VOLUME 33 NUMBER 29 OCTOBER 10 2015

JOURNAL OF CLINICAL ONCOLOGY R E V I E W A R T I C L E

Nasopharyngeal Cancer: Molecular Landscape

Jeff P. Bruce, Kenneth Yip, Scott V.
Bratman, Emma Ito, and Fei-Fei Liu,
University Health Network; and Scott V.
Bratman, Emma Ito, and Fei-Fei Liu,
University of Toronto, Toronto, Ontario,
Canada.
Published online ahead of print at
www.jco.org on September 8, 2015.
Authors disclosures of potential
conflicts of interest are found in the
article online at www.jco.org. Author
contributions are found at the end of
this article.
Corresponding author: Fei-Fei Liu, MD,
Department of Radiation Oncology,
Princess Margaret Cancer Center/On-
tario Cancer Institute, 610 University
Ave, Toronto, Ontario, Canada M5G
2M9; e-mail: Fei-Fei.Liu@rmp.uhn.on.ca.
2015 by American Society of Clinical
Oncology
0732-183X/15/3329w-3346w/$20.00
DOI: 10.1200/JCO.2015.60.7846
Jeff P. Bruce, Kenneth Yip, Scott V. Bratman, Emma Ito, and Fei-Fei Liu
A B S T R A C T
Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy arising from the superior aspect
of the pharyngeal mucosal space, associated with latent Epstein-Barr virus infection in most cases.
The capacity to characterize cancer genomes in unprecedented detail is now providing insights
into the genesis and molecular underpinnings of this disease. Herein, we provide an overview of
the molecular aberrations that likely drive nasopharyngeal tumor development and progression.
The contributions of major Epstein-Barr virus encoded factors, including proteins, small RNAs,
and microRNAs, along with their interactions with pathways regulating cell proliferation and
survival are highlighted. We review recent analyses that clearly define the role of genetic and
epigenetic variations affecting the human genome in NPC. These findings point to the impact of
DNA methylation and histone modifications on gene expression programs that promote this
malignancy. The molecular interactions that allow NPC cells to evade immune recognition and
elimination, which is crucial for the survival of cells expressing potentially immunogenic viral
proteins, are also described. Finally, the potential utility of detecting host and viral factors for the
diagnosis and prognosis of NPC is discussed. Altogether, the studies summarized herein have
greatly expanded our knowledge of the molecular biology of NPC, yet much remains to be
uncovered. Emerging techniques for using and analyzing well-annotated biospecimens from
patients with NPC will ultimately lead to a greater level of understanding, and enable improve-
ments in precision therapies and clinical outcomes.
J Clin Oncol 33:3346-3355. 2015 by American Society of Clinical Oncology

INTRODUCTION EPIDEMIOLOGY AND ETIOLOGY

Nasopharyngeal carcinoma (NPC) is an epithelial
malignancy arising from the most superior por-
tion of the pharynx, extending from the upper
surface of the soft palate to the base of the skull.
This disease is characterized by a unique set of
geographic, etiologic, and biologic features dis-
tinct from other head and neck cancers. The mo-
lecular landscape of NPC is defined by an array of
familial and somatic genetic and epigenetic varia-
tions, which, in most NPCs, are intermingled with
latent Epstein-Barr virus (EBV) infection, result-
ing in a malignant phenotype. The events precip-
itating the development of NPC appear to occur
in a stepwise manner from normal nasopharyn-
geal epithelium to a malignant carcinoma.1 Given
that most NPCs are associated with EBV infec-
tion, EBV-negative NPC remains largely under-
represented in the current literature; as such, this
review will focus primarily on the molecular biol-
ogy of EBV-positive NPC. Specifically, we will
review the pertinent aberrancies within these ma-
lignant tissues, and describe how they affect cellu-
lar pathways in promoting the development and
progression of NPC.
The most common causal agent in NPC is EBV. The
EBV genome is present in approximately 90% of
NPCs observed in endemic regions, and is consid-
ered to play an important role in many aspects of
NPC development.1 Interestingly, human papillo-
mavirus infection has recently been identified in
EBV-negative NPCs in nonendemic regions; how-
ever, the true biologic implications of these observa-
tions remain to be fully characterized.2,3
Other environmental risk factors associated
with NPC have also been identified, such as alco-
hol consumption and tobacco smoking.4 Notably,
a stronger association between tobacco smoking
and NPC is observed in populations outside en-
demic regions than within high-risk populations.5
Concordantly, this occurs with differentiated ver-
sus undifferentiated or EBV-positive versus EBV-
negative NPC.
A variety of dietary habits have also been asso-
ciated with NPC development, such as consump-
tion of salt-preserved foods.4 The preservation
process is thought to increase levels of carcinogenic
nitrosamines, leading to an approximately two-fold
higher incidence of NPC in regions of frequent

