Immunodetection of EGF-receptor, c-erbB oncogenes and HSV-1 antigene in oral mucosal swab preparation from kretek (clove cigarette) smokers

Smoking is the biggest factor for oral cavity malignancy. An oral cavity mucous swab was performed to 30 smokers (mild, moderate and severe) and 10 non smokers which was followed by immunocytochemistry smear using antibody for EGF-R, c-erbB, and HSV-1 antigenes to reveal the effect of smoking on the occurence of oral cavity malignancy. �he results from this e�periment are analy�ed statistically using one-way Anava. It is discovered that based on the assessment of the e�pression level towards EGF-R, and c-erbB ��c��e�e ��ere ��� �� �er�� ��������c���� ����ere�ce be��ee� ��e �����er �r���� ���� ��� �����er �r���� (��<0.01). Me������le ��e re���l��� �r�� ��e ������e�����e�� �����r��� HSV-1 ��c��e�e �� ��� ����� ��������c���� differences between the smoker group (mild, moderate and severe) and the non smoker group (p>0.05). �he results of this study shows that the more a person smokes, the higher possibility of oral cavity malignancy and that the antibody for EGF-R and c-erbB can be used as a marker for early detection of oral cavity malignancy caused by smoking.


INTRODUCTION
Malignant tumors may be found in all parts of the human body including the oral cavity.Reports from various pathology center in Indonesia shows that around 3.2% of the malignant tumors are oral cavity malignant tumors. 1 Oral cavity malignant tumors, just as other body part malignant tumors, are triggered by intrinsic or e�trinsic risk factors.Epidemiologically, it has been shown that smoking is the biggest risk factor for oral cavity malignant tumor. 2,3�he hot smoke that is inhaled continuously into the mouth is a physical thermal stimulant that brings bad effects on the oral tissue.On the other hand, the cigarette burned contains around 4,000 chemicals including some carcinogenic chemicals such as tar, nicotine and nitrosamine.It has been stated frequently that nitrosamine can trigger malignancy in human. 4nother factor that plays the role for oral cavity malignancy is virus. 5Herpes simple� virus in certain environment can be oncogenic and causes cell transformation. 6Several reports show that herpes simple� virus with nitrosamine found in tobacco can cause oncogene transformation. 7n severe tumor cells, it will be shown that EGFreceptors are found on the cell membrane surface and are considered as a product from the c-erbB oncogene.�he EGF-receptor may be used as an important marker in making an early diagnosis of oral cavity malignant tumor. 8It has also been reported that antibody towards HSV-1 increases in oral cavity malignant tumor patients 9,10 and that oral cavity malignant tumor cells contain HSV-1 antigen on its cell membrane surface. 11ntil now, routine e�aminations such as histopathological e�amination using hemato�ylin eosin dye and cytological e�amination using Papanicolaou dye are frequently used to make the diagnosis.However, due to the accuracy in identifying various tumor/antigen markers, the use of immunohistochemistry/immunocytochemistry methods have been introduced in the pathology ��el� ��r��� ��e l����� ��� �ec���e��.T�e��e �e������ can be used to manage various problems in diagnosing undiagnosed neoplasma using the routine e�aminations above.
Based on that reason, this study is performed to discover whether there is an increased e�pression of EGF-receptor and c-erbB oral cavity mucous cell swab retrieved from kretek smoker's oral cavity and there is an increased e�pressions of HSV-1.
By considering the fact that smoking is one of the biggest risk factors for oral cavity malignant tumor development 2,3 , there is a big possibility that an increased e�pression of EGF-receptor oncogenes, c-erbB and HSV-1 will be found in the mucous cell from oral swab in kretek smoker's oral cavity in line with the amount of cigarette consumption.

MATERIALS AND METHODS
�his study is a laboratory study with "e� post facto" design.�hirty staff of Faculty of Dentistry, Universitas Padjadjaran, who are smokers were recruited as subjects for this study.�he inclusion criteria include male, 40 years old or above who has been smoking from 5 years or more, and who is clinically proven as not having oral cavity abnormality.�hese smokers were grouped into mild smokers (less than 10 cigarettes a day), moderate smokers (between 11-20 cigarettes a day) and severe smokers (more than 20 cigarettes a day).As a comparison, 10 non smoker staff was also included in the study.

Antibody
Antibodies used in this study were antibodies for EGF-receptor, c-erbB and HSV-1 antigene.�he monoclonal antibody for EGF-R was purchased from Dako M866, Mo a Hu; klon EGFR-1; was diluted with a ratio of 1:10.Monoklonal antibody for c-erbB, was purchased from Dako A-0485, Mo a Hu diluted with a ratio of 1:50.Antibody for HSV-1 antigene, was purchased from Dako N-1562 without diskated.LSAB kit pero�idase was purchased from Dako KO-681.

RESULT
Oral cavity mucous swabs were performed to 30 staff of the Faculty of Dentristry Universitas Padjadjaran, who are smokers and 10 non smoker staff.Of this swab, an immunocytochemistry smear was performed using EGF-receptor, c-erbB and HSV-1 antigenes.

