biomarker of ebv-npc
TRANSCRIPT
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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 unpublished data. 2012.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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