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 Molecular Landscape of NPC
consumers. Additional environmental risk factors include alcohol
drinking, wood-fire smoke, occupational exposures, and some herbal
medicines.4 In contrast, high consumption of fresh fruits and vegeta-
bles has been shown to decrease an individuals risk of NPC,4 which
could be attributed to many components within these food products,
such as fiber, vitamins, folate, and carotenoids. Although EBV is likely
an early event in EBV-associated nasopharyngeal tumorigenesis, there
is also mounting evidence suggesting that preexisting genetic abnor-
malities (eg, loss of chromosomal regions on 3p and 9p) may be
required to support the persistent infection of premalignant or malig-
nant epithelial cells of the nasopharynx.6,7
MOLECULAR CONSEQUENCES OF EBV INFECTION
EBV is an enveloped, double-stranded human DNA herpesvirus asso-
ciated with several human malignancies, including Burkitts lym-
phoma, Hodgkin lymphoma, gastric carcinoma, and NPC. NPC cells
exhibit a type II EBV latency profile, expressing several protein-coding
(Epstein-Barr nuclear antigen 1 [EBNA1], Bam H1 reading frame 1,
and latent membrane proteins [LMPs] 1, 2A, and 2B) and non
protein-coding (Epstein-Barr encoded small RNAs [EBERs] 1 and 2
and Bam H1-A region rightward transcript [BART] microRNAs) EBV
genes with a variety of oncogenic properties.8
LMP1
Perhaps the most well-characterized EBV-encoded protein in the
context of NPC is LMP1, which acts, in part, as a viral mimic of the
tumor necrosis factor (TNF-) receptor family members TNF
receptor (TNFR) 1 and CD40.9 Although LMP1 shares minimal se-
quence homology with these receptors, it modulates several TNF-
responsive signaling proteins, including TNFR-associated factors
(TRAFs) and the TNFR-associated death domain protein.9 Unlike
TNFRs, however, LMP1 does not depend on ligand activation, but is
constitutively activated, consequently promoting tumor development
and progression via several signaling pathways9 (Fig 1 provides sum-
mary schema).
Early studies demonstrated that LMP1 transformed immortal-
ized rodent cells, and inhibited differentiation in human epithelial
cells.10 Subsequent studies showed that LMP1 is capable of promoting
all of the hallmarks of cancer.11 Specifically, LMP1 increased NPC cell
proliferation and survival by inducing expression of mitogenic recep-
tors, such as epidermal growth factor receptor and c-Met, as well as
activating progrowth and antiapoptotic mediators, such as survivin,
nuclear factor B (NF-B), phosphatidylinositol 3-kinase (PI3K)/
Akt, and mitogen-activated protein kinase (MAPK) pathways.9 Al-
though genetic aberrations of CDKN2A/p16 are common in NPC,
LMP1 can also decrease p16 expression by cytoplasmic sequestration
of E2F4/5 and Ets2 transcription factors.12 LMP1 also increased met-
astatic potential of NPC cells through induction of epithelial-to-
mesenchymal transition, as well as increased expression and release of
matrix metalloproteinases through activation of NF-B, specificity
protein 1, activator protein 1, and extracellular regulated kinase
(ERK)MAPK pathways.9 Evidence also indicates that LMP1 pro-
motes angiogenesis in the NPC microenvironment through the in-
duction of hypoxia-inducible factor 1 and vascular endothelial
growth factor.9 Notably, high LMP1 expression in primary NPC sam-
ples has been associated with regional and distant metastasis.13 Yet
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another activity of LMP1 is modulation of epigenetic marks on the
host genome.9 Specifically, LMP1 can induce the expression and ac-
tivity of DNA methyltransferases 1, 3a, and 3b, resulting in promoter
hypermethylation of tumor-suppressor genes, such as E-cadherin.14
Despite its many functions in NPC development and progres-
sion, LMP1 expression is heterogeneous within and among NP
tumors.15 Indeed, using current techniques, LMP1 expression is
undetectable in approximately 50% of NPC specimens.13 As such,
additional EBV-encoded genes must function in concert with
LMP1 to promote NPC oncogenesis through overlapping and
independent mechanisms.
LMP2A/B
In contrast to LMP1, LMP2A is consistently expressed in almost
all NPCs, whereas LMP2B mirrors LMP1, with expression observed in
approximately 40% of samples.15 Although LMP2A and 2B do not
appear to transform normal cells alone, they do regulate several onco-
genic pathways in NPC cells.16
LMP2A-transfected epithelial cells exhibit increased prolifera-
tion, epithelial-mesenchymal transition, invasion, and migration
through the activation of several signaling cascades, including Notch,
ERK, Syk, PI3K/Akt, and Wnt/-catenin.9 LMP2A has also negatively
regulated the B-cell receptor in epithelial cells, resulting in inhibition
of several B-cell receptorregulated processes, including intracellular
calcium release, phospho-tyrosine signaling, and the switch from la-
tent to lytic EBV replication.17 Although slightly less well understood,
LMP2B also activated PI3K/Akt signaling, and promoted cell motility
through disruption of cellular adhesion.18 Finally, both LMP2A and
LMP2B imparted resistance to antiviral interferon (IFN) signaling in
NPC cells through the increased turnover and degradation of IFN-
responsive receptors, IFN-/ receptor (IFNAR) and IFN- receptor
(IFNGR).19
EBNA1
EBNA1 is the only EBV-encoded protein expressed in all EBV-
associated tumors, whose expression is both necessary and sufficient
for the stable maintenance of EBV episomes in dividing cells through
its interactions with EBV oriP (origin of plasmid replication).8 EBNA1
is also capable of inducing expression of other EBV-encoded genes
through transcriptional activation of the Cp and LMP promoters.
Aside from its role in the maintenance of the EBV genome and mod-
ulation of other EBV genes, EBNA1 also directly promotes oncogen-
esis in that its knockdown via RNA interference reduced proliferation
and survival in an NPC-derived cell line. In turn, its ectopic expression
promoted tumor growth and metastasis in NPC xenografts. In fact, a
variety of EBNA1-host interactions result in modulating numerous
cellular pathways, and altering cellular homeostasis.8 Interestingly,
EBNA1 has induced reactive oxygen species in NPC cells, possibly
through the expression of the reactive oxygen speciesproducing
nicotinamide-adenine dinucleotide phosphate, reduced form, oxi-
dase 2, which has been demonstrated to cause DNA damage in B
cells20; if extended to NPC, this could presumably result in additional
oncogenic mutations. EBNA1 also directly modulated a myriad of
signaling cascades, including tumor growth factor (TGF-), signal
transducer and activator of transcription 1, and NF-B, leading to
increased growth, survival, metastasis, and angiogenesis.8
2015 by American Society of Clinical Oncology 3347
 Bruce et al

