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

INTRODUCTION EPIDEMIOLOGY AND ETIOLOGY

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

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Molecular Landscape of NPC

consumers. Additional environmental risk factors include alcohol another activity of LMP1 is modulation of epigenetic marks on the
drinking, wood-fire smoke, occupational exposures, and some herbal host genome.9 Specifically, LMP1 can induce the expression and ac-
medicines.4 In contrast, high consumption of fresh fruits and vegeta- tivity of DNA methyltransferases 1, 3a, and 3b, resulting in promoter
bles has been shown to decrease an individuals risk of NPC,4 which hypermethylation of tumor-suppressor genes, such as E-cadherin.14
could be attributed to many components within these food products, Despite its many functions in NPC development and progres-
such as fiber, vitamins, folate, and carotenoids. Although EBV is likely sion, LMP1 expression is heterogeneous within and among NP
an early event in EBV-associated nasopharyngeal tumorigenesis, there tumors.15 Indeed, using current techniques, LMP1 expression is
is also mounting evidence suggesting that preexisting genetic abnor- undetectable in approximately 50% of NPC specimens.13 As such,
malities (eg, loss of chromosomal regions on 3p and 9p) may be additional EBV-encoded genes must function in concert with
required to support the persistent infection of premalignant or malig- LMP1 to promote NPC oncogenesis through overlapping and
nant epithelial cells of the nasopharynx.6,7 independent mechanisms.

MOLECULAR CONSEQUENCES OF EBV INFECTION LMP2A/B


In contrast to LMP1, LMP2A is consistently expressed in almost
EBV is an enveloped, double-stranded human DNA herpesvirus asso- all NPCs, whereas LMP2B mirrors LMP1, with expression observed in
ciated with several human malignancies, including Burkitts lym- approximately 40% of samples.15 Although LMP2A and 2B do not
phoma, Hodgkin lymphoma, gastric carcinoma, and NPC. NPC cells appear to transform normal cells alone, they do regulate several onco-
exhibit a type II EBV latency profile, expressing several protein-coding genic pathways in NPC cells.16
(Epstein-Barr nuclear antigen 1 [EBNA1], Bam H1 reading frame 1, LMP2A-transfected epithelial cells exhibit increased prolifera-
and latent membrane proteins [LMPs] 1, 2A, and 2B) and non tion, epithelial-mesenchymal transition, invasion, and migration
protein-coding (Epstein-Barr encoded small RNAs [EBERs] 1 and 2 through the activation of several signaling cascades, including Notch,
and Bam H1-A region rightward transcript [BART] microRNAs) EBV ERK, Syk, PI3K/Akt, and Wnt/-catenin.9 LMP2A has also negatively
genes with a variety of oncogenic properties.8 regulated the B-cell receptor in epithelial cells, resulting in inhibition
of several B-cell receptorregulated processes, including intracellular
LMP1 calcium release, phospho-tyrosine signaling, and the switch from la-
Perhaps the most well-characterized EBV-encoded protein in the tent to lytic EBV replication.17 Although slightly less well understood,
context of NPC is LMP1, which acts, in part, as a viral mimic of the LMP2B also activated PI3K/Akt signaling, and promoted cell motility
tumor necrosis factor (TNF-) receptor family members TNF through disruption of cellular adhesion.18 Finally, both LMP2A and
receptor (TNFR) 1 and CD40.9 Although LMP1 shares minimal se- LMP2B imparted resistance to antiviral interferon (IFN) signaling in
quence homology with these receptors, it modulates several TNF- NPC cells through the increased turnover and degradation of IFN-
responsive signaling proteins, including TNFR-associated factors responsive receptors, IFN-/ receptor (IFNAR) and IFN- receptor
(TRAFs) and the TNFR-associated death domain protein.9 Unlike (IFNGR).19
TNFRs, however, LMP1 does not depend on ligand activation, but is
constitutively activated, consequently promoting tumor development
EBNA1
and progression via several signaling pathways9 (Fig 1 provides sum-
EBNA1 is the only EBV-encoded protein expressed in all EBV-
mary schema).
associated tumors, whose expression is both necessary and sufficient
Early studies demonstrated that LMP1 transformed immortal-
for the stable maintenance of EBV episomes in dividing cells through
ized rodent cells, and inhibited differentiation in human epithelial
its interactions with EBV oriP (origin of plasmid replication).8 EBNA1
cells.10 Subsequent studies showed that LMP1 is capable of promoting
is also capable of inducing expression of other EBV-encoded genes
all of the hallmarks of cancer.11 Specifically, LMP1 increased NPC cell
proliferation and survival by inducing expression of mitogenic recep- through transcriptional activation of the Cp and LMP promoters.
tors, such as epidermal growth factor receptor and c-Met, as well as Aside from its role in the maintenance of the EBV genome and mod-
activating progrowth and antiapoptotic mediators, such as survivin, ulation of other EBV genes, EBNA1 also directly promotes oncogen-
nuclear factor B (NF-B), phosphatidylinositol 3-kinase (PI3K)/ esis in that its knockdown via RNA interference reduced proliferation
Akt, and mitogen-activated protein kinase (MAPK) pathways.9 Al- and survival in an NPC-derived cell line. In turn, its ectopic expression
though genetic aberrations of CDKN2A/p16 are common in NPC, promoted tumor growth and metastasis in NPC xenografts. In fact, a
LMP1 can also decrease p16 expression by cytoplasmic sequestration variety of EBNA1-host interactions result in modulating numerous
of E2F4/5 and Ets2 transcription factors.12 LMP1 also increased met- cellular pathways, and altering cellular homeostasis.8 Interestingly,
astatic potential of NPC cells through induction of epithelial-to- EBNA1 has induced reactive oxygen species in NPC cells, possibly
mesenchymal transition, as well as increased expression and release of through the expression of the reactive oxygen speciesproducing
matrix metalloproteinases through activation of NF-B, specificity nicotinamide-adenine dinucleotide phosphate, reduced form, oxi-
protein 1, activator protein 1, and extracellular regulated kinase dase 2, which has been demonstrated to cause DNA damage in B
(ERK)MAPK pathways.9 Evidence also indicates that LMP1 pro- cells20; if extended to NPC, this could presumably result in additional
motes angiogenesis in the NPC microenvironment through the in- oncogenic mutations. EBNA1 also directly modulated a myriad of
duction of hypoxia-inducible factor 1 and vascular endothelial signaling cascades, including tumor growth factor (TGF-), signal
growth factor.9 Notably, high LMP1 expression in primary NPC sam- transducer and activator of transcription 1, and NF-B, leading to
ples has been associated with regional and distant metastasis.13 Yet increased growth, survival, metastasis, and angiogenesis.8

