biomarker of ebv-npc

7/29/2019 Biomarker of EBV-NPC

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RECENT UPDATE ON EBV

AS ETIOLOGICAL FACTOR OF

NASOPHARYNEAL CARCINOMA

Sofia Mubarika24 February 2013


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Nasopharyngeal carcinoma (NPC)

Tumor arising from the epithelial cells covering

nasopharyngeal surface. Remarkable geographical incidence; frequently occurs in a

well-defined regions. Strong association to Epstein-Barr virus (EBV) infection.

http://nasopharyngealcancer.com/images/head.jpg;IARC publication; Chang ET & Adami HO. Cancer Epid Biomarkers Prev. 2006.
http://nasopharyngealcancer.com/images/head.jpghttp://nasopharyngealcancer.com/images/head.jpg


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CT-scan

MRI

Nasendoscopy

Clinical manifestation and diagnosis

Nasal symptoms Aural symptoms

Headache,diplopia

Upper neck mass

Predominant symptoms atfirst presentation

Histopathology(gold standard)

Lee JT et al. Otolaryngol Head Neck Surg. 2005; Wei WI & Sham JS. Lancet. 2005; Adham M et al. Chin J Cancer. 2012.


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Problems in Indonesia

Incidence rate: 6.2/100,000persons/year.

The most prevalent head and neck

cancer; fourth prevalent canceroverall.

Male : female = 3 : 1.

Most of cases come at late stage.

No test for NPC screening available.Soeripto. Berita Kedokteran Masyarakat. 1998.Fachiroh J et al. J Clin Microbiol. 2006.Adham M et al. Chin J Cancer. 2012.Hariwiyanto B et al. unpublished data. 2012.


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Epstein-Barr virus (EBV)

Double stranded DNA.

Infects 90% infects world population.

In most individuals, EBV stays for life time.

EBV relates with types of malignancies =

class 1 human carcinogenic virus.

Nasopharyngeal carcinoma (~100%)

Gastric adenocarcinoma

Non-Hodgkins lymphoma Burkitts lymphoma

Hodgkins disease

Post-transplant lymphoproliferative disorders

International agency of research on cancer (IARC) monograph. 1997.Middeldorp JM et al. Crit Rev Oncol Hematol. 2003.


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Biodiversity

Human genome :

Methylation of TSG

EBV Human interaction:

Antibody reponses to EBV proteins :

IgG, IgA (VCA-p18 + EBNA-1), EA

EBV : Type A , B and Mixed

LMP1, LMP2 ( CTL Epitope : HLA A2, HLA-A11,HLA-A 22)

BARF


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Epstein-Barr Virus

Familiy of Herpes viruses class 1; no.1 carcinogene ( 7 cancers)

EBV related carcinomas : NPC, lymphomas, gastric cancer virusgenome found in tumor cells

In healthy individuals ( 90% world population) virus reside , and hidelatenly in the cytoplasm of circulating B lymphocytes.

In normal individual

the virus latenly found in the cytoplasma of B lymphocytes EBV expressed limited viral proteins unrecognised by immune

system Escape immune surveillance

Viral load about 1-10/106 per B cell.

Ring 1994; Farrel 1995; WHO-IARC 1997; Rickinson&Kieff 2001


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EBV strongly associates to NPC

Viral proteins in all tumor cells.

Characteristic IgG and

IgA response to EBV proteins

paralelling tumor outgrowth

Abnormal EBV-DNA loads and

viral mRNA in circulation and

nasopharyngeal brushings

Fachiroh J et al. J Inf Dis. 2004; Stevens et al. Int J Cancer. 2006; Fachiroh J et al. J Clin Microbiol. 2006;Fachiroh J et al. J Clin Microbiol. 2008; Paramita DK et al. Clin Vaccine Immunol. 2009.

Recently EBV-based assays have been developed for NPC screening.


