidence rate compared to other ethnic groups living in
the same geographical regions. Among the high-risk population,
incidence of NPC peaks between 45 and 54 years old and declines
thereafter. For unknown reason, men are roughly 2–3 folds more
frequently affected than women [3–5]. The remarkable racial and
geographic distribution of NPC suggests a strong association of
NPC with genetic susceptibility and environmental factors.

http://dx.doi.org/10.1016/j.oraloncology.2014.02.006
1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
 S.W. Tsao et al. / Oral Oncology 50 (2014) 330–338
Interestingly, the incidence rate of NPC has been decreasing con-
tinuously in Hong Kong from 1980 (28.5/100,000 in males and
11.2/100,000 in females) to 2009 (14.0/100,000 in males and 4.6/
100,000 in females), a mark decrease of over 50% in the past
30 years [6,7]. The decreasing trend in this high-risk region may re-
flect the change of certain risk factors related to dietary habits and
lifestyle. The continuous immigration of Chinese from non-ende-
mic region to Hong Kong may also contribute partly to the decreas-
ing incidence rate of NPC [8]. Similar decreasing trend was also
reported in Taiwan [9] and Singapore [10]. The decreasing trend
of incidence of NPC in the past 20–25 years was however not ob-
served in other regions of Guangdong province, including Sihui
and Cangwu counties, where traditional dietary habits were still
maintained until early 1990’s [11].
Histopathology of NPC
The nasopharynx is a tubular space situated at the base of the
skull. It represents a transitional area between the nasal cavities
and the oropharynx, forming part of the Waldeyer ring of lymphoid
tissues. The nasopharyngeal mucosa shows numerous folds and
crypts. It consists of a special type of stratified squamous epithe-
lium referred as intermediate or transitional epithelium [12]. Var-
iable amount of mixed stratified squamous epithelium and ciliated
epithelium are present at the lateral and posterior wall of the naso-
pharynx. Aside from sero-mucous glands, abundant infiltration of
lymphocytes are found in the underlying stroma of the nasopha-
ryngeal epithelium. NPC is commonly developed from the lateral
wall of nasopharynx, especially at the fossa of Rosenmüller and
superior posterior wall [13]. The WHO classification in 1978 recog-
nized three histological subtypes of nasopharyngeal carcinoma:
squamous cell carcinoma (WHO type 1), nonkeratinizing carci-
noma (WHO type 2), and undifferentiated carcinoma (WHO type
3) which was modified in 1991. The WHO type 1 NPC was retained,
while the WHO type 2 and type 3 are combined into a single cate-
gory of ‘‘nonkeratinizing carcinoma’’, which was further subdi-
vided as ‘‘differentiated’’ or ‘‘undifferentiated’’ [14]. The
histological features of keratinizing squamous cell carcinoma of
NPC are similar to squamous cell carcinoma arising from other
sites of the head and neck regions with well-differentiated histo-
logical features including presence of intercellular bridges, keratin
production and epithelial pearl formation. NPC in the endemic
areas of Southern China is mainly of the nonkeratinizing carcinoma
which occupies up to 99% of all cases. The nonkeratinizing carci-
noma lacks keratinization features and is ubiquitously associated
with EBV infection [4,15].
Etiological factors of NPC
The epidemiological studies reveal distinctive ethnic and geo-
graphic distribution of NPC strongly indicating that genetic suscep-
tibility plays a major contributing role. The aforementioned
decreasing trends of NPC incidence reported in Hong Kong, Taiwan
and Singapore population indicate that alteration of dietary habits
and change in environment factors may also alter incidence rate of
NPC. The clonal origin of EBV infection and its ubiquitous presence
in NPC strongly indicates its involvement in NPC pathogenesis [2].
EBV infection, together with genetic susceptibility and environ-
mental factors are considered as the three major etiological factors
of NPC among southern Chinese in endemic areas of NPC.