A Activation, gain, or upregulation Deactivation, loss, or downregulation EBV proteins or miRNAs

WNT WIF1 Methylation

LMP2A/B CMET EGFR LMP1 FZD

Copy
number
TRAFs Copy RASSF1A loss,
number methylation,
loss, mutation
P16
methylation,
Copy mutation
number
PI3K gain,
mutation
EBNA1
Copy
AKT Cyclin D1 number
gain

APC
RAS/MAPK P27 GSK-3 CDK4/6 STAT1 TGF CDC20

Proliferation/Cell Cycle Progression

Activation, gain, or upregulation Deactivation, loss, or downregulation EBV proteins or miRNAs

B LMP2A/B LMP1 LTBR

Copy number gain
EBNA1

EBV BART
TRAFs Copy number loss, miRNAs
hypermethylation, USP7
mutation
NFB
Copy P14
number
RAS/MAPK gain, PI3K
mutation
MDM2
miR-218
Pro-apoptotic Anti-apoptotic AKT Hypermethylation
Pro-apoptotic
CASP8/9 MCL1 P53
BIM BCL-XL PUMA
BAD BIM
HID BAD FOXO SURVIVIN hTERT TOMM22

BCL2

Inhibition of Apoptosis/Promotion of Survival

Fig 1. Alterations of cellular pathways. (A) Proliferation/cell cycle progression. Epstein-Barr virus (EBV) latent membrane protein (LMP) 1 can promote cellular proliferation and
progression of the cell cycle via multiple mechanisms, such as mimicking tumor necrosis factor (TNF-) receptor family members, subsequently activating TNF receptor (TNFR)
associated factors (TRAFs). LMP1-TRAF activation induces phosphatidylinositol 3-kinase (PI3K) mediated phosphorylation and activation of Akt, which then inactivates and/or
downregulates the cyclin-dependent kinase inhibitor p27. In addition, LMP1-TRAF can activate mitogen-activated protein kinases (MAPKs), which can also inhibit p27. Moreover, LMP1
can induce the expression of mitogenic receptors epidermal growth factor receptor (EGFR) and c-Met. EBV-encoded LMP2A/B proteins also activate PI3K and MAPKs to promote
growth. Epstein-Barr nuclear antigen 1 (EBNA1) directly activates several proliferation signals, including signal transducer and activator of transcription 1 (STAT1) and tumor growth
factor (TGF-). CDKN2A (p16), a cyclin-dependent kinase inhibitor that inhibits CDK4/CDK6, is inactivated in nasopharyngeal carcinoma (NPC) via multiple mechanisms leading to cell
cycle progression. LMP1 can also decrease p16 expression by causing the cytoplasmic sequestration of E2F4/5 and Ets2 transcription factors. Further promoting CDK4/6 activation
and progression past the G1/S checkpoint in NPC is the cyclin D1 (CCND1), which is commonly amplified in NPC. Ras association domain family member 1 (RASSF1) is frequently
deleted, methylated, or mutated in NPC. RASSF1 downregulation can lead to overexpression of members of the TGF- signaling pathway. Suppression of RASSF1 can also promote
mitotic progression through the loss of direct inhibition of the anaphase-promoting complex (APC) cell-division cycle protein 20 complex (CDC20), which functions to promote
microtubule stability. Wnt pathway aberrations are also common in NPC: Wnt is commonly overexpressed and the Wnt inhibitor, WIF1, is commonly underexpressed, in part via
promoter hypermethylation; this possibly leads to increased activation of the canonical Wnt pathway via frizzled (FZD) receptors. Furthermore, EBV proteins LMP1 and LMP2A activate
AKT to phosphorylate and inactivate glycogen synthase kinase 3, which results in nuclear accumulation of the oncogenic -catenin. (B) Antiapoptosis/prosurvival. EBV-encoded
LMP1/LMP2A activation of AKT can lead to Forkhead Box subclass O (FOXO) inhibition, thereby preventing the transcription of several proapoptotic genes (eg, FasL, Bim, Bcl6,
TNFR-associated death domain protein, and TNF-related apoptosis-inducing ligand). In addition, AKT phosphorylates and inactivates Bad, consequently enabling Bcl-2 to inhibit
apoptosis. LMP1 may directly or indirectly upregulate Bcl-2, possibly via nuclear factor B (NF-B). LMP1s activation of the NF-B pathway can also promote the translocation of
telomerase (human telomerase catalytic subunit) to the nucleus, via direct binding with NF-B p65. EBV Bam H1-A region rightward transcript (BART) microRNAs induce the
degradation of several proapoptotic targets, including proapoptotic effector p53 upregulated modulator of apoptosis (PUMA), Bim, and translocase of outer mitochondrial membrane
22 homolog (TOMM22). Activation of the RAS/MAPK pathway by LMP1 and LMP2A/B can also activate several antiapoptotic proteins (eg, Mcl-1 and Bcl-xL) and inhibit proapoptotic
proteins (eg, caspase-8/-9, Bim, Bad, and Hid). LMP1 can also upregulate survivin (via p53), which functions to inhibit caspases and regulate mitotic spindles. Furthermore, aberrant
methylation and subsequent downregulation of miR-218 in NPC can lead to upregulation of survivin. Although most NPCs contain wild-type p53, it is functionally disrupted by
inactivation of p14ARF, which enables MDM2 to ubiquitinate p53, signaling it for degradation. Moreover, EBNA1 interacts with the ubiquitin-specific protease (USP) 7, which
deubiquitinates and subsequently stabilizes p53. Thus, inhibition of USP7 by EBNA1 prevents the deubiquitination of p53, resulting in decreased p53 accumulation in response to DNA
damage, resulting in reduced apoptosis.