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1. EBV protein immobilised by

western-blot transfer EBV strips2. Recognition of IgG and IgA to EBVlatent and lytic antigens (~ 80 proteins)

3. IgG/ EBV antigens of Indonesia(Yogyakarta), Chinese (Hongkong) andCaucasian (Netherlands) NPC and

normal panel are SIMILAR

4. Development of ELISA by employingthe most suitable EBV antigens

normal


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0

1

2

3

0

1

2

3

Normal healthy (n=254) NPC (n=147)

EBNA1 VCA-p18 Combination EBNA1 VCA-p18 combination

Early screening method: ELISA : IgA- EBNA1 + VCA-p18

OD

450

nm

( J. Fachiroh et al, 2006)


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EBV-based marker-1 : EBV-IgA ELISA

Non-invasive, simple, economical.

Good diagnostic value

sensitivity 90%

specificity 85%.

Use for diagnostic confirmation.

Normal Control NPC

Basic principle:

IgA level increases in NPC cases.

IgA detectable prior to onset of disease.

Fachiroh J et al. J Clin Microbiol. 2006; Fachiroh J et al. J Clin Microbiol. 2008.

Optimation in prick test method for usein remote area.


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-EBV-DNA load (EBNA1 protein) in whole blood ofNPC

- metode : real-time PCR

- Result:a. the use of 2 primers (213-bp and 99-bp)b. 213bp : 72.5% NPC positive with 29.5% above

cut-off

99bp : 85.9% NPC positive with 60.4% above cut-offSuggesting that EBV-DNA isfragmented in circulation

c. No correlation between DNA load and IgAin circulation


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The role of BARF1

NPC cells

Full lengthBARF1

sBARF1

c-MYC Telomerase Bcl-2

Sel B

Sel epitel

2Para/autocrinegrowth factor

1Imortalisation /

Tumorigenic

Transformation

hCSF-1antagonist

(c-fms homolog)

3Imunomodulasi & immune

evasion

Strocbine 1998, Cohen 1999, Decausin 2000, Danve 2001, Ooka 2005,Wei 2005,

sBARF1

sBARF1

R d M f BARF1 h NPC


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Road Map of BARF1 research on NPC

(FM UGM/ VUmc, NL)

Research Publication Grants

BARF1 gene detected from

peripheral blood

Stevens, Hariwiyanto,

Harijadi, Fachiroh,

Paramita, Tan, Mubarika,

MiddeldorpJ. Clin Microb 05

- KWF NL (2005)

- EU /AsiaLink (2003- 2006)

BARF1 expression onnaspharyngeal epitel

-for diagnostic

Stevens, Verkuijlen,

Hariwiyanto, Harijadi,

Paramita, Fachiroch,

Adam, Tan, Mubarika,

Middeldorp .IntJ Cancer, 06

KWF NL (2006)

The Sekuens gen BARF1 asnew marker for NPC

Hutajulu, Stevens,

Verkuijlen, Hariwiyanto,

Indrasari, Fachiroch,

Harijadi, Kusumo,

Mubarika, Middeldorp

(in preparation)

- KWF, NL (2007)

- Nuffic/NFP

-Frontier Research UGM 07

- UGM young scientist grant

-Professor Grant UGM 2008


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80% NPC sampel

Genetic diversity(= compared to wild type)

mutation 6-12 nukleotides

Mutastion 1-3 amino acid

V29A (valinealanine)

W72G (tryptophan

glycine)H130R (histidinearginine)

Hutajulu , Mubarika (in preparation)

BARF1 Protein Sequence on NPC


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Mutasi

codon

NPC Healthy

individuals

S12T 1.8 0.0

V29A 62.0 40.0

W72R 1.8 0.0

W72G 14.3 0.0

S128F 1.8 0.0

H130R 62.0 40.0

Comparison mutation of BARF1 at protein

level . NPC 80% VS healthy indivudual 40%

(p= 0,04)


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EBV-based marker

Genetic

Epigenetic

Inflam-

mation

Epstein-Barr virus

Environ

ment

Epigenetic marker

NPC markers

Esteller M. N Engl Med J. 2008; Hutajulu SH et al. Mol Cancer. 2011; Hutajulu SH et al. Int J Cancer. 2012 (submited).