Genetic susceptibility
The distinct ethnicity of NPC indicates the important contribu-
tion of genetic susceptibility to the pathogenesis of NPC. As stated
331
earlier, the incidence of NPC is 20–50 folds higher in southern Chi-
nese compared to populations in Western countries. Notably, the
secondary and third generations of the southern Chinese who emi-
grated to low incidence area in the USA still retains a higher risk of
NPC than the resident population despite of cultural assimilation
[16]. Familial clustering of the disease was observed at a frequency
around 10% among Chinese patients [17]. Sharing common envi-
ronmental risk factors may also account for familial aggregation
of NPC and may be difficult to distinguish from inherited genetic
susceptibility. Nonetheless, genetic susceptibility loci to NPC in
high-risk population have been reported notably the association
with the HLA class I genes in the MHC locus at chromosome
6p21 [18]. The HLA class I genes encode proteins to identify and
present foreign antigens, including EBV-encoded peptides, to the
cytotoxic T cells to trigger the host immune response against vi-
rally infected cells. The variable degree of susceptibility to NPC
among different ethnic populations may reflect the differential
ability of HLA haplotypes in controlling EBV infection in infected
populations. Individuals with specific HLA alleles may be less effi-
cient in mounting a cytotoxic immune response against EBV-in-
fected cells and may contribute to increased susceptibility to
NPC. An early study in affected sib pairs from southern China iden-
tified a NPC susceptible locus closely linked to the HLA region [19].
A large scale genome-wide association studies (GWAS) reported
recently also revealed that genes within the HLA region on chro-
mosome 6p21 are strongly associated with NPC [20]. Independent
and strong association signals in the HLA-A and HLA-B/C loci were
observed. Other studies have also confirmed the consistent associ-
ation of NPC risk with HLA regions [21]. However, several non-HLA
genes including GABBR1 and MICA in the HLA region also show
strong association on NPC susceptibility [22,23]. Apart from the
genes in HLA region, significant association with MDS1-EVI1 at
3q26.2, CDKN2A/2B at 9p21.3, and TNFRSF19 at 13q12.12 was also
reported [20]. Identification of the causal variants in these loci and
elucidation of their functions should enhance our understanding
on the contribution of genetic factors in NPC pathogenesis.
In addition to GWAS, three pedigree-based linkage studies per-
formed in high-risk NPC Chinese families have also identified NPC
associated genes with high penetrance. Using microsatellite poly-
morphic markers, three disease susceptibility loci on chromosomes
3p21.3, 4p15.1-q12 and 5p13 were identified from high-risk NPC
Chinese pedigrees [24–26]. However, the susceptibility locus iden-
tified in each of these familial studies is diverse. These non-concor-
dant findings may be due to genetic heterogeneity and limited
number of the Chinese familial NPC cases in these studies. Fine-
mapping and functional characterization of these susceptibility
genes are warranted.
Case-control studies have demonstrated a link between genetic
polymorphism and NPC risk by influencing the individual suscep-
tibility to EBV infection and/or chemical carcinogen-induced cell
transformation. By candidate gene-based approach, increased risks
of NPC were shown to be associated with genetic polymorphisms
involved in nitrosamine metabolism (CYP2E1, CYP2A6), detoxify-
ing carcinogenic electrophiles (GSTM1), DNA repair (XRCC1,
hOGG1, NBS1), EBV entry into nasopharyngeal epithelium (PIGR),
interleukins (IL1A, IL1B, IL2, IL8 and IL10) and toll-like receptors
(TLR3, TLR4, TLR10) [27–36]. Significant correlation of variants of
cancer-associated genes, including TP53, MDM2, CCND1 and
NEDD4, was also reported to be associated with increased NPC risk
among southern Chinese [37–41].
Environmental factors
A number of agents in the environment have been postulated to
be associated with NPC risk. It has been reported in earlier studies
that the ingestion of Cantonese-style salted fish, especially during
332 S.W. Tsao et al. / Oral Oncology 50 (2014) 330–338
childhood, correlates with increased risk of NPC in endemic popu-
lations [5,42–47]. Experimental studies have shown that nasal and
paranasal malignant tumors were developed in rats fed with
salted-fish [48,49]. Other salted and preserved food items includ-
ing shrimp paste and preserved vegetables may represent indepen-
dent risk factors for NPC among Chinese [5,42–44]. Volatile
nitrosamines in some of the traditional food items of southern Chi-
nese were postulated as the putative carcinogens for NPC develop-
ment [50,51]. The active carcinogenic metabolite of these
nitrosamines may induce DNA damage and chronic inflammation
in nasopharyngeal mucosa of genetically susceptible individuals.