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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- NAs have been identified, both viral and host. Viral targets include
specific protease USP7, to prevent the deubiquitination and conse- LMP1, putatively targeted by several microRNAs from the BART
quent stabilization of p53. This relationship consequently reduces p53 region (ebv-miR-BARTs 1, 9, 16, and 17)25; and LMP2A, identified as
accumulation in response to DNA damage, thereby decreasing apo- a target of ebv-miR-BART22.26 Subsequent functional experiments
ptosis and increasing radioresistance in vitro.8 In addition, EBNA1 has indicated that BART-microRNA modulation of these virally encoded
been shown in NPC cell lines to disrupt promyelocytic leukemia proteins influences multiple cellular properties, including prolifera-
nuclear bodies, which are also involved in p53 activation, apoptosis, tion, survival, and evasion of host immune response.25,26 Host targets
and DNA repair.21 Hence, EBNA1 clearly mediates a complexity of of BART-microRNAs include proapoptotic effectors p53 upregulated
cellular processes in nasopharyngeal oncogenesis. modulator of apoptosis,27 Bcl-2 interacting mediator of cell death,28
and translocase of outer mitochondrial membrane 22 homolog,29 as
EBERs well as several genes thought to influence host immune response,
The EBV-encoded genes EBER1 and EBER2 encode small RNAs including major histocompatibility complex class Irelated chain B,30
(167 and 172 nucleotides long, respectively) that are highly expressed importin 7,29 and Dicer.31 Overall, EBV-encoded microRNAs play a
in NPC cells, with up to 1 million copies detected per cell.22 Evidence complementary role to the viral proteins expressed in NPC: promot-
suggests that EBERs can function within NPC cells to promote growth ing survival and proliferation of NPC cells and contributing to evasion
and survival and modulate host immunity through a variety of mech- of the host immune response.
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 GENOMIC ALTERATIONS IN NPC
(IGF-1), possibly through activation of the IGF-1 promoter. More-
over, IGF-1 expression was frequently detected in NPC biopsy speci- Most genomic studies on NPC have focused on targeted sequencing
mens, indicating that the EBER/IGF-1 axis may contribute to the analysis of candidate genes or cytogenetic techniques, such as
development of NPC in vivo. In addition, preliminary data suggest G-banding, comparative genomic hybridization, and array compara-
that this induction of IGF-1 occurs through the interaction of EBERs tive genomic hybridization, for gross chromosomal analyses to detect
with the double-stranded RNA sensing toll-like receptor 3.22 copy number events and translocations. In August 2014, a landmark
study described the genomic analysis of 128 NPC samples using
EBV-Encoded microRNAs whole-exome sequencing, targeted deep sequencing, and single-
To date, 25 EBV-encoded precursor microRNAs processed into nucleotide polymorphism arrays.32 This report plus previous studies
44 mature microRNA sequences have been verified (miRBase [www. have identified several key genomic alterations that promote NPC
mirbase.org], Release 20). They emanate from two major regions of development and progression via a variety of mechanisms (Table 1).
the EBV genome: the BARTs and the open reading frame of the
BHRF1 gene. Originally cloned from EBV-infected Burkitts lym- Copy Number Alterations
phoma cells,23 BHRF1-microRNAs do not appear to be expressed in Important examples of recurrent chromosomal events in NPC
EBV-positive primary NPC tissues.24 Several targets of EBV microR- 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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and 16q. These have been identified and confirmed in multiple stud- of 144 primary NPC samples interrogated using reverse transcriptase
ies40,48,50,55; however, many of the specific tumor-suppressor targets in polymerase chain reaction.64 Functional analysis demonstrated that
these regions remain to be identified. One important region is the the resultant chimeric protein promoted proliferation and colony-
9p21.3 cytoband, originally observed to exhibit copy number losses in forming ability in vitro, as well as tumor formation in vivo.64 A second
most (61%), and a homozygous deletion in one of the evaluated study identified and validated three in-frame gene fusions (YAP1-
primary NPC samples.55 Contained within this region are two genes MAML2, PTPLB-RSRC1, and SP3-PTK2) in two NPC samples using
that encode three tumor suppressors: p16-INK4a, ARF (gene high-throughput sequencing of circularized DNA fragments and a
CDKN2A), and p15-INK4b (gene CDKN2B). Hypermethylation of novel computational algorithm.65 Subsequent investigation using flu-
the p16 promoter was also frequently detected in NPC samples, indi- orescent in situ hybridization analysis of 196 NPC samples observed
cating that this was the target of copy number loss in this region.33,34 In that structural rearrangements in the three involved genes were de-
subsequent studies, the functional implication of p16 loss in NPC was tected at low frequencies (MAML2 in 3, PTK2 in 6, and SP3 in 2 of 196
examined, demonstrating that p16 reexpression led to G0/G1 arrest cases), although none of the originally identified partners were simi-
along with reduced in vivo tumorigenicity, most likely mediated via its larly fused in the validation set.65
canonical role as an inhibitor of interaction between cdk4/cdk6 and
cyclin D1 (CCND1).56 Moreover, p16 deletion was a critical event in Small Insertions/Deletions and
human telomerase catalytic subunit immortalization of normal naso- Single-Nucleotide Variants
pharyngeal epithelial cells,57 further providing support for p16 inacti- Before the recent report on whole-exome sequencing of NPC,32
vation as an early event in NPC development.56 the detection of single-nucleotide variants (SNVs) and small insertion
Similar to p16, Ras association domain family member 1 and deletions (Indels) in NPC had been limited to targeted analysis of
(RASSF1) was identified as an important tumor suppressor in NPC specific tumor suppressors or oncogenes. These analyses indicated
because of its frequent deletion at 3p21.3, its promoter methylation, that the frequency of SNVs is low in NPC (1 to 342 coding mutations/
and somatic mutation in a few NPC samples (9.5%).38,39,48 Since its sample), even when compared with other virally associated head and
initial cloning in 2000,58 multiple putative functions have been as- neck tumors, such as human papillomaviruspositive head and neck
cribed to RASSF1, although its precise role remains controversial.59 In squamous cell or EBV-positive gastric adenocarcinomas (Fig 266,67).
the few studies focused on NPC, stable reexpression of RASSF1A in Nonetheless, several key genes and pathways do appear to be targeted
C666-1 cells resulted in growth inhibition both in vitro and in vivo by small genomic alterations, often overlapping with those that are
mediated through TGF- signaling (activin E and Id2).60 RASSF1A also frequently amplified or deleted at the copy number level, or
also plays an important role in regulating mitotic progression through epigenetically altered.
its direct inhibition of the anaphase-promoting complex cell- TP53, the most widely mutated gene in human cancers, is rarely
division cycle protein 20 complex, which functions to promote micro- mutated in NPC (0% to 8%); however, this proportion increases
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 (n = 56) (n = 35) (n = 21) (n = 200) (n = 244)
Several oncogenes are also activated in NPC via copy number
Somatic Mutation Frequency (/Mb)

