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International Journal of Biological & Medical Research

Int J Biol Med Res. 2014; 5(3): 4350-4354

HIA-a24 Gen Allele At Peripheral Blood Samples And Nasopharyngeal

Cytobrush In Nasopharyngeal Carcinoma Patients In Makassara b c d

Eka Savitri *, Freddy George Kuhuwael , Abdul Qadar Punagi , Imelda Gunawati Agus

a,b,c Department of Ortholaryngology, Faculty of Medicine, Hasanuddin University, Kampus Tamalanrea, Jl. Perintis Kemerdekaan Km.10, Makassar, South Sulawesi, Indonesia.

A R T I C L E I N F O A B S T R A C T

Keywords:

Epstein - Barr virusNasopharyngealCarcinomaHLA - A24Gene.

Original Article

Nasopharyngeal cancer (NPC) is a malignancy that is highly associated with Epstein - Barr virus infection (EBV), highly oncogenic, easily transmitted through saliva and is a multifactorial genetic disease with endemic character. EBV infection will cause an immune response in the patient themselves to recognize and eliminate foreign antigens which is associated with the ability of the antigen in presenting HLA molecules to cytotoxic T cells. Some of the genes under study which are genes that influence the occurrence of NPC are HLA genes. Aims: This study aims to determine the presence of the HLA- A24 allele in peripheral blood samples and nasopharyngeal cytobrush NPC patients in Makassar. Methods: The study design was a cross sectional analytic observational test of Fischer's Exact test on 21 samples of NPC patients who come in Dr. Wahidin Sudirohusodo Hospital. The examination was conducted by BOOM'S method for DNA isolation, and followed by PCR with primers forward and reverse HLA - A24. Result: The results showed the presence of the HLA allele A-24 in peripheral blood samples (37.5%) and nasopharyngeal cytobrush (42.9%) and stated that by performing the same method for taking specimen and examination, HLA-A24 existing in nasopharyngeal cytobrush was also found in peripheral blood. Conclusion: It is concluded that the HLA A-24 was obtained in patients with NPC in Makassar.

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1. Introduction

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Nasopharyngeal cancer (NPC) is a malignant epithelial

neoplasm that occurs in upper tract autodigestive and a

multifactorial genetic disease with endemic character. There are

approximately 80,000 new cases each year with the highest

incidence of 2500 cases in South China per year. The highest

incidence of NPC is in the Mongoloid race with a fairly high

frequency in South China, Hong Kong, Vietnam, Thailand, Malaysia, 1,2,3.Singapore and Indonesia

NPC is in the 4th rank among all cancers in Indonesia after

uterine, breast, and skin cancer, with incidence of 4.7 per 100,000

population of DR.Wahidin Sudirohusodo Hospital reported in 10-

years period (2000-2009) and there were 362 cases (57.28%) of 4,5the malignant tumors of head and neck .

Viruses that play a role in increasing the risk of NPC are the

Epstein Barr Virus (EBV). After EBV infects B lymphocytes, it could

be latent and persistent for life in the host6. In some researches, the

presence of EBV DNA from patients' peripheral blood samples

is also found. This shows how an EBV avoids the immune system of 7the human body and is stabilized at lymphocytes B6, .

B cells infected by EBV will express some latent antigens such

as LMP1 and LMP2 (LMP2A and LMP2B)8,9,10. LMP2A is a latent

antigen expressed in B lymphocytes that are infected with EBV. 8,10,12,13One of the most suspicious genetic factors is the HLA genes .

These genes are inherited through heterozygous process and are

co-dominant. As a result, groups with particular HLA will face the

risk of certain diseases. Some diseases are presumably related to

HLA, so people with certain diseases will have specific HLA 14,15gene .

According to research on Southeast Asian and Caucasian 17,18populations, HLA - A24 and HLA - A2 are commonly found .

Indigenous Indonesian people who have HLA - A24 possess higher

possibility to NPC19. According to Middleton et al. (2003), allele

HLA- A2402 is allele of the HLA - A24 gene that is widely available 19in Asian populations . Immunogenetic studies in Jakarta in 1997

found HLA - A24 and HLA - B63. Both class I antigen phenotypes

are suspected as a causative factor for the indigenous Indonesian 20NPC .

* Corresponding Author : Eka Savitri

Jl. Perintis Kemerdekaan Km.10, Makassar, South Sulawesi, Indonesia. e-mail : ekasapan@yahoo.com

Copyright 2010 BioMedSciDirect Publications. All rights reserved.c

Eka Savitr et.al Int J Biol Med Res. 2014; 5(3): 4350-4354

4351

This study aims to determine the presence of the HLA- A24

allele in peripheral blood and nasopharyngeal cytobrush in NPC

patients in Makassar.