Consuming this dietary carcinogen during childhood may lead to
accumulation of aberrant genetic lesions and development of field
cancerization in nasopharynx at early age, predisposing them to
EBV infection and thereby increase the risk of developing NPC.
The consumption of salt fish among Hong Kong population has
dropped dramatically for the past 20 years. It remains to be deter-
mined if the change of dietary habits is accountable for the contin-
uous decreasing trend of NPC incidence reported in Hong Kong for
the past 30 years [6,7].
The use of traditional herbal medicines has also been reported
to be a unique factor associated with elevated NPC risk in Asian
populations [5,52–54]. Some Chinese herbal plants may contribute
to NPC development by inducing EBV lytic antigen expression
[55,56]. In contrast to the preserved foods and herbal medicines,
consumption of fresh fruit and leafy vegetables, especially during
childhood, has been reported to be protective factors against NPC
in Chinese [57].
Non-dietary risk factors associated with increased NPC risk
have also been reported. Occupational exposures to formaldehyde,
wood dust, fumes and chemicals have been recognized as NPC risk
factors by causing chronic inflammation in nasopharynx
[3,5,53,58]. Cigarette smoking has been reported to be correlated
with well-differentiated NPC in low-risk population; its association
with undifferentiated or nonkeratinizing carcinoma in the endemic
area is controversial [57].
Epstein–Barr virus (EBV) infection
Despite the close association of EBV infection with NPC, the role
of EBV infection in NPC pathogenesis remains enigmatic. An intri-
cate interplay of EBV with host stroma and genetic alteration in in-
fected host cells is likely to be involved in the pathogenesis of NPC
(Fig. 1).
The association of EBV and human malignancies dated back to
1960’s with the discovery of EBV infection in Burkitt’s lymphoma,
a childhood cancer seen in Africa [59]. In vitro, EBV infection readily
drives proliferation and immortalization of B lymphocytes. EBV
infection accounts for 90% of infectious mononucleosis, driving
the proliferation of infected B-lymphocytes in vivo [60,61]. The lym-
phoblastoid proliferation is self-limiting and will eventually subside
as the host-immune defense mechanisms are activated, driving EBV
into latent infection in memory B-lymphocytes. EBV infection in
premalignant nasopharyngeal epithelial cells harboring genetic
alterations may drive their malignant transformation as proposed
in Burkitt’s lymphoma, Hodgkin’s disease and gastric carcinoma.
The association of EBV infection and NPC was first indicated
based on the serological evidence that NPC patients had higher
antibody titers against viral capsid antigen and early antigen than
healthy controls [62]. Elevated IgA and anti-DNase antibodies
against EBV were shown to have strong association with subse-
quent development of cancer in a large cohort [63]. A more recent
study also showed that pretreatment levels of EBV DNase-specific
neutralizing antibody could be used as a prognostic marker to com-
plement TNM classification in nasopharyngeal carcinoma [64]. In
almost all nonkeratinizing NPC, EBV genome could be detected with
viral gene expression profile distinctive of type II latency [65]. Anal-
ysis of the TR repeats in EBV episomes isolated from premalignant
nasopharyngeal epithelium and NPC showed clonal origin of EBV
infection [66] suggesting that NPC is derived from the expansion
of a single EBV-infected cell [67]. Using microdissected tissues, alle-
lic deletion of 3p and 9p could be detected in premalignant and his-
tologically normal nasopharyngeal epithelial tissues free of EBV
infection suggesting that genetic alterations in premalignant naso-
pharyngeal epithelium are present prior to EBV infection [68,69].