gains. Frequent amplification of 11q13 has been observed in multiple


100
cytogenetic studies, with CCND1 identified as the likely target.48,49
50
Moreover, CCND1 was observed to be overexpressed in more than
90% of primary NPC samples by immunohistochemical staining, and
10
its knockdown in NPC cell lines significantly decreased prolifera-
5
tion.49 Similarly, recurrent amplification of 12p13 was determined to
target lymphotoxin- receptor, overexpression of which was con-
1
firmed to contribute to cell survival and tumor growth in NPC models 0.5
via activation of NF-B.54 Likewise, an increase in copy number of
phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit al-
0.1
pha (PIK3CA) at 3q26.1 has been observed in 70% to 75% of primary 0.05
NPC samples,50,51 and immunohistochemical evidence indicated that
the PI3K/Akt pathway was activated in approximately 85% of NPC
EBV+ Gastric
Adenocarcinoma

EBV- Gastric
Adenocarcinoma
HPV+ HNSCC
EBV+ NPC

HPV- HNSCC

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 Fig 2. Somatic coding mutation frequency (from exome sequencing, normal-
translocations, have been identified in NPC. By using paired-end ized to covered area) in nasopharyngeal carcinoma (NPC), compared with other
RNA-sequencing data from C666-1 cells, a fusion was first detected 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
between ubiquitin protein ligase E3 component n-recognin 5 and zinc were obtained from The Cancer Genome Atlas.66,67 EBV, Epstein-Barr virus;
finger protein 423, which was subsequently corroborated in 12 (8.3%) HPV, human papilloma virus.