This is an observational analytic study. NPC patients who agree

to follow this study were upheld by histopathologic examination.

Venous blood was taken and brushed with guidance of

nasoendoscopy nasopharynx. The study was conducted at

Dr.Wahidin Sudirohusodo Hospital Makassar and Biomolecular

Engineering Laboratory Faculty of Medicine in October - November

2013. The study population was patients with NPC who came to the

ENT clinic at Dr.Wahidin Sudirohusodo Hospital Makassar for two

months. Sampling was conducted by consecutive sampling.

Inclusion criteria were patients with NPC based on the results of

histopathological examination included in the study that were

willing to sign a statement to participate in the study after receiving

an explanation (informed consent). Exclusion criteria were patients

uncooperative and refused to participate in this study.

Patients with inclusion criteria and agreed to participate in this

study, their blood samples were taken from venous blood samples

and then brushing nasopharyngeal guided endoscopic

visualization. Further isolation of DNA was done through BOOM'S

method. Subsequently, PCR with forward and primary reserve for

HLA-A24 with the amount of product 464 bp was performed.

During October to December 2013 has done the research to see

HLA-A24 gene allele from peripheral blood and nasopharyngeal

cytobrush in 21 patients with NPC.

Characteristics of the study sample are presented in Table 1.

Based on patient age nasopharyngeal carcinoma, the most in the

range of 40-49 years as much as 38.0%. 16 males (76.2%) and 5

women (23.8%) with a ratio of 3.2:1 consists of 9 Bugis people

(42.8%), 5 Makassar people (23.8%), and 1 person for all other

tribes of South Sulawesi (4.7%).

Histopathological distribution of NPC patients according to

WHO obtained showed the highest type III is 17 people (80.9%),

WHO type II by 4 people (19.1%), and WHO type I obtained in this

study can be seen in Table 2.

PCR results of peripheral blood samples show DNA bands

appeared on several samples with varying thickness and led to 6

samples (W2, W6, W8, W10, W12, W16) of 16 samples (37.5%) and

9 samples (B02, B04, B06, B08, B10, B12, B16, B19, B20) of 21

samples (42.9%) in nasopharyngeal cytobrush.

Based on statistical tests (Fischer's Exact test) in 16 paired

samples, it suggests that HLA-A24 presenting in 100% of

nasopharyngeal cytobrush samples was also found in the

peripheral blood of NPC patients and HLA-A24 contained in

peripheral blood was also found in the nasopharynx cytobrush.

Materials and Methods

Brush of HLA Positive

Negative

6

0

0

10

HLA Blood

Positive Negative

Patients

Table 3. Comparison of HLA-A24 existing in peripheral

blood samples and nasopharyngeal cytobrush

Type I (carcinoma of squamosa cell with

ceratinized)

TypeII (Differentiated carcinoma with no ceratinized)

Type III (Undifferentiated carcinoma/ anaplastic)

0

4

17

0

19,1

80,9

Histopathology result (WHO)

Patients %

Table 2. Distribution of patients by histopathology test

(WHO Types)

Table 1. Characteristic of the study samples

Sex

Men

Women

Age

< 20

20-29

30-39

40-49

50-59

>60

Tribe

Bugis

Makassar

Toraja

Bali

Buton

Ternate

Luwuk Banggai

Kaili

16

5

0

2

1

8

5

5

9

5

1

1

1

1

1

1

76,2

23,8

0

9,5

4,7

38,0

23,8

23,8

42,8

23,8

4,7

4,7

4,7

4,7

4,7

4,7

Characteristic Patient %

Results

4352

Figure 1. The results of HLA-A24 PCR in peripheral blood

samples

Graph 1. Evaluation of HLA-A24-positive samples by tribe

Discussion

Graph 2. Evaluation of HLA-A24-positive samples by

histopathology

Figure 2. The results of HLA-A24 PCR in cytobrush

nasofaring samples

0

2

4

6

8

10

12

14

PCR (+) PCR (-)

bugis

makassar

mandar

toraja

lain-lain

0

2

4

6

8

10

12

PCR (+) PCR (-)

WHO type I

WHO type II

WHO type III

Graph 1

In the evaluation of 21 samples of peripheral blood and

nasopharyngeal cytobrush, it is found that most HLA-A24 positive

at 4 samples in Bugis tribe (19%), 2 samples in Makassar tribe

(9%), 1 sample in Mandar tribe (4%), and 2 samples beyond South

Sulawesi (9%).

Graph 2.

Based on Graph 2, it can be seen that 7 of the 21 samples

(33.3%) were positive for HLA-A24 with WHO type III and 2

samples (9%) with WHO type II.