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 Molecular Landscape of NPC

EBNA1 also reduces p53 levels by interacting with the ubiquitin-
specific protease USP7, to prevent the deubiquitination and conse-
quent stabilization of p53. This relationship consequently reduces p53
accumulation in response to DNA damage, thereby decreasing apo-
ptosis and increasing radioresistance in vitro.8 In addition, EBNA1 has
been shown in NPC cell lines to disrupt promyelocytic leukemia
nuclear bodies, which are also involved in p53 activation, apoptosis,
and DNA repair.21 Hence, EBNA1 clearly mediates a complexity of
cellular processes in nasopharyngeal oncogenesis.
EBERs
The EBV-encoded genes EBER1 and EBER2 encode small RNAs
(167 and 172 nucleotides long, respectively) that are highly expressed
in NPC cells, with up to 1 million copies detected per cell.22 Evidence
suggests that EBERs can function within NPC cells to promote growth
and survival and modulate host immunity through a variety of mech-
anisms. Studies in the EBV-positive NPC cell line C666-1 revealed that
EBERs promote cell growth through an autocrine mechanism by
inducing the expression and release of insulin-like growth factor-1
(IGF-1), possibly through activation of the IGF-1 promoter. More-
over, IGF-1 expression was frequently detected in NPC biopsy speci-
mens, indicating that the EBER/IGF-1 axis may contribute to the
development of NPC in vivo. In addition, preliminary data suggest
that this induction of IGF-1 occurs through the interaction of EBERs
with the double-stranded RNA sensing toll-like receptor 3.22
EBV-Encoded microRNAs
To date, 25 EBV-encoded precursor microRNAs processed into
44 mature microRNA sequences have been verified (miRBase [www.
mirbase.org], Release 20). They emanate from two major regions of
the EBV genome: the BARTs and the open reading frame of the
BHRF1 gene. Originally cloned from EBV-infected Burkitts lym-
phoma cells,23 BHRF1-microRNAs do not appear to be expressed in
EBV-positive primary NPC tissues.24 Several targets of EBV microR-
NAs have been identified, both viral and host. Viral targets include
LMP1, putatively targeted by several microRNAs from the BART
region (ebv-miR-BARTs 1, 9, 16, and 17)25; and LMP2A, identified as
a target of ebv-miR-BART22.26 Subsequent functional experiments
indicated that BART-microRNA modulation of these virally encoded
proteins influences multiple cellular properties, including prolifera-
tion, survival, and evasion of host immune response.25,26 Host targets
of BART-microRNAs include proapoptotic effectors p53 upregulated
modulator of apoptosis,27 Bcl-2 interacting mediator of cell death,28
and translocase of outer mitochondrial membrane 22 homolog,29 as
well as several genes thought to influence host immune response,
including major histocompatibility complex class Irelated chain B,30
importin 7,29 and Dicer.31 Overall, EBV-encoded microRNAs play a
complementary role to the viral proteins expressed in NPC: promot-
ing survival and proliferation of NPC cells and contributing to evasion
of the host immune response.
GENOMIC ALTERATIONS IN NPC
Most genomic studies on NPC have focused on targeted sequencing
analysis of candidate genes or cytogenetic techniques, such as
G-banding, comparative genomic hybridization, and array compara-
tive genomic hybridization, for gross chromosomal analyses to detect
copy number events and translocations. In August 2014, a landmark
study described the genomic analysis of 128 NPC samples using
whole-exome sequencing, targeted deep sequencing, and single-
nucleotide polymorphism arrays.32 This report plus previous studies
have identified several key genomic alterations that promote NPC
development and progression via a variety of mechanisms (Table 1).
Copy Number Alterations
Important examples of recurrent chromosomal events in NPC
include focal losses on chromosomes 3p and 9p, plus 11q, 13q, 14q,

