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GlobalIlluminators FULL PAPER PROCEEDING Multidisciplinary Studies

Full Paper Proceeding ETAR-2014, Vol. 1, 566-572

ISBN: 978-969-9948-23-7

*All correspondence related to this article should be directed to , Eka Savitri, Hasanuddin University, Makassar, Indonesia. Email: [email protected]

© 2014 The Authors. Published by Global Illuminators. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer-review under responsibility of the Scientific & Review committee of ETAR-2014.

ETAR 2014

Viral Load From Tissue Brushing And Peripheral Blood Of Nasopharyngeal

Carcinoma From Makassar Before And After Treatment

Eka Savitri1*, Sofia Mubarak Haryana2, Anwar Mallongi3

1,2,3

Hasanuddin University, Makassar, Indonesia

Abstract

The aim of this study is to compare the plasma of Ebstein Barr Virus (EBV) DNA load and viral load of tissue

brushing correlate of Nasopharyngeal Carcinoma (NPC) from Makassar before and after treatment. This

research applied an analytic study to all 39 NPC’s cas es that have been informed to be recru ited and every case

have signed their informed consent. EBV DNA plasma was examined using RT-PCR as written by the

manufacturer. Primer used in this study were QP1, QP2, and internal hybridization probes fluorigenic LC N

EBNA and EBNA FLN (TIBMOL Biol, Berlin, Germany). Real-t ime PCR reagents from Roche Diagnostics

(Almere, Netherlands). The d istribution of cases was based on anaplastic histopathological status. Results

indicated that distribution of cases based on pathology anatomy are: type I (Carcinoma of squamosa cell with

ceratinized) was 1 case, type II (Differentiated carcinoma without ceratinized) was 13 cases, and type III

(Anaplastic) was 25 cases. The absolut specificity indicated that EBV viral load fro m brushing is more specific

for NPC compared to serum. Sensitivity (67.6%), Specificity (100%) and accuracy (69,2%) indicated that

brushing is quite sensitive to be used as diagnostic for NPC compare to serum. The average of viral load from

brushing before tretment was 370959680.9 and viral load plasma was 109324.6. The average of viral load from

brushing after treatment was 192995.3, and viral load plasma was 981.6. In conclusion, EBV DNA of viral load

can be used as a marker for the presence of NPC and the progression of the disease.

© 2014 The Authors. Published by Global Illuminators. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the Scientific & Review committee of ETAR-2014.

Keywords : Brushing, Nasopharyngeal Cancer, EBV, Viral Load, Diagnostic.

.

Introduction

Nasopharyngeal cancer (NPC) is a malignant epithelial neop lasm with most frequently incidence at the upper aerodigestif tract. Epidemiology of NPC is very unique as it

is rarely found in European populations, but some are found in China (particularly southern China), Southeast Asia, and Africa1,2. In Indonesia, NPC incidence rate is 4.7/100,000 population per year with a comparison between men and women of 2-3:1.3,4. In Indonesia,

almost 80% of the NPC’s patients are diagnosed at the age of 30-59 years old,5 with an

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Eka Savitri /ETAR-2014/Full Paper Proceeding Vol. 1, 566-572

International Conference on “Emerging Trends in Academic Research” (ETAR- 2014)

567

increase of incidence trend followed by age. Incidence in Makassar, South Sulawesi, from

January 2004 to June 2007 was recorded about 33% of malignancies in the ear, nose and throat.6

Epstein-Barr virus (EBV) is closely related to the incident of NPC 7,8,9. The production of protein of viral gene can be detected in the NPC tissue of majority of sufferers,

namely latent membrane protein 1(LMPI), latent membrane protein (LMP2) 2, EBNA 1-6 (Epstein-Barr Nuclear Antigen1-6)10. It was also found an increase of titer of immunoglobulin A antibody (IgA) viral proteins (viral capsid antigen/VCA and early-

antigen/EA) (IgA (VCA-p18 + EBNA-1)11,12. From Serological test conducted previously have been proven that the test can be used as early detection and follow up nasopharyngeal

cancer13. In addition to that, viral load can be used to test the disease progression 7. Serological test have been proven can be used as early detection and follow up of NPC. Its sensitivity and specificity was increased using viral load test, can be used to see the disease

progression. Very interesting, that viral load in Makassar was 866.400 million, meanwhile in Yogyakarta was 48.840 million.14 The significant difference of the viral load interest us to

carry out this research. based on the nature of viral infection, EBV infection can activate immune system of the body. Therefore, we assume that EBV infection associated with the high occurrence of NPC cases Makassar may be related to the low antigenicity of EBV virus

