IN SITU HYBRIDIZATION, WITH OR WITHOUT TYRAMIDE SIGNAL AMPLIFICATION, IN EVALUATION OF HUMAN PAPILLOMAVIRUS STATUS INEARLY STAGE CERVICAL CARCINOMA
Kubelka-Sabit KB*, Prodanova ILj, Zografski GD, Basheska NT
*Corresponding Author: Katerina Kubelka-Sabit, M.D., M.Sci., Department of Histopathology and Clinical Cytology, Institute of Radiotherapy and Oncology, Medical Faculty, Vodnjanska 17, 1000 Skopje, Republic of Macedonia; Tel.: +389-2-3147-902; Fax: +389-2-3111-430 ; E-mail:catkubelka@yahoo.co.uk
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INTRODUCTION

 

In the 1980s, zur Hausen [1] revealed that human papilloma virus (HPV) could be a possible etiologic agent in cervical carcinogenesis. The association of high-risk HPV types with malignant transformation of epithelial cells of the uterine cervix is now accepted [2-9]. Human papillomavirus type 16 is the most common high-risk HPV type [10-13], being detected in nearly half of all cervical carcinoma and cervical intraepithelial neoplasia (CIN) 2/3 lesions [14-16], and is followed by HPV type 18, which is detected in 14-25% of invasive cervical carcinomas [14,15]. Human papillomavirus types 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 are also included in the high-risk group [17]. Many molecular methods for the detection of HPV DNA have been developed, which are generally based on polymerase chain reaction (PRC) or in situ hybridization (ISH) [18]. Introduced by Gall and Pardue in 1969 [19], ISH detects nucleic acids, thus enabling evaluation 42 of results in preserved tissue or cells. By increasing the cellular permeability, contact between probe and target nucleic sequence is enhanced, the morphology of cells or tissue is completely preserved [20- 22], and approximative estimation of the number of copies of viral DNA in the host nucleus [21,23] and characterization of the state of the viral DNA, whether in episomal or integrated form [24], are permitted. Conventional in situ hybridization (CISH) also allows recognition of multiple HPV infection by detecting the concomitant presence of DNA from more than one type of HPV, but has relatively low sensitivity. With non isotopic indirect fluorescent in situ hybridization (FISH) on cytological material, the sensitivity of CISH is limited to the detection of 1 kb of target DNA [23]. In routine work on formalin- fixed tissue, the sensitivity is limited to the detection of 40 kb of target DNA and 10-20 copies of mRNA or viral DNA [6,21]. Many modifications to the protocol have been introduced to increase the sensitivity of the CISH. Some are based on amplification of the target nucleic acid sequence, while others are based on amplification of the hybridization signal [21,23,25,26]. In situ hybridization with tyra mide signal amplification (ISH TSA) [21] uses biotinyl ated tyramide and its interaction with horseradish peroxid ase (HRP). After an initial binding of streptavidin-peroxid ase to the biotinylated probe, the peroxidase oxidizes the biotinyl tyramide, which then binds to nuclear proteins that contain aromatic amino acids, such as tyrosine, trypto phan or phenylalanine, thereby preventing diffusion of the amplification products [26]. We have compared CISH with ISH