DISCUSSION
Malignant tumors can occur in all human body parts, including the oral cavity.Reports from various pathology center in Indonesia shows that around 3.2% of the malignant tumors are oral cavity malignant tumors. 1 Oral cavity malignant tumors, just as other body part malignant tumors, are triggered by intrinsic or e�trinsic risk factors.Epidemiologically, it has been shown that smoking is the biggest risk factor for oral cavity malignant tumor. 2,3he result of a study done by Schwant� 12 ������� ����� ��ere ��� �� ��������c���� rel������������ between smoking and malignancy in various part of oral cavity. 12A research in United States reveals that the risk for oral cavity malignant tumor for smokers is 5 times higher than the non smokers.�he smoking habit has been proven to increase the risk of oral cavity malignancy such as stated by Wynder et al. who show that around 97% of oral cavity malignant tumor patients are smokers.�he risk of oral cavity malignant tumor will be bigger for those who are 40 years old or more and heavy smokers. 3hen the cigarette end is lighted in a high temperature, the cigarette that contains air, steam, gas, liquids, imperfectly burned particles is a very comple� organic and inorganic compound mi�ture.�he hot smoke that is inhaled continuously into the mouth is a physical thermal stimulant that brings bad effects on the oral tissue.On the other hand, the cigarette burned contains around 4,000 chemicals including some carcinogenic chemicals, i.e. substances that trigger tumor development, such as tar, nicotine and nitrosamine.�he burned cigarette also containes co-carcinogen, i.e. substances that enhance tumor development but do not start it; cilioto�ic agent, i.e. substances that disturb or destroy protective activities of the cilia in the respiratory tract, such as acrolein and hydrogen cyanide and nitrogen o�ide. 13It is also stated that nitrosamine may create malignancy in human. 4nother factor that plays the role for oral cavity malignancy is virus. 5Herpes simple� virus in certain environment can be oncogenic and causes cell transformation. 6Several reports show that herpes simple� virus with nitrosamine found in tobacco can cause oncogene transformation. 7 this research, an immunochemistry e�amination is performed to assess the e�pression level of EGF-R, c-erbB and HSV-1 oncogenes in EGF-R, c-erbB and HSV-1 oncogenes in , c-erbB and HSV-1 oncogenes in oral mucous smear cells related to various levels of kretek smokers.�he immunocytochemistry e�amination results show that there is a relationship between the e�pression level of EGF-R and c-erbB EGF-R and c-erbB and c-erbB oncogene with various levels of kretek smokers.However, there is no relationship between the e�pression level of HSV-1 antigene e�pression with various levels of kretek smoker level.
EGF-receptor (EGF-R) is a 170 kD EGF-R) is a 170 kD ) is a 170 kD �r������e�br���e ���r�����e-����e�����c �����������������e found in chromosome-7.In a normal condition, EGF-R can be found in basal cell layer.However, can be found in basal cell layer.However, in oral cavity malignancy, they can be found in all layers. 6In malignant tumor cells, EGF-receptor will be shown in the cell membrane surface and can be used as an important marker in making an early diagnosis for oral cavity malignant tumor. 8In this research result, there will be increased EGF-EGF-R e�pression level in mild, moderate and severe e�pression level in mild, moderate and severe smoker groups compared to the non smoker group (�ab.1).�his shows that the more someone smokes, the higher the number of EGF-r oncogene EGF-r oncogene oncogene because there is a bigger possibility of oral cavity malignancy.
c-erbB is a homologous proto-oncogen with EGF-R that codes 185 kD and produces putative that codes 185 kD and produces putative growth factor glicoprotein transmembrane of the tyrosinekinase family.Proto-oncogen c-erbB, when it is mutated, will change into an oncogene whose product will be overe�pressed.Several studies stated that the inhibiton of apoptosis by the mutation and overe�pression of c-erbB oncogene have a big contribution to the tumor growth because the cell survival and proliferation will increase. 14�he apoptosis inhibition is mostly thought as a result of the missing physiological apoptotic signal/cell death needed.From the result of this research, it seems that there is an increased c-erbB e�pression level in mild, moderate and severe smokers compared to the non smoker group (�ab.2).�his shows that the more someone smokes, the higher the c-erbB oncogene level due to the apoptosis inhibition and, as a result, there will be higher chance that an oral cavity malignancy will occur.
It has been reported that the antibody for HSV-1 has increased in oral cavity malignant tumor patients 9,10 and the oral cavity malignant tumor cells also contain HSV-1 antigene on the cell membrane surface. 11HSV-1 in certain environment can cause the cell to transformed and being and oncogene. 7Some reports show that HSV-1 can work synergically with chemical carcinogenes and causes oncogene transformation. 7HSV-1 may be a cocarcinogen with tobacco.From this research, there is no increased e�pression of HSV-1 antigenes in oral cavity mucosal swab cells in the smoker groups, including the mild, moderate and severe smokers, compared to non smoker group (�ab.3).�his is different from the results of other studies that may be due to the small sample si�e and the small number of Indonesians who are infected by HSV-1 compared to the western.However, the results from a study performed by Shillitoe et al. 15 also show that not all studies show the e�istence of HSV in malignancy.
In this research, there is also no clear relationship between the involvement of herpes simple� virus and the occurrence of oral cavity malignancy.�his implies that despite the unclear involvement of the virus, the oncogene transformation from nitrosamine still happens.So, smoking makes a malignancy more probable compared to virus involvement.
From all results in this study it is apparent that there is EGF-R and c-erbB oncogenes activation EGF-R and c-erbB oncogenes activation and c-erbB oncogenes activation in smoker oral cavity mucous swab preparation.According to several studies, it is revealed that the oncogen activation is related to a genetic change that is responsible for malignant change in the future, including oral cavity malignancy. 16From this research it is concluded that moderate and severe smoking habit may cause genetic changes or mutation that may create changes towards oral cavity malignancy.

CONCLUSION
�he more cigarettes consumed by a smoker, the bigger the possibility for oral cavity malignancy.No clear relationship between smoking with Herpes Simple� virus involvement and the occurence of oral cavity malignancy.Smoking is an important possibility for oral cavity malignancy.Antibody towards EGF-R and cerb-B oncogene can become EGF-R and cerb-B oncogene can become and cerb-B oncogene can become a marker for early detection of the possibility of oral cavity malignancy caused by smoking.

Figure
Figure 3. Cells that are positive for c-erbB oncogene c-erbB oncogene oncogene (200x).