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Molecular Landscape of NPC

to approximately 20% when somatic copy number loss is


IMMUNE EVASION IN NPC
included.32,35-37 Similarly, although the major mechanisms of RASSF1
deactivation are copy number loss or promoter hypermethylation, Evasion of host immune response is a recognized hallmark of cancer.11
mutations have been also detected in approximately 10% of NPC Immunologic recognition of nonself-antigens provides a strong se-
samples.38 Activating mutations and amplification of members of the lective pressure during tumorigenesis.76 Consequently, cancer cells
oncogenic PI3K and receptor tyrosine kinase pathways have also been have developed strategies that allow for their continued survival. This
identified.32,68,69 Interestingly, Lin et al32 reported frequent alterations phenomenon is particularly important in EBV-associated NPC, with
in genes involved in chromatin modification pathways (ARID1A, both viral and host factors contributing to the survival of NPC cells
BAP1, KMT2D/3, TSHZ3, and TET1/2/3), whereby patients harbor- during tumor initiation and progression.
ing such mutations experienced significantly inferior overall survival
than their wild-type counterparts. Other pathways that appear to be
Viral Factors Affecting Immune Response
targeted by SNVs/Indels in NPC include the NF-B,68 cell cycle, ad-
Although EBV infection is associated with robust immune infil-
hesion, differentiation, and autophagy pathways.32
trates, viral factors repress the activity of antitumor lymphocytes,
permissive for NPC cell survival.77 Several EBV gene products directly
Epigenetic Alterations interfere with the antigen-processing machinery and immune recog-
As discussed above, EBV infection, environmental factors, and nition. Surface expression of major histocompatibility complex mol-
genetic mutations targeting cellular modulators of epigenetics appear ecules and loading of viral peptide antigens are reduced by viral
to play integral roles in the modulation of epigenetic marks in NPC. proteins expressed during lytic replication, including BGLF5, BILF1,
These precipitating events can lead to a variety of functional outcomes, and BNLF2a78 (Fig 3). The interleukin-10 homolog BamHI-C frag-
including DNA methylation, and to a lesser extent, histone modifica- ment rightward reading frame 1 (BCRF1) can also affect antigen
tions, ultimately leading to alterations in gene expression. presentation through its direct inhibitory effects on T cells.79 During
Both RASSF1 and CDKN2A are tumor-suppressor genes whose latency, the expression of most gene products is suppressed; EBNA1
promoters are frequently methylated in NPC, in addition to being and LMP2A/B are the most ubiquitously expressed proteins during
targets of copy loss and SNV/Indels.32,34,37-39,48 The promoter region latency, yet do not elicit strong immune responses.80 Consequently,
of Wnt inhibitory factor 1 has also been reported to be frequently tumor-infiltrating lymphocytes are often incapable of effectively elim-
hypermethylated in NPC (85% to 90%).70 Furthermore, in vitro stud- inating EBV-associated NPC cells. Moreover, a recent report sug-
ies demonstrated that this hypermethylation leads to downregulation gested that exosomes released from EBV-positive NPC cells can
of Wnt inhibitory factor 1 expression with subsequent increase in recruit regulatory T cells and enhance their inhibitory activity, thereby
clonogenic survival.70 Cell adhesion molecule 1 (aka, TSLC1) and providing another mechanism by which NPC evades the host im-
Thy-1 cell surface antigen, both located at 11q23, have also been mune response.81
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 Host Factors Affecting Immune Response
Other genes whose promoters are frequently hypermethylated in Recognition and elimination of EBV-associated NPC cells by
NPC mediate a variety of pathways, including cell invasion/migration infiltrating lymphocytes depend on effective antigen presentation.