– that immune system / Cytotoxic T lymphocytes cannot eliminate the virus infected cells therefore virus can escape from immunosurvelilance 14

The nature of EBV is, once the virus infects human being, virus will stay long life in the form of episom in the cytoplasma of B lymphocytes 15. If the per son immunocompromised (

Unable to give normal fnction), episom can reactivated from become linear. Subsequently, viral replication will occur. Therefore, Viral DNA in circulating blood can be used as clinical indcators.Viral Loadis the best marker for prognostic factors of NPC. In addition to that this

can e use as important marker of diseases progression and recurrency monitoring of NPC KNF.

Material And Methods

This research is analytic study to compare viral load from brushing and serum of NPC

cases from Makassar before and after teatment.Ethical clearance was approved from The Institutional Ethical Committee from faculty of Medicine Hasanuddin University, Makassar.

Number 0232/Ho.4.8.4.5.31/PP36 KOMETIK/2008, September 3rd 2008. Research was conducted at the hospital of Dr. WahidinSudirohusodo, Makassar, South Sulawesi, Indonesia from November 2006 to June 2008. The samples must fulfill these criterias : 1) all patients

were clinicallydiagnosed as NPC’s patients, agreed to be involved in this and signed the informed consent; 2) They were already a confirmed through anamnesis, physical

examination of ear, nose and throat (ENT), histopathology, radiology, nasopharyngeal CT-Scan, thoracic images, liver ultrasound, and bone survey; 3) Patient were not limited to age, sex, ethnicity and race.

The steps of working are as follows;venous blood samples of patients were taken as 6

cc to check the viral load . EBV DNA load in blood is checked by a quantitative Light Cycler (LC) real-time PCR. Primers used QP1 and QP2, and internal hybridization probes fluorigenic LCN EBNA and EBNA FLN (TIBMOL Biol, Berlin, Germany), has been



568

previously described (Stevens). LC-based PCR amplification of 99-bpEBNA1 (location of

213-bp QP1-QP2 amplicon) is used for DNA fragments quantification.

In the experiments, reagents and hybridization probes are on the 99-bp LC-based PCR-bp

LC identical 213-based PCR, except primer (forward primer QP3 15-CCACAATGTCGTCTTACACC-3)and reverse primer (QP4 15-

ATAACAGACAAFGGACTCCCTL-3J). Real-time PCR reagents are obtained from Roche Diagnostics (Almere, The Netherlands).

Results

The study was conducted on 39 patients with nasopharyngeal carcinoma. Patients’

blood samples were taken for checking the Viral load and brushing examination. Blood tests were examined to assess IgA (VCA-p18 + EBNA-1), Viral Load, in the case of NPC before and after therapy were included in the remaining 39 inclusion criteria. Based on the result,

only 39 patients with NPC cases were examined, while the remaining patient failed to follow the research because a few were dead and left without reasons.Based on the progressive

incidence, it resulted two groups.Characteristic sample of this study is presented in Table 1.

Table 1. The Characteristics of Sample

Characterictic Group

Cases Control

Sex

- Men 26 (66,7%) 14 (48,3%)

- Women 13 (33,3%) 15 (51,79%)

Stadium

2 5 (12,8%)

3 15 (38,5%)

4 19 (48,7%)

Age 45,32 ± 13,72 27,35 ± 8,60

Tribe

Bugis 16 ( 41 %) 9 (31,03%)

Makassar 10 ( 25,6 %) 8 (27,59%)

Poso 1 ( 2,56 %) 1 (3,45%)

Mandar 1 ( 2,56 %) 1 (3,45%)

Toraja 7 ( 17,95 %) 3 (10,34%)

Buton 1 ( 2,56 %) 2 (6,90%)

Tionghoa 1 ( 2,56 %) 2 (6,90%)

Jawa 2 ( 5,13 %) 2 (6,90 %)

Bali 1 ( 3,45%)

Table 1 showed that NPC’s patient of men is more than women, the higher stadium of NPC’s

patient is stadium 3, and from ethnical result we found the high cases from Buginese. Nowadays NPC cases are also found in children and younger people.Distribution of cases based on histopathological status is presented in Table 2 are as follow:

Table 2. Distribution of cases based on histopathological status



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Status of histopathology (WHO) n %

Type I : Carcinoma of squamosa cell with ceratinized 1 2,6

Type II: Differentiated carcinoma not ceratinized 13 33,33

Type III: Anaplastic 25 64,1

Table 2 showed that 64,1% are type III (Anaplastic) from all 39 patients.