Table 1. Genes Frequently Altered by Genetic and Epigenetic Mechanisms

Gene Symbol Chromosomal Location Molecular Alteration References

Tumor-Suppressor Genes

ARID1A 1p36 SNV/indel and CNV Lin et al32

CDKN2A/B 9p21 SNV/indel, CNV, and methylation Lin et al,32 Lo et al,33 Kwong et al34
TP53 17p13 SNV/indel and CNV Lin et al,32 Spruck et al,35 Sun et al,36 Effert et al37
RASSF1 3p21 SNV/indel, CNV, and methylation Lo et al33,38,39
SYNE1 6q25 SNV/indel Lin et al32
THY1 11q23 CNV and methylation Hui et al,40 Lung et al41
CADM1 11q23 CNV and methylation Hui et al,40,42 Lung et al43
DLC1 8p22 Methylation Seng et al44
PTPRG 3p14 Methylation Guan et al45
SOX1 13q34 Methylation Li et al46
DAB2 5p13 Methylation Tong et al47
Oncogenes

CCND1 11q13 CNV Lin et al,32 Lo et al,48 Hui et al49

PIK3CA 3q26 SNV/indel and CNV Lin et al,32 Hui et al,50 Or et al,51 Jiang et al,52 Zhang et al53
LTBR 12p13 CNV Or et al54

NOTE. Genes herein have been limited to those that are either altered through SNV/indel in 5% of nasopharyngeal carcinoma (NPC) samples studied, or
frequently altered by copy number variant (CNV) or epigenetic mechanisms and demonstrated to play a functional role through experimentation in NPC models.
Abbreviations: indel, small insertion and deletions; SNV, single-nucleotide variant.

