Nasopharyngeal Cancer Workshop EBV Biology in Nasopharyngeal Cancer 

25 – 26 January 2019 Prof Lawrence S Young

Epstein-Barr virus Biology

in Nasopharyngeal Carcinoma:
new insights and future challenges
Nasopharyngeal Carcinoma Workshop
25-26 January 2019, Singapore

Professor Lawrence S. Young

Pro-Dean, Warwick Medical School
University of Warwick
UK

Nasopharyngeal Carcinoma - a unique tumour Epstein-Barr Virus

• Site of origin - Fossa of Rosenmuller (pharyngeal recess), • Herpesvirus, infects > 90% of human population.
Waldeyer’s tonsillar ring.

• Life-long virus carrier state, usually asymptomatic.

• Histology - ‘lymphoepithelioma’, undifferentiated SCC (WHO
II/III)
• DNA tumour virus: associated with lymphoid /epithelial
cancers.
• Epidemiology - Genetic susceptibility, Environmental factors
(Cantonese salted fish), Epstein-Barr virus (EBV).
• EBV displays dual tissue tropism:
• Genetics - Somatic genetic and epigenetic changes, familial.
Infection of B lymphocytes:
growth transformation in vitro (LCL)
• Cell Biology - EBV infection, stem cells, stromal interactions,
metastases. establishment of latency
colonisation of host
• Diagnosis - EBV DNA, antibodies, miRNAs, methylated DNA. Infection of squamous epithelial cells:
virus replication linked to differentiation
• Therapy - Conventional vs new approaches. dissemination of virus to other hosts ?
establishment of latency ?

EBV-ASSOCIATED TUMOURS
Malignancies in immunosuppressed host
Post-transplant lymphomas: Polyclonal B cell proliferations
Monoclonal non-Hodgkin’s lymphomas

HIV-associated lymphomas: Immunoblastic lymphoma

Diffuse large B cell lymphoma
Primary CNS lymphoma
Burkitt’s lymphoma
Pleural effusion lymphoma

Malignancies in immunocompetent host

B cell lymphomas: Burkitt’s lymphoma
Hodgkin’s lymphoma

T/NK lymphomas: Polyclonal T cell proliferations

Lethal midline granuloma

Carcinomas: Nasopharyngeal carcinoma

Gastric adenocarcinoma

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Nasopharyngeal Cancer Workshop EBV Biology in Nasopharyngeal Cancer 
25 – 26 January 2019 Prof Lawrence S Young

NASOPHARYNGEAL CARCINOMA EBV latent gene expression in NPC

1. Patients show elevated anti-EBV antibodies and circulating
EBV DNA. Plasma EBV DNA as a primary screen.

2. Consistently EBV genome positive - monoclonal episomes.

3. Restricted EBV latent gene expression - EBNA1, LMPs,

BARTS, BARF1 but also some lytic gene expression.

4. Presence of EBV in high grade pre-invasive lesions. EBERs

EBERs EBNA1
Key Questions
Relevance of epithelial infection to EBV’s natural history?
Role of EBV strain variation?
Precise contribution of EBV latent and lytic genes?
Contribution of the microenvironment?
EBNA1

Targeting EBV for therapeutic/prophylactic intervention? LMP1 LMP2A

EBV strain variation – lots more genomes

What’s special about the NPC cell and its progenitor?

What’s special about the type of EBV in NPC cells?

Why can’t we detect more premalignant disease?

When does EBV infection occur in the carcinogenic process?

Where does EBV replicate in vivo?

How important is tumour heterogeneity and what are the

clinical implications?

Heterogeneity – EBV and virus gene expression, genetics and

epigenetics, immune microenvironment
Palser et al., J. Virol. 92:e01132-18, 2015r

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Nasopharyngeal Cancer Workshop EBV Biology in Nasopharyngeal Cancer 
25 – 26 January 2019 Prof Lawrence S Young

B SNP variation across the EBV genome ZpV3 polymorphism: higher frequency in NPC and enhances EBV lytic reactivation