The result of EBV DNA viral load in brushing and plasma groups is presented in Table 3.

Table 3. EBV DNA Viral Load in Brushing and Plasma Groups

Result Number of samples Average of viral load and Standard of deviation

Group of Brushing 39 88956.2364

(184540.43607)

Group of Plasma 39 65222177.7169

(152875457.69133)

Table 3showed that brushing and blood for viral load and VCA can be interchangeable.

Result of analysis of sensitivity and specificity of EBV DNA Viral Load in Plasma can be seen in Table 4. This table showed that plasma of EBV viral load positive from brushing

method is 67,6%.

Table 4. Analysis of Sensitivity and Specificity EBV DNA Viral Load in Plasma

EBV Viral Load Brushing

Total Positive Negative

Positive Count 25 0 25

Plasma % within Brushing 67,6 0,0 64,1

Negative Count 12 2 14

% within Brushing 32,4 100 35,9

Total Count 37 2 39

% within Brushing 100 100 100

Sensitivity : 67.6%, Specificity : 100.0%, Accuracy : 69,2%

Variable values of viral load brush and plasma before and after treatment is presented in Table 5 are as follow:

Table 5. Variable values of viral load brush and plasma before and after treatment



570

Variables that analyzed Average value before treatment

Average value after treatment

p

Viral load brush 370959680.9 192995.3 0,008

Group of Plasma 109324.6 981.6 0,013

Discussion

Prognostic marker is needed besides the VCA-p18 and EBNA-1. In our preliminary

study, we found that the number of copies of EBV DNA viral load in NPC’s patients from Makassar2 was very high. Based on the specificity of NPC cases of Makassar, we further

analyze the relationship between EBV DNA viral load with IgA (VCA-p18 + EBNA -1), for use as a prognostic biomarker for NPC’s patients from Makassar.

The relationship between DNA EBV Viral Load with progressifity of NPC

This research is conducted with 39 NPC patients based on the

histopathologicexamination to see the relation of viral load with disease progression. Examination of viral load was conducted by Real Time PCR (RT- PCR) in NPC patients

using samples from brushing and peripheral blood (plasma). Three histopathological types are recognized in the World Health Organization (WHO) classifications. Type I is squamous cell carcinoma (SCC), type II is non-ceratinizing carcinoma and type III is undifferentiated

carcinoma.16 The distribution of the untreated NPC patients accord ing histopathology one WHO type I had Viral Load more than cut off although lower than patient WHO Type II

and WHO type III (252.320 copies/ml).

The results showed that the viral load in NPC patients after therapy both by brushing

and blood method decreased significantly. The research in Malaysia17 also showed the reduction is significantly from EBV DNA of viral load of NPC’s patients after the therapy. It can be concluded from the result of this research that the viral load in plasma may represent

brushing either before and after therapy, so the viral load can be checked easier, cheaper and more convenient for patients especially after therapy. In addition, it was concluded also that

EBV DNA of viral load can be used as a good prognostic indicator of disease progression.18,19,20

Therefore, noninvasive methods using peripheral blood can be proposed to replace methods of invasive brushing both for Viral Load and for VCA. Some researches on DNA

viral load through brushing have been done by Steven et al. (2005) in the University of Gajah Mada Yogyakarta but those researches on DNA viral load through plasma have not been conducted.4 Research on viral load of NPC’s patients in Makassar and Yogyakarta has shown

that the highest viral load of Makassar’s patients (866.400 million) is 40 times bigger than Yogyakarta’s patients (48.84 million). The viral load NPC patients from Makassar and

Yogyakarta are significantly different.14

The conclusions of this research are as follows: Plasma of EBV DNA of viral load is

equivalent with EBV DNA of viral load from smears brushing. Therefore, plasma EBV DNA of viral load can be used as a follow up with minimal invasive with relatively cheaper cost

and also as a replacement of DNA EBV of viral load brushing.



571

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