(CDH1, ZMYND10, and miR-148a), response to DNA damage The affinity of different HLA allele products for EBV antigens can
(GADD45), apoptosis (DAPK1, UCHL1, and miR-218), cell cycle/ influence tumor immunoediting (reviewed by Hildesheim and
proliferation (DLC1, HIN1, PTPRG, and RARB), MAPK/ERK Wang82). Certain HLA haplotypes are associated with reduced risk for
(RASAL and DAB2), and Wnt/-catenin (SOX1).34,44-47,71 developing NPC. For example, the HLA-A*0201 allele is underrepre-
Aberrancies in other epigenetic mechanisms, such as his- sented among patients with NPC in certain populations, possibly a
tone modification, have also been noted in NPC. EZH2, a result of efficient binding and presentation to T cells of conserved
histone-lysine N-methyltransferase, has been demonstrated to epitopes from EBV proteins.83 In East Asia, an elevated risk for devel-
play an important role in NPC by several groups.72-74 Altera- oping NPC has been attributed to the HLA-A*0207 allele84 (Fig 3).
tions in histone methylation resulting from EZH2 upregulation Aside from the HLA genes, additional risk loci have been uncovered by
effect a myriad of phenotypes in NPC cells, including increased genome-wide association studies,82,84 suggesting that other compo-
migration/invasion, antiapoptosis/cell survival, and angiogen- nents might contribute to differential immune recognition of EBV
esis. Finally, DNA methylation and modification of histones antigens. With the recent emergence of novel immune-modulatory
within the EBV genome itself also play important roles by therapies,85 such approaches definitely warrant examination in EBV-
restricting the expression of EBV genes associated with the type associated NPC.
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 MOLECULAR BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS
(Qp, LMP, EBER, and Bam HI-A transcript promoters).75 These
collective observations raise the intriguing possibilities regard- Several different types of diagnostic and prognostic molecular bio-
ing the potential targeting of such alterations or pathways, using markers for NPC have been investigated, including both blood- and
RTK inhibitors or epigenetic (histone deacetylase) modulators tissue-derived factors, that promise to have profound impact on the
in NPC treatment. individualized management of patients with this disease.

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Copyright 2017 American Society of Clinical Oncology. All rights reserved.
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 NPC biology is a complex interplay between cellular, viral, stromal,
Within NP tumor tissue and the surrounding stroma, several and host components. Although much has been learned regarding
factors have the potential to serve as indicators of clinical outcome. the development and progression of NPC, more remains to be un-
For example, tumor expression of caspase-3, survivin,86 c-Met,87 raveled. The emergence in recent years of high-throughput
BUB1b, CENPF,88 ERBB3,89 MTDH,90 and FADD91 may each have molecular characterization methods (eg, massively parallel se-
prognostic significance. In addition, two tissue-based microRNA quencing and single-nucleotide polymorphism/methylation
expression signatures have been identified, which are associated microarrays) has resulted in significant inroads into our under-
with survival and distant metastasis in NPC.92,93 Within the standing of the genomic underpinnings of cancer biology. To date,
stroma, increased expression of periostin and decreased number of however, there has been only one report of comprehensive se-
tumor-infiltrating lymphocytes have both been linked with poor quencing analysis of a modest number (n 56) of NPC samples.32
survival.94,95 Additional independent validation of each of these 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

nasopharyngeal carcinoma: Evidence from a meta- 26. Lung RW, Tong JH, Sung YM, et al: Modu-
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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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