Mean number SNPS per 1kb

30
25
20
15
10
5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

ND
0

BHLF1
BFLF2
BFLF1

BORF1
BORF2
BaRF1

BSLF2_BMLF1
BSRF1
BLRF1
BLRF2

BZLF2
BZLF1
BRLF1

BKRF2
EBNA-3C
LMP-2A
LMP-2B
BNRF1
BCRF1
BCRF2
EBNA-LP
BWRF1
BWRF1
BWRF1
BWRF1
BWRF1
BWRF1
BWRF1
EBNA-2
BHRF1

BFRF1A
BFRF1
BFRF2
BFRF3
BPLF1
BOLF1

BMRF1
BMRF2
BSLF1
BLLF3

BLLF1
BLLF2
EBNA-3A
EBNA-3B

BRRF1
BRRF2
EBNA-1
Mean number SNPS per 1kb

30
25 Gene name
20
15
10
5

LF3 ND
0
BKRF3
BKRF4
BBLF4
BBRF1
BBRF2
BBLF2_BBLF3
BBRF3
BBLF1
BGLF5
BGLF4
BGLF3.5
BGLF3
BGRF1_BDRF1
BGLF2
BGLF1
BDLF4
BDLF3.5
BDLF3
BDLF2
BDLF1
BcLF1
BcRF1
BTRF1
BXLF2
BXLF1
BXRF1
BVRF1
BVLF1
BVRF2
BdRF1
BILF2
LF2
LF1
RPMS1
BILF1
BALF5
BALF4
A73
BALF3
BARF0
BALF2
BALF1
BARF1
BNLF2b
BNLF2a
LMP-1
Gene name

16 Synonymous
Mean number SNPS per 1kb

Non‐synonymous
14
12
10
8
C 6
4
Correia et al., J. Virol. DOI 10.1128/JVI.01132-18, 2018r
2
0
Latent Early lytic Late lytic

Bristol et al., PLoS Path 14 (7):e1007179, 2018r

Expression of BILF1 in NPC

RT-QPCR analysis revealed BILF1 expression in primary NPC tissues (n=44), EBV-
Genome Diversity of EBV – key questions infected nasopharyngeal epithelial cell lines (n=9) and C666-1
LCM Primary NPC 
Primary NPC (Malaysia) (JA) EBV‐infected cell lines

• Are there clear geographic variants of EBV?

C125855 

HO NE 1_I

SU N E 1_I
N P460_I

TW O 1_I
TW O 4_I
CN E1_I
CN E2_I
N PC 15

N PC 23
N PC 26
N PC 28
N PC 31
N PC 32
N PC 33
N PC 35
N PC 41

N PC 43
N PC 44
N PC 46

N PC 53

N PC 55

C666_I
QEH10
QEH11
QEH14
QEH15
QEH22
QEH23
QEH26
QEH33
QEH38
TS E 10
TS E 14

TS E 20

TS E 42

TS E 47
TS E 52

TS E 54

M CAV
N PC 2
N PC 7

M KEC

HK1_I
XY3‐6
QEH3
QEH8

E10
E11
E15
E23
E62
E63
Wp
Cp

• What is wild type EBV? Basis for comparison with disease associated EBV? QUK
UK
EBNA2.1
EBNA2.2
EBNA3A
EBNA3B

• Multiple infections in a single individual? EBNA3C

LMP1 B95
LMP1 CAO
LMP1 TR
LMP1 TR CAO
LMP2 ex 6

• Different sites? Saliva vs blood vs tumour

LMP2A ex 1
LMP2B ex 1
LMP2 TR
BHRF1 W2HF
BHRF1 Y2HF
BHRF1 H2HF
BZLF1

• Case-control studies needed. BRLF1

BMLF1
BMRF1
BALF1
BALF2
BARF1

• Functional consequences: NPC-derived M81 epitheliotropic and more lytic; BGFL5

BNLF2A
BNLF2B

EBNA1 from NPC compromised for episomal mainentance, more lytic. ZpV3 BILF1
BILF2
BNRF1
BVRF2
GP350
BALF4

• Therapeutic consequences: vaccine design, targeted drugs.

FU
FUK
LF1
LF2
LF3
BART1‐3
BART2‐3
EBER1
EBER2
B2m
PGK1
GAPDH

RNAscope analysis demonstrates BILF1 expression in a subset of primary NPC
What if the prevalent form of EBV in NPC and in China and
7TM GPCR; activates G-protein signaling; downregulates MHC-1 expression via exocytic and endocytic
pathways; attenuates PKR phosphorylation; interacts with and impairs CXCR4 function; transforms NIH 3T3 South-East Asia is a more replication-competent virus?
fibroblasts.
6 out of 28 primary NPC cases are BILF1‐positive (21.4%) 
Could this explain:
with a mean H‐score of 100 (max.300)