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 Bruce et al
and 16q. These have been identified and confirmed in multiple stud-
ies40,48,50,55; however, many of the specific tumor-suppressor targets in
these regions remain to be identified. One important region is the
9p21.3 cytoband, originally observed to exhibit copy number losses in
most (61%), and a homozygous deletion in one of the evaluated
primary NPC samples.55 Contained within this region are two genes
that encode three tumor suppressors: p16-INK4a, ARF (gene
CDKN2A), and p15-INK4b (gene CDKN2B). Hypermethylation of
the p16 promoter was also frequently detected in NPC samples, indi-
cating that this was the target of copy number loss in this region.33,34 In
subsequent studies, the functional implication of p16 loss in NPC was
examined, demonstrating that p16 reexpression led to G0/G1 arrest
along with reduced in vivo tumorigenicity, most likely mediated via its
canonical role as an inhibitor of interaction between cdk4/cdk6 and
cyclin D1 (CCND1).56 Moreover, p16 deletion was a critical event in
human telomerase catalytic subunit immortalization of normal naso-
pharyngeal epithelial cells,57 further providing support for p16 inacti-
vation as an early event in NPC development.56
Similar to p16, Ras association domain family member 1
(RASSF1) was identified as an important tumor suppressor in NPC
because of its frequent deletion at 3p21.3, its promoter methylation,
and somatic mutation in a few NPC samples (9.5%).38,39,48 Since its
initial cloning in 2000,58 multiple putative functions have been as-
cribed to RASSF1, although its precise role remains controversial.59 In
the few studies focused on NPC, stable reexpression of RASSF1A in
C666-1 cells resulted in growth inhibition both in vitro and in vivo
mediated through TGF- signaling (activin E and Id2).60 RASSF1A
also plays an important role in regulating mitotic progression through
its direct inhibition of the anaphase-promoting complex cell-
division cycle protein 20 complex, which functions to promote micro-
tubule stability.61 Hence, when RASSF1A expression was suppressed
in immortalized nasopharyngeal epithelial cells, microtubule struc-
tures were disrupted. Chromosomal instability ensued, leading to
enhanced tumorigenicity.62
Several oncogenes are also activated in NPC via copy number
gains. Frequent amplification of 11q13 has been observed in multiple
cytogenetic studies, with CCND1 identified as the likely target.48,49
Moreover, CCND1 was observed to be overexpressed in more than
90% of primary NPC samples by immunohistochemical staining, and
its knockdown in NPC cell lines significantly decreased prolifera-
tion.49 Similarly, recurrent amplification of 12p13 was determined to
target lymphotoxin- receptor, overexpression of which was con-
firmed to contribute to cell survival and tumor growth in NPC models
via activation of NF-B.54 Likewise, an increase in copy number of
phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit al-
pha (PIK3CA) at 3q26.1 has been observed in 70% to 75% of primary
NPC samples,50,51 and immunohistochemical evidence indicated that
the PI3K/Akt pathway was activated in approximately 85% of NPC
samples.63 Hence, given that PIK3CA point mutations are rarely de-
tected in NPC, amplification is most likely the major mechanism of
constitutive activation of this pathway.51
Translocations
Recently, several fusion transcripts, resulting from genomic
translocations, have been identified in NPC. By using paired-end
RNA-sequencing data from C666-1 cells, a fusion was first detected
between ubiquitin protein ligase E3 component n-recognin 5 and zinc
finger protein 423, which was subsequently corroborated in 12 (8.3%)
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of 144 primary NPC samples interrogated using reverse transcriptase
polymerase chain reaction.64 Functional analysis demonstrated that
the resultant chimeric protein promoted proliferation and colony-
forming ability in vitro, as well as tumor formation in vivo.64 A second
study identified and validated three in-frame gene fusions (YAP1-
MAML2, PTPLB-RSRC1, and SP3-PTK2) in two NPC samples using
high-throughput sequencing of circularized DNA fragments and a
novel computational algorithm.65 Subsequent investigation using flu-
orescent in situ hybridization analysis of 196 NPC samples observed
that structural rearrangements in the three involved genes were de-
tected at low frequencies (MAML2 in 3, PTK2 in 6, and SP3 in 2 of 196
cases), although none of the originally identified partners were simi-
larly fused in the validation set.65
Small Insertions/Deletions and
Single-Nucleotide Variants
Before the recent report on whole-exome sequencing of NPC,32
the detection of single-nucleotide variants (SNVs) and small insertion
and deletions (Indels) in NPC had been limited to targeted analysis of
specific tumor suppressors or oncogenes. These analyses indicated
that the frequency of SNVs is low in NPC (1 to 342 coding mutations/
sample), even when compared with other virally associated head and
neck tumors, such as human papillomaviruspositive head and neck
squamous cell or EBV-positive gastric adenocarcinomas (Fig 266,67).
Nonetheless, several key genes and pathways do appear to be targeted
by small genomic alterations, often overlapping with those that are
also frequently amplified or deleted at the copy number level, or
epigenetically altered.
TP53, the most widely mutated gene in human cancers, is rarely
mutated in NPC (0% to 8%); however, this proportion increases
(n = 56) (n = 35) (n = 21) (n = 200) (n = 244)
Somatic Mutation Frequency (/Mb)
100
50
10
5
1
0.5
0.1
0.05
EBV+ Gastric
Adenocarcinoma
EBV- Gastric
Adenocarcinoma
HPV+ HNSCC
EBV+ NPC
HPV- HNSCC
Fig 2. Somatic coding mutation frequency (from exome sequencing, normal-
ized to covered area) in nasopharyngeal carcinoma (NPC), compared with other
relevant or virally driven solid malignancies. NPC data were generated from Lin et
al32; head and neck squamous cell carcinoma (HNSCC) and gastric adenoma data
were obtained from The Cancer Genome Atlas.66,67 EBV, Epstein-Barr virus;
HPV, human papilloma virus.
JOURNAL OF CLINICAL ONCOLOGY
 Molecular Landscape of NPC
to approximately 20% when somatic copy number loss is
included.32,35-37 Similarly, although the major mechanisms of RASSF1
deactivation are copy number loss or promoter hypermethylation,
mutations have been also detected in approximately 10% of NPC
samples.38 Activating mutations and amplification of members of the
oncogenic PI3K and receptor tyrosine kinase pathways have also been
identified.32,68,69 Interestingly, Lin et al32 reported frequent alterations
in genes involved in chromatin modification pathways (ARID1A,
BAP1, KMT2D/3, TSHZ3, and TET1/2/3), whereby patients harbor-
ing such mutations experienced significantly inferior overall survival
than their wild-type counterparts. Other pathways that appear to be
targeted by SNVs/Indels in NPC include the NF-B,68 cell cycle, ad-
hesion, differentiation, and autophagy pathways.32
Epigenetic Alterations
As discussed above, EBV infection, environmental factors, and
genetic mutations targeting cellular modulators of epigenetics appear
to play integral roles in the modulation of epigenetic marks in NPC.
These precipitating events can lead to a variety of functional outcomes,
including DNA methylation, and to a lesser extent, histone modifica-
tions, ultimately leading to alterations in gene expression.
Both RASSF1 and CDKN2A are tumor-suppressor genes whose
promoters are frequently methylated in NPC, in addition to being
targets of copy loss and SNV/Indels.32,34,37-39,48 The promoter region
of Wnt inhibitory factor 1 has also been reported to be frequently
hypermethylated in NPC (85% to 90%).70 Furthermore, in vitro stud-
ies demonstrated that this hypermethylation leads to downregulation
of Wnt inhibitory factor 1 expression with subsequent increase in
clonogenic survival.70 Cell adhesion molecule 1 (aka, TSLC1) and
Thy-1 cell surface antigen, both located at 11q23, have also been
hypermethylated in NPC.42,43 Interestingly, expression of both Thy-1
cell surface antigen and cell adhesion molecule 1 was more frequently
lost in lymph-node metastases than primary NPCs.42,43
Other genes whose promoters are frequently hypermethylated in
NPC mediate a variety of pathways, including cell invasion/migration
(CDH1, ZMYND10, and miR-148a), response to DNA damage
(GADD45), apoptosis (DAPK1, UCHL1, and miR-218), cell cycle/
proliferation (DLC1, HIN1, PTPRG, and RARB), MAPK/ERK
(RASAL and DAB2), and Wnt/-catenin (SOX1).34,44-47,71
Aberrancies in other epigenetic mechanisms, such as his-
tone modification, have also been noted in NPC. EZH2, a
histone-lysine N-methyltransferase, has been demonstrated to
play an important role in NPC by several groups.72-74 Altera-
tions in histone methylation resulting from EZH2 upregulation
effect a myriad of phenotypes in NPC cells, including increased
migration/invasion, antiapoptosis/cell survival, and angiogen-
esis. Finally, DNA methylation and modification of histones
within the EBV genome itself also play important roles by
restricting the expression of EBV genes associated with the type
II latency expression program.75 Evidence suggests that meth-
ylation of the Wp and Cp promoters leads to suppression of
EBNA2-6, whereas other key promoters remain unmethylated
(Qp, LMP, EBER, and Bam HI-A transcript promoters).75 These
collective observations raise the intriguing possibilities regard-
ing the potential targeting of such alterations or pathways, using
RTK inhibitors or epigenetic (histone deacetylase) modulators
in NPC treatment.
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Copyright 2017 American Society of Clinical Oncology. All rights reserved.
IMMUNE EVASION IN NPC
Evasion of host immune response is a recognized hallmark of cancer.11
Immunologic recognition of nonself-antigens provides a strong se-
lective pressure during tumorigenesis.76 Consequently, cancer cells
have developed strategies that allow for their continued survival. This
phenomenon is particularly important in EBV-associated NPC, with
both viral and host factors contributing to the survival of NPC cells
during tumor initiation and progression.
Viral Factors Affecting Immune Response
Although EBV infection is associated with robust immune infil-
trates, viral factors repress the activity of antitumor lymphocytes,
permissive for NPC cell survival.77 Several EBV gene products directly
interfere with the antigen-processing machinery and immune recog-
nition. Surface expression of major histocompatibility complex mol-
ecules and loading of viral peptide antigens are reduced by viral
proteins expressed during lytic replication, including BGLF5, BILF1,
and BNLF2a78 (Fig 3). The interleukin-10 homolog BamHI-C frag-
ment rightward reading frame 1 (BCRF1) can also affect antigen
presentation through its direct inhibitory effects on T cells.79 During
latency, the expression of most gene products is suppressed; EBNA1
and LMP2A/B are the most ubiquitously expressed proteins during
latency, yet do not elicit strong immune responses.80 Consequently,
tumor-infiltrating lymphocytes are often incapable of effectively elim-
inating EBV-associated NPC cells. Moreover, a recent report sug-
gested that exosomes released from EBV-positive NPC cells can
recruit regulatory T cells and enhance their inhibitory activity, thereby
providing another mechanism by which NPC evades the host im-
mune response.81
Host Factors Affecting Immune Response
Recognition and elimination of EBV-associated NPC cells by
infiltrating lymphocytes depend on effective antigen presentation.
The affinity of different HLA allele products for EBV antigens can
influence tumor immunoediting (reviewed by Hildesheim and
Wang82). Certain HLA haplotypes are associated with reduced risk for
developing NPC. For example, the HLA-A*0201 allele is underrepre-
sented among patients with NPC in certain populations, possibly a
result of efficient binding and presentation to T cells of conserved
epitopes from EBV proteins.83 In East Asia, an elevated risk for devel-
oping NPC has been attributed to the HLA-A*0207 allele84 (Fig 3).
Aside from the HLA genes, additional risk loci have been uncovered by
genome-wide association studies,82,84 suggesting that other compo-
nents might contribute to differential immune recognition of EBV
antigens. With the recent emergence of novel immune-modulatory
therapies,85 such approaches definitely warrant examination in EBV-
associated NPC.
MOLECULAR BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS
Several different types of diagnostic and prognostic molecular bio-
markers for NPC have been investigated, including both blood- and
tissue-derived factors, that promise to have profound impact on the
individualized management of patients with this disease.
2015 by American Society of Clinical Oncology 3351
 Bruce et al