Our recent study shows that genetic alterations (e.g. cyclin D1 over-
expression) in premalignant or dysplastic nasopharyngeal epithe-
lium supports persistence of EBV episomes in infected cells [70].
EBV readily infects and transforms primary B cells but not primary
nasopharyngeal epithelial cells
EBV infection is rarely detected in vivo in nasopharyngeal epi-
thelium except in premalignant nasopharyngeal dysplastic lesions
and nonkeratinizing NPC [4]. Most children in the developing world
are infected with EBV before the age of three and mostly asymp-
tomatic in nature [60]. EBV infects B lymphocyte through binding
to the CR2 (CD21) receptor on the surfaces of B lymphocytes [71].
The CR2 receptor is generally low or absent in mucosal epithelium
lining the head and neck regions as detected by immunocytochem-
istry. Nonetheless, mRNA for CR2 receptor could be detected in ton-
sillar epithelial cells [72]. A low% (1 in 105 cells) of EBV-infected
epithelial cells was reported in ex vivo cultures of asymptomatic
EBV carriers [73]. EBV replication was reported in 1.3% of tongues
mucosa [74]. These findings support the involvement of oropharyn-
geal epithelial cells in the production of infectious EBV in vivo.
Events that facilitate entry and establishment of EBV infection in
human nasopharyngeal epithelial cells are only beginning to be
understood [75]. Recent studies showed that human epithelial cells
could be infected in vitro, through the formation of specialized B
cell-epithelial cell conjugates involving the EBV viral envelope gly-
coprotein, GP350, and the CR2 receptor of B lymphocytes [76,77].
This cell-to-cell method of EBV infection of nasopharyngeal epithe-
lium may take place in vivo. EBV-infected B cells invariably may be
present in the nasopharyngeal mucosa and have chance to come
into close contact with the polarized nasopharyngeal epithelial
cells to transmit the EBV. Cell-free EBV virions may also enter the
epithelial cells through the basolateral membranes involving inter-
action of the EBV glycoprotein, BMRF-2, with the integrin a5b1 of
the epithelial cells which may represent a transmission route of
EBV virus from infected epithelial cells across the lateral mem-
branes to infect adjacent epithelial cells [78]. EBV entry into epithe-
lial cells may also be achieved via the IgA (against EBV-VCA)/Sc
(secretory component) complex present on the surface of mucosal
epithelial cells through endocytosis [79,80].
Genetic alterations in premalignant nasopharyngeal epithelium
support stable EBV infection
Different mechanisms are involved in facilitating entry of EBV
into epithelial cells and persistence of EBV episomes post infection.
EBV infection of primary nasopharyngeal epithelial cells does not in-
duce their proliferation, which contrast greatly to EBV infection in B-
lymphocytes [81]. However, infection of established epithelial cells
harboring multiple genetic alterations may promote growth and
confer tumorigenicity in infected cells [82]. In nasopharyngeal epi-
thelial cells immortalized by telomerase, EBV infection induces
senescence and growth arrest phenotypes [70]. Overexpression of
cyclin D1 in the immortalized nasopharyngeal epithelial cells could
overcome the growth arrest phenotype induced by EBV infection
and support long-term propagation of EBV-infection in infected
cells. Cyclin D1 overexpression is common in dysplastic nasopha-
ryngeal epithelium. Suppression of p16 expression also supports
propagation of EBV infection in immortalized nasopharyngeal epi-
 S.W. Tsao et al. / Oral Oncology 50 (2014) 330–338
Figure 1. Tumorigenesis model of pathogenesis of EBV-associated nasopharyngeal carcinoma.
thelial cells. Hence, genetic alterations in premalignant nasopharyn-
geal epithelium which support EBV persistence of EBV may facilitate
their transformation into cancer cells. Counterintuitively, we failed
to observe growth advantage in EBV-infected nasopharyngeal epi-
thelial cells or NPC cells in vitro [75]. It is also well-documented that
EBV-infected NPC cells readily lost their EBV genomes upon propa-
gation in culture showing that EBV infection per se does not confer
growth advantage in vitro. The C666-1, which was established from
a xenografted NPC passaged in nude mice for prolonged period, is
the only NPC cell line which continues to harbor EBV upon prolonged
propagation. A defect in the lytic reactivation of EBV genome in
C666-1 cells and/or unique host factors supporting EBV persistence
may be involved.