1. Elevated antibody titres to late viral antigens have diagnostic and

prognostic value in NPC.
Photomicrographs showing RNA in situ
hybridisation for BILF-1 (RNAscope,
Advanced Cell Diagnostics). (A) Akata 2. Elevated virus load is frequently observed before the onset of NPC.
EBV positive cells show a few BILF-1
positive cells. (B) Akata-induced cells
have numerous BILF-1 positive cells. 6 of 3. Lytic cycle antigens more frequently seen in NPC than previously thought.
28 NPCs showed evidence of BILF-1
transcripts, examples of weak (C) and
moderate staining (D) for BILF-1 RNA. 4. Lytic and virion-associated viral products interfere with cellular functions
that could contribute to oncogenesis.

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Nasopharyngeal Cancer Workshop EBV Biology in Nasopharyngeal Cancer 
25 – 26 January 2019 Prof Lawrence S Young

Does EBV induce malignant transformation of epithelial cells?

Replicative EBV infection in differentiating layers of tongue epithelium
Why don’t we see more pre-malignant disease?
1. Difficulty in establishing EBV-positive NPC cell lines in vitro and in vivo.

2. Loss of EBV from cultured NPC cells – HONE1 [Glaser et al. Int J Cancer, 1990]
EBV replication
3. Loss of EBV from in vitro infected epithelial cell lines. EBV DNA ISH in 1.3% normal
samples
(217 patients)

Oral Hairy Leukoplakia

HIV infection, post-transplant

BZLF1

Frangou et al., J. Infect. Dis. 191, 238-242 (2005).

EBV INFECTION OF EPITHELIAL CELLS

Squamous basal cell, Stem cell,
Initiated cell - Bcl2, ∆Np63, cyclin D,
3p/9p loss?

EBV

Epithelial cells unable

Normal differentiating epithelium to differentiate

EBV replication EBV latent infection

PNAS 111:16544-9, 2014

Analysis of Plasma Epstein-Barr Virus DNA to Screen for Nasopharyngeal Cancer

Chan et al., NEJM 377:513-522, 2017

Why was pre-malignant/dysplastic disease not detected?

Rapid progression/regression?

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Nasopharyngeal Cancer Workshop EBV Biology in Nasopharyngeal Cancer 
25 – 26 January 2019 Prof Lawrence S Young

FUTURE PERSPECTIVES AND PRIORITIES

1. Contribution of EBV strain variation and virus-encoded genes (lytic)

2. Role of cancer stem cells and tumour microenvironment.

3. Other co-factors? Other viruses, microbiome, chemical carcinogens.

4. More detailed characterization of early and metastatic disease.

5. Development of model systems - in vitro and in vivo.

6. Patient stratification - predictive biomarkers of response and metastases.

7. Identification and therapeutic targeting of driver signalling pathways.

8. Targeted therapy – EBV latent proteins, epigenetic, gene and immunotherapy.

9. Prevention - prophylactic vaccination, diet.

Institute of Cancer & Genomic Sciences Division of Biomedical Sciences

College of Medical and Dental Sciences Warwick Medical School EBV occurs in the precancerous lesions of
University of Birmingham University of Warwick
nasopharyngeal carcinoma
NPC and HL project Data Analytics Collaborators
EBV Sequencing
Chris Dawson Jean Crabtree
Anne Palser Nasir Rajpoot
Paul Murray Ingemar Ernberg
Paul Kellam Sascha Ott
Graham Taylor Lori Frappier
Nigel Dyer Dysplasia Dysplasia
John Arrand Lindsey Hutt-Fletcher
Martina Vockerodt Maria Lung Normal
Wenbin Wei Gerald Niedobitek Normal

Chunfang Hu Rob White Kenzo Takada Carcinoma

in situ Carcinoma
Jia Junying Martin Allday Angela Lo Carcinoma in situ
Sim Sihota Paul Farrell KW Lo in situ
Yuk Ting Ma George Tsao Cancer Carcinoma
Kaisheng Wen Lee Fah Yap Cancer in situ
Ciaran Woodman Ian Paterson
Musheng Zeng Cancer Cancer
Yi-Xin Zeng

HE staining EBERs
28

KLF4/BLMP1
Epithelial infection
EBV Rp/LMP1
Normal differentiating epithelium
EBV replication

EBV
Super-Replicator
Strain

Progression
Genetic NPC
Germline predisposition Epigenetic
changes

Regression
Selection for stable EBV infection &
Stem cell in transformation zone
recruitment of stromal cells

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