biomarkers, however, must be performed before any clinical im-

plementation can be contemplated.
LMP1 NPC Cell
BARF1
Circulating EBV DNA
Latency Cancer cells release DNA into the circulation, and levels of

A*0207
Phase LMP2
circulating tumor DNA reflect disease burden.96 EBV DNA is
EBNA1
detectable in plasma and serum from patients with NPC,97 which
exists as short fragments, likely reflecting apoptotic processes, as
opposed to intact virions.98 The DNA is cleared rapidly following
A*0207 surgery, radiation, or chemotherapy.99,100 Although the mecha-
nisms of clearance remain undefined, the half-life of elimination
may be linked with clinical outcome. In the pretreatment setting,
TCR

plasma EBV DNA has been proposed as a diagnostic and prognos-

tic tool for NPC,101-103 as well as a screening test in certain high-risk
populations.104 Moreover, detectable post-treatment EBV DNA
CD8+ T Cell levels identify patients with recurrent or residual disease101,105 and
may predict clinical benefit from adjuvant chemotherapy after
concurrent chemoradiotherapy.106 Robust implementation of
plasma EBV DNA detection within clinical trials will benefit from
TCR

harmonized laboratory methods.107

BCRF1
A*0201

EBV Serology
Antibodies reactive to EBV epitopes have been observed to be
significantly elevated in patients with NPC compared with healthy
BILF1 BNLF2a
controls in numerous studies.97,101,108 Anti-EBV immunoglobulin A
and/or G levels have been proposed to precede clinical presentation of
NPC and may therefore be useful as a screening or diagnostic
A*0201

Lytic
BGLF5 BCRF1
Phase test.109,110 Persistent elevation of anti-EBV antibodies after radiother-
NPC apy is a poor prognostic indicator, but its predictive power is super-
Precursor Cell seded by plasma EBV DNA.111