Growth advantages of EBV-infected NPC cells in vivo
In contrast to the rapid loss of EBV-infected NPC cells in vitro,
EBV-infected NPC cells are actively selected and maintained
in vivo. EBV episome could be detected in practically all cancer
333
cells in undifferentiated NPC from patients. Furthermore, NPC
xenografts (xeno666, xeno2117, C15 and C17) which have been
passaged in immune deficient animals for over 20 years still retain
their EBV episomes. The nature of these selection advantages of
EBV-infected NPC in vivo is unclear but may include (a) enhanced
survival against apoptotic stimuli, (b) enhanced growth in inflam-
matory stroma and (c) evasion from host immune surveillance.
Elucidation of growth advantages of EBV infection in NPC cells
in vivo may reveal the role of EBV in NPC pathogenesis.
EBV infection confers a stress–resistant phenotype in infected cells.
We were able to establish stable EBV infection in multiple lines of
immortalized nasopharyngeal epithelial cells and NPC cells [70].
The most consistent cellular phenotype observed is their resistance
to nutrient and growth factor deprivation. Compared to uninfected
cells, EBV-infected cells often reveal a robust phenotype in surviv-
ing nutrient deprivation. Detail mechanisms involved are not com-
pletely elucidated but activation of PI3 K and Akt pathways are
334 S.W. Tsao et al. / Oral Oncology 50 (2014) 330–338
involved [75,83]. Stress–resistance phenotype could also be ob-
served in nasopharyngeal epithelial cells expressing the EBV-en-
coded LMP1 [84]. LMP1 expression was shown to inhibit the
AMPK/LKB1 signaling pathways to promote cellular growth and
survival [85]. In EBV-infected cells, LMP1 expression could be in-
duced upon nutrient and growth factor deprivation to enhance cell
survival (unpublished observation). The ability to withstand stress
by EBV-infected cells may provide a growth advantage in vivo as
ischemia is common during exponential growth of tumor.
Contribution of stromal inflammation to growth of EBV-infected NPC
cells. A characteristic histopathological feature of NPC is the pres-
ence of exceptionally rich inflammatory stroma. EBV-infected
NPC cells in patients are continuously confronted by inflammatory
cytokines present in the tumour stroma including TNF-a, TGF-b,
IL6, IL8, etc. [86]. The effects of these inflammatory cytokines on
the growth and malignant properties of EBV-infected NPC cells
are not fully understood. These inflammatory cytokines may sup-
port the growth of EBV-infected NPC cells. Among these inflamma-
tory cytokines, the IL6 was shown to support latent infection of
EBV through activation of STAT3 signaling [87]. STAT3 activation
in EBV-infected cells upregulates the activity of the EBV Qp pro-
moter for EBNA1 transcription which is essential for latent infec-
tion of EBV and maintenance of EBV episome in infected cells.
The latent EBV gene, LMP1, could also upregulate transcription of
IL6 in EBV-infected cells which activates STAT3 to upregulate
LMP1 transcription in EBV-infected NPC cells, constituting a posi-
tive feedback loop involving STAT3, LMP1 and IL6 to sustain activa-
tion of STAT3 activation and latent EBV infection in NPC cells
[87,88]. The action of STAT3 activation to enhance survival and
malignant properties of human cancer cells is well-documented
[89]. We recently reported that prolonged propagation of EBV-in-
fected immortalized nasopharyngeal epithelial cells (NP460) con-
ferred enhanced sensitivity to IL6 activation of STAT3 [90]. The
enhanced responsiveness of EBV-infected NP460 cells to IL6 activa-
tion of STAT3 was shown to be largely due to overexpression of IL6
receptor (IL-6R). IL-6R overexpression was detected in NPC speci-
mens. Overexpression of IL-6R also enhanced in vivo growth and
tumorigenicity of C666-1, an EBV-positive NPC cell line, in immune
deficient animals.