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Other biomarkers for NPC :

* Methylation of tumor supr genesPLUS

* VCAp18 + EBNA-1 IgA

> 90% sensi and specificity

Susana Hutajulu et al., 2011, 2012


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Early stage (stage I-II)

Treatment: radiation

Good cure rate: >90%

Difficult to diagnose

non-specific early signs

hidden location of thenasopharynx

Late stage (stage III-IV)

Early identification of NPC is

crucial

Lee AW et al. Int J Radiat Oncol Biol Phys. 2005.Hariwiyanto B et al. unpublished data. 2012.

Treatment: radiation andchemotherapy.

Cure rate progressif,recurrence and complications.

Represents >85% patientsvisiting the local center


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Non-invasive screening tools highly

needed

Early detection may need long term monitoring. Bottleneck of screening:

expensive clinical work up.

invasive procedure for standard

diagnosis. Non-invasive screening assays are clinically

relevant and desirable.

Ji MF et al. Br J Cancer. 2007.

Cao SM et al. Plos One. 2011.Hutajulu SH et al. Int J Cancer. 2012 (submited).

Eff ti i th d t t


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Effective screening method targets

NPC risk factor

EBV-based

tumor marker tests

Genetic

Epigenetic

Inflam-

mation

Epstein-Barr virus

Environ-

ment

Stevens SJS et al. Int J Cancer. 2006.Fachiroh J et al. J Clin Microbiol. 2006.

Fachiroh J et al. J Clin Microbiol. 2008.Paramita DK et al. Clin Vaccine Immunol. 2009.Hutajulu SH et al. J Med Virol. 2011.


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EBV-based marker

Genetic

Epigenetic

Inflam-

mation

Epstein-Barr virus

Environ

ment

Epigenetic marker

NPC markers

Esteller M. N Engl Med J. 2008; Hutajulu SH et al. Mol Cancer. 2011; Hutajulu SH et al. Int J Cancer. 2012 (submited).

l ti l h i C


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Translational Research in EBV NPC

Basic Clinic - PH

Literature Review

Virusgenomic

Host virusinteraction

Antigen/peptidesynthesis

Diagnostickit

Fieldhospital

Epidemi

ology

ABGC

Routineclinical

application

P i h l bl d


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Peripheral bloodIgA to EBV proteins is specific marker for NPC serodiagnosis

NPC response better to multiple EBV markers

Sensitivity & Specificity ~90%

Combination of 2 ELISA s: Sensitivity & Specificity > 95%

Diagnosis Confirmation

2 x ELISAs are expensive and inefficient


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Epigenetic promoter DNA methylation

Covalent addition ofa methyl group to the DNA

Promoter DNA methylationleads to gene silencing.

Worm J & Guldberg P. J Oral Pathot Med. 2002; Groanbaek K. APMIS. 2007.


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WIF1

DLC1

H1N1

UCHL1DAB2

RASSF1A

RASAL1

tp53

Gene silencing of tumor suppressor genes

contributes to neoplastic growth.

Cell cycle regulator Cell adhesionApoptosis regulatorDNA damage

NPCLi L et al. Chin J Cancer 2011.

bi d h l i k h


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Combined methylation marker shows a

good diagnostic performance

Sensitivity 98%

Specificity 96%

CHFR 60% 0% 0%

RIZ1 57% 0% 0%

WIF1 61% 18% 0%p16 66% 27% 0%

RASSF2A 29% 0% 0%

RASSF1A 76% 0% 4%

DAPK1 79% 41% 48%

DLC1 77% 41% 64%

CDH13 77% 73% 64%

CADM1 70% 36% 72%

Tumor Suppressor NPC High Risk Healthy

Gene

Hutajulu SH et al.Mol Cancer 2011.


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Conclusion

Biodiversities from EBV and immunological

responses to EBV protein were found on NPCcases in Indonesia

Several biomarkers have been shown very

promising to be used as biomarkers for diagnosis (VCA-p18+EBNA-1) IgA, EA (IgA) as confirmatorytest;

Variations on oncogenic part of EBV BARF alsohave been found; methylation of TSG cancontribute to oncogenesis of NPC

How this variations will affect the clinical

performance need to be explored further


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biomarker of ebv-npc

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