Other Circulating Biomarkers

Fig 3. Immune evasion in nasopharyngeal carcinoma (NPC). In the pathogen- Several EBV-encoded factors have been identified in the circula-
esis of NPC, Epstein-Barr virus (EBV) containing cells use multiple mechanisms
for immune evasion. During latency, minimally immunogenic viral proteins
tion of patients with NPC, including LMP1 and Bam H1 reading
(Epstein-Barr nuclear antigen 1 [EBNA1], Bam H1 reading frame 1 [BARF1], and frame 1112 proteins, as well as noncoding RNA molecules EBER1,
latent membrane proteins [LMPs] 1, 2A, and 2B) are expressed, leading to EBER2, and BART microRNAs.113,114 Human nucleic acid, protein,
reduced cell surface presentation of viral peptides (red triangles) by major
histocompatibility complex (MHC; gray boxes). Host factors, including HLA
and metabolite biomarkers have also been proposed for diagnostic
haplotype, can affect the affinity of viral peptide binding: the HLA-A*0207 allele and prognostic use in patients with NPC. These include human
predisposes individuals to developing NPC, whereas the HLA-A*0201 allele is microRNAs,115 hypermethylated DNA fragments from promoter re-
protective. In the lytic phase of replication, seen during acute EBV infection
within NPC precursor cells in the nasopharyngeal epithelium, secreted factors
gions of several known tumor-suppressor genes,116 and circulating
(eg, the viral interleukin-10 homolog, BamHI-C fragment rightward reading cytokines, including TGF-117 or IFN-.77 More studies will clearly be
frame 1 (BCRF1)) inhibit CD8 cytotoxic T-cell activity, whereas other viral necessary before these potential biomarkers can be incorporated into
proteins interfere with MHC expression on the cell surface and loading of viral
antigens (BamHI I leftward reading frame 1 [BILF1], BamHI N leftward reading clinical practice.
frame 2a [BNLF2a], and BamHI G leftward frame 5 [BGLF5]). TCR, T-cell
receptor.
CONCLUSION AND FUTURE DIRECTIONS

Tissue-Based Tumor Biomarkers
Within NP tumor tissue and the surrounding stroma, several
factors have the potential to serve as indicators of clinical outcome.
For example, tumor expression of caspase-3, survivin,86 c-Met,87
BUB1b, CENPF,88 ERBB3,89 MTDH,90 and FADD91 may each have
prognostic significance. In addition, two tissue-based microRNA
expression signatures have been identified, which are associated
with survival and distant metastasis in NPC.92,93 Within the
stroma, increased expression of periostin and decreased number of
tumor-infiltrating lymphocytes have both been linked with poor
survival.94,95 Additional independent validation of each of these
NPC biology is a complex interplay between cellular, viral, stromal,
and host components. Although much has been learned regarding
the development and progression of NPC, more remains to be un-
raveled. The emergence in recent years of high-throughput
molecular characterization methods (eg, massively parallel se-
quencing and single-nucleotide polymorphism/methylation
microarrays) has resulted in significant inroads into our under-
standing of the genomic underpinnings of cancer biology. To date,
however, there has been only one report of comprehensive se-
quencing analysis of a modest number (n 56) of NPC samples.32
One reason for the limited study of NPC using high-throughput

3352 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

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Copyright 2017 American Society of Clinical Oncology. All rights reserved.
 Molecular Landscape of NPC

genomic technologies is a lack of sizeable fresh/frozen specimens existing targeted therapies to improve outcome for future patients
because of the limited surgical role in the management of NPC. with NPC.
Consequently, most tissue specimens are small formalin-fixed
paraffin-embedded biopsy samples. However, significant advances
are being achieved to reduce both the quantity and quality of DNA, AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
RNA, and protein necessary for many high-throughput epig- OF INTEREST
enomic, genomic, transcriptomic, and proteomic technologies,
rendering thousands of archival tissue specimens useful for inter- Disclosures provided by the authors are available with this article at
www.jco.org.
rogation. Further investigation of additional NPC specimens using
techniques such as whole-exome, whole-genome, bisulfite, and
RNA sequencing will further broaden our understanding of the
AUTHOR CONTRIBUTIONS
molecular characteristics of NPC, particularly when linked to clin-
ical outcome databases. Such studies will provide insight into less
Manuscript writing: All authors
frequently targeted genes, improve the power with which key Final approval of manuscript: All authors
targets altered by copy number changes can be resolved, and iden- Conception and design: All authors
tify novel mechanisms promoting nasopharyngeal oncogenesis. Collection and assembly of data: All authors
Ultimately, these insights will lead to evaluations of novel and Data analysis and interpretation: All authors

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2015 by American Society of Clinical Oncology 3355
 Bruce et al

AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Nasopharyngeal Cancer: Molecular Landscape

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are
self-held unless noted. I Immediate Family Member, Inst My Institution. Relationships may not relate to the subject matter of this manuscript. For more
information about ASCOs conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.
Jeff P. Bruce Emma Ito
No relationship to disclose No relationship to disclose
Kenneth Yip Fei-Fei Liu
No relationship to disclose No relationship to disclose
Scott V. Bratman
Patents, Royalties, Other Intellectual Property: Patent licensed to Capp
Medical

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Copyright 2017 American Society of Clinical Oncology. All rights reserved.