The IL8 (CXCL8) also supports spheroid growth and stemness
properties of EBV + ve NPC cells, C666-1 [91,92]. Overexpression
of IL8 is common in NPC tissues. Interestingly, the C666-1 cells also
secrete IL8. An autocrine stimulating pathway may be involved to
promote growth of EBV-infected NPC cells.
Constitutive NF-jB signaling, which is proinflammatory in nat-
ure, is common in NPC and could be activated by either LMP1
expression or genetic alterations in EBV-positive NPCs. The major
NF-jB signals in NPC are p50/p50/BCL3 and p50/RELB [93]. By
microarray analysis of c666-1 cells with p50 knockdown, we iden-
tified multiple NF-jB downstream targets, including oncogenes
(MYB, BCL2), chemokines and chemokine receptors (CXCL9,
CXCL10, CX3CL1 and CCL20) [94]. NPC is characterized by heavy
infiltration of tumour infiltrating lymphocytes (TILs). This has
raised the possibility that chemokines induced by NF-jB activation
in NPC cells may recruit TILs to the tumour microenvironment and
interact with the NPC cells in various ways to support their growth
in vivo. The expression of CCL20 may also contribute to the escape
of EBV-infected NPC cells from host anti-viral immune response by
recruiting memory regulatory T-cells. The inflammatory stroma
could play important roles to promote growth and progression of
NPC and may represent a druggable target for effective treatment
of the disease.
Evasion from immune surveillance. Immunosurveillance plays a vi-
tal role in elimination of tumour cells at early stage of
tumorigenesis. EBV infection may support infected premalignant
or malignant nasopharyngeal epithelial cells to escape immuno-
surveillance in vivo. Multiple mechanisms may be involved. Pre-
sentation of antigenic peptides on MHC class I molecules is a
signal for CD8 + T cells to recognize non-self antigens in defending
parasite infection. EBV infection may suppress expression of MHC I
in infected-NPC cells to interfere with the presentation of viral or
tumour antigen to activate host innate and adaptive immunity. A
genome-wide expression profiling in laser-captured, microdissect-
ed NPC tissues and normal nasopharyngeal epithelium showed
strong correlation with the expression of EBV genes with inhibition
of expression of multiple MHC class I HLA genes [95]. Similarly,
down-regulation of locus-specific HLA Class I expression was also
reported in EBV-associated gastric cancer [96]. The EBV proteins
could also directly impede the function of HLA molecules. The
Gly-Ala repeat (Gar) within the EBV EBNA1, which is expressed
in all latent EBV-infected cells, inhibits MHC class 1 restricted anti-
gen presentation [97]. The EBV BNLF2A could bind to the TAP
transporter to inhibit the delivery of the antigenic peptide to HLA
molecules for presentation [98]. Furthermore, the EBV BILF1,
which function as a G protein-coupled receptor, increases endocy-
tosis and lysosomal degradation of MHC class I molecules [99]. A
recent study also reported that the EBV BILF1 impedes presenta-
tion of viral antigen and cytotoxic T cell recognition of the infected
cells [100]. The EBV BGLF5 also suppresses HLA antigen expression
as part of its action to shut off host immune function [101].
The EBV-encoded miRNAs have also been reported to partici-
pate in the immune evasion of EBV-infected cells. The ebv-miR-
BART2-5p and ebv-miR-BART3 target MICB and IPO7 respectively
[102]. The MICB is a cellular ligand for NKG2D which is a potent
activating receptor presents on NK cells. The ebv-miR-BART2-5p
suppresses the expression of MICB in EBV-infected NPC cells pro-
tecting them from recognition and killing by NK cells. The IPO7
(importin 7) is a nuclear importer receptor which contributes to
cytokine and early response gene expression in activated T cells.
The transportation of EBV-encoded miRNA from NPC tumor cells
to neighboring TILs may impair their cytotoxic function through
inhibiting IPO7 expression.
Another study reported that EBV-infected cells commonly used
exosomes secretion to communicate with neighboring cells [103].