In the study of 16 peripheral blood samples and 21 samples of

nasopharyngeal cytobrush, it is found that there are more men

(76.2%) compared to women (23.8%) with a ratio of 3.2 : 1 , while

on age and ethnicity of Nasopharyngeal Carcinoma patients , in

which most of them is in the range of 40-49 years by 38.0 %,

consisting of 9 Bugis people (42.8%) and 5 Makassar people

(23.8%).

Eka Savitr et.al Int J Biol Med Res. 2014; 5(3): 4350-4354

4353

Distribution of NPC patients according to WHO histopathology,

type III is found to be the highest that consists of 17 people (80.9%).

According to Pieter (2013) in Makassar, from 50 patients with NPC,

WHO type III is obtained by 82.36 % 21.

The presence of HLA - A24 picture of DNA bands in 6 of 16

samples of patients with NPC are similar to those obtained by Islami

(2011) in Jogjakarta. The study also found the presence of HLA -

A2402 in peripheral blood samples of patients with NPC22.

Likewise, the research on immunogenetic in Jakarta in 1997 found

HLA - A24 and HLA - B63, as well as a research conducted by

Judajana (2007) which supports HLA - A24 , HLA - A2 and HLA - A11

HLA class, which is the main target as a genetic marker systems 20,23immune to the NPC .

Cytobrush samples also obtained presence of HLA - A24, 9

samples from 21 patients with NPC sample. Moningka's research

(2012) also obtain HLA - A24 in cytobrush samples of nasopharynx.

Based on these studies, DNA bands in some samples of peripheral

blood and cytobrush suggests the possibility of a few virus peptide 24or latent antigens are not amplified in the examination .

According to Munir (2007), the presence of susceptibility genes

and protective alleles are different at each location and ethnicity,

allegedly due to the binding molecule and peptide epitopes of

different renderers. In addition it is also due to the different

sampling, a different distribution of HLA genes in each population

and due to the strong influence of environmental factors on the 25,26incidence of NPC in these locations .

Based on statistical tests, the presence of HLA - A24 that are in

cytobrush 100% can also be found in peripheral blood samples.

This indicates the presence of HLA - A24 in NPC tissues can also be

found or represented through peripheral blood samples.

Tribal evaluation of PCR positive HLA - A24 were Bugis (19 %)

as the highest rate, then Makassar (9 %) and Mandar (4 %).

According to Munir (2007), Indonesia's population is unique due to

the process of racial integration and as a result of migration or gene

flow. Some research suggests the similarity patterns of HLA genes

with the Chinese Indonesian population. In population genetic

studies in Jakarta, it is concluded that the HLA antigens on

Indonesian population is classified in the group of Mongoloid25.

Indonesia has three different Subras, namely Subras

Paleomongoloid Proto Malays, Subras Deutro Paleomongoloid and 27,28Subras Proto Malanesia . Bugis and Makassar is categorized as

Paleomongoloid Subras Deutro Malay. Javanese population is

categorized as Subras Paleomongoloid Malay Deutro which also get

HLA - A 24 HLA - A2 and HLA - A1123.

Histopathological evaluation on a sample of HLA - A24 positive

PCR obtained WHO type III (33.3 %) and WHO type II (9 %) from 21

samples. According to Thompson (2004), EBV has been detected in

nasopharyngeal carcinoma in situ, before undifferentiated

carcinoma (WHO type III ), and it also determines the progression of 29malignancy . This shows that EBV infection occurs before

neoplasia. Therefore, there is no presence of the HLA relationship

with histopathological types but there is a confirmation about

the presence of EBV in WHO type III which is 100 % correlated with

EBV and reported a high incidence rate in Southeast Asia30.

EBV express latent antigens type II also produces undifferentiated

carcinoma ( WHO type III ) . Additionally, undifferentiated

carcinoma (WHO type III) is also caused by abnormal cytogenetic

related to deletions in chromosome 3 short chain loci 3p25 and 293p14 .

Based on this research, HLA-A24 is found in the peripheral

blood and nasopharyngeal cytobrush. By using the technique of

collecting samples and examination methods with Fischer Exact

test trials, the HLA-A24 found in cytobrush were also present in the

peripheral blood. It was concluded that the HLA-A24 can be found

in the peripheral blood and cytobrush nasopharyngeal in patients

with nasopharyngeal carcinoma in Makassar.

Further research needs to be done to look for other HLA alleles

in order to find genetic risk factors in Makassar.

We acknowledged to Dr. dr. Burhanuddin Bahar, M.S. from

Faculty of Public Health, Hasanuddin University, Makassar for his

help and supervised during the research.

Conclusion

Acknowledgement

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ISSN: 0976:6685.c

Eka Savitr et.al Int J Biol Med Res. 2014; 5(3): 4350-4354