Exosomes isolated from EBV-infected lymphoblasts (LCL) are en-
riched in small RNAs and EBV-miRNAs which are delivered to
and internalized by monocyte-derived dendritic cells to repress
some of the EBV-targeted genes, including CXCL11/ITAC, which is
an immunoregulatory gene down-regulated in primary EBV-asso-
ciated lymphomas [104]. NPC cells could produce exosomes con-
taining high amount of galectin-9, a ligand of the membrane
receptor Tim-1, to induce apoptosis in mature Th1 lymphocytes
[105]. Recombinant galectin-9 could induce apoptosis in EBV-spe-
cific CD4 + T cells and may play a role in the immune evasion of
EBV-infected NPC cells. Immune invasion may confer a distinct
in vivo advantage of EBV-infected NPC cells.
Contribution of latent and lytic EBV infection in NPC carcinogenesis
Latent EBV infection is believed to be involved in tumorigenesis
as lytic reactivation of EBV infection in infected cells will lead to
cell death. In NPC, the lytic infection of EBV is generally believed
to be abortive in nature without production of infectious viral par-
ticles. Recent studies showed that both lytic and latent EBV genes
may contribute to the carcinogenesis of NPC.
The role of the latent infection of EBV in human carcinogenesis
has long been postulated. EBV readily established type II latency in
NPC cells and expression of EBV genes, including EBERs, EBNA1,
LMP1, LMP2 and recently EBV-encoded miRNAs, have been shown
to be involved in tumorigenesis. Furthermore, latent infection of
EBV may drive epigenetic changes in host cell genome to promote
 S.W. Tsao et al. / Oral Oncology 50 (2014) 330–338
tumorigenesis [106]. The LMP1 is considered as a classical viral
oncogene which activates multiple cell signaling, including NF-
jB, MAPK and PI3K, to drive tumorigenesis [107]. A role of LMP1
in driving epigenetic changes has been reported. In NPC cells, the
LMP1 was shown to downregulate E-cadherin via activation of
DNA methyltransferases [108]. LMP1 could also directly activate
the DNMT1-P1 promoter via the JNK-AP-1 pathway [109]. In gas-
tric cancer, LMP2A was reported to induce DNMT1 overexpression
through STAT3 activation and PTEN inactivation. The EBV EBNA1,
which is ubiquitously expressed in EBV-associated human malig-
nancies, could induce DNA damage through induction of reactive
oxygen species [110]. EBER-overexpressing EBV promotes in vivo
growth of infected NPC cells [111]. The BART transcript is abun-
dantly expressed in NPC [95]. The BART miRNAs encoded by EBV
are expressed at particularly high levels in EBV associated epithe-
lial malignancies undergo type II latency [112]. The BART miRNAs
may have an important role in driving growth and malignant
transformation in infected NPC cells. Multiples studies have re-
vealed that the EBV-encoded BART miRNAs target various cellular
and viral proteins involved in maintenance of viral latency, im-
mune evasion, and cell survival in NPC [113].
While EBV infection in NPC cells is largely latent in nature, EBV
lytic genes, such as BZLF1, are invariably detected in clinical NPC
specimens [95]. The impact of lytic EBV infection in NPC carcino-
genesis is not fully understood. Lytic infection of EBV has been
shown to be important in the development of EBV-induced lym-
phoma. A mutant EBV incapable to induce lytic infection is much
less potent in induction of lymphoma in humanized mice reconsti-
tuted with human fetal CD34 + hematopoietic stem cells [114].
Expression of lytic EBV genes may contribute to genomic instabil-
ity in infected cells. The immediate-early protein, BZLF1 proteins,
which plays an integral role in activation of lytic EBV infection
could induce G2 and mitotic blocks in cells [115]. The BZLF1 pro-
tein could also bind to chromosome during mitosis and may
sequester other BZLF-1 interacting proteins in cells to alter chro-
mosome compaction and chromatin structure [116]. The BZLF1
also increases the level of acetylated histone and translocates
CBP to mitotic chromosomes. Another EBV lytic gene, BGLF4 could
induce early DNA condensation and abnormal mitosis [117].
Recurrent induction of lytic infection of EBV contributed to an en-
hanced tumorigenic phenotype in EBV-infected HONE1 cells [118].
Prolonged DNA damage response in normal cells usually induces
apoptosis. Expression of multiple latent EBV genes, notably
LMP1, prevents apoptosis and enhances survival of EBV-infected
cells [107]. By analogy to the classical two-stage carcinogenesis
model, EBV lytic infection may play an initiator role while EBV la-
tent infection plays a role as promotor in carcinogenesis of NPC.
The inflammatory cytokines in the tumour stroma may play an
important role in regulating latent and lytic infection of EBV. The
TGFb and its downstream mediators, could activate Zp promoter
to activate transcription of the lytic EBV transactivator protein,
BZLF1 protein, to initiate lytic infection of EBV [119]. In contrast,
the IL6 and possibly other cytokines induce STAT3 activation to
support latent EBV infection. The interplay of stromal cytokines
in the regulation of latent and lytic infection of EBV may contribute
to the tumorigenic transformation and progression of EBV-infected
nasopharyngeal epithelial cells.
Conclusion and future directions
The pathogenesis of NPC is a multi-stage event and involves
multiple etiological factors. The unique geographical distribution
and ethnicity of NPC incidence implicate strongly the involvement
of genetic susceptibility. A close association with HLA locus has
been identified in high risk NPC families. The environmental
335
factors, including consumption of salted fish and preserved food,
may contribute to the generation of genetic alterations in prema-
lignant nasopharyngeal epithelial cells in high risk NPC popula-
tions. EBV infection is consistently demonstrated in
undifferentiated NPC. Genetic changes in premalignant nasopha-
ryngeal epithelial cells support EBV infection and its persistence
after infection. The local stromal inflammatory cytokines have pro-
found effects on growth of EBV-infected nasopharyngeal epithelial
cells. Multiple EBV genes, latent and lytic in nature, may contribute
to malignant transformation of premalignant nasopharyngeal epi-
thelial cells. EBV infection and expression of viral genes including
the EBV-encoded miRNAs may enable the virally infected NPC cells
to evade immune surveillance and confer selective advantages for
their growth in vivo. An intricate interplay of these factors is be-
lieved to contribute to NPC pathogenesis (Fig. 1).
With recent advances in the genomic studies, comprehensively
deciphering the gene-environment interactions may provide new
opportunities for early diagnosis as well as therapeutic opportuni-
ties for NPC. In most of the endemic regions, screening of NPC is
commonly limited to the patient’s family members. Identification
of NPC-susceptibility loci and NPC-associated HLA using GWAS
analysis and next generation sequencing of a large panel of pa-
tients with individual risk factor information will help to define
the high-risk population for further diagnostic evolution and may
improve the chance of early cancer detection. Because of the con-
sistent association of EBV infection and NPC, EBV-specific anti-
body-based assays have been commonly used as screening and
diagnostic markers in high-risk region. The development of real-
time quantitative PCR analysis of EBV DNA in plasma has highly
improved the sensitivity and specificity for early detection of
NPC. A recent study on a large cohort of healthy individuals has
demonstrated that plasma DNA analysis is useful in screening for
early NPC [120]. By targeting on the high risk population with de-
fined genetic factors will further enhance the effectiveness of early
detection of NPC. The success in the early screening program in en-
demic regions will greatly improve the current clinical manage-
ment of this deadly disease.
Conflict of interest statement
None declared.
Acknowledgements
The authors acknowledged the generous support of the various
funding sources from the Research Grant Council, Hong Kong: GRF
(777809, 779810, 780911, 779312; 470708, 471709, 471610,
471211, 470312); CRF (CUHK8/CRF/11R); AoE NPC Grant (AoE/M-
06/08) and Theme-Based Research Scheme (T12-401/13-R). The
authors would also like to acknowledge funding from the HMRF
Grant, Hong Kong (12110942 and 13120872) and the CRCG Grant
from the University of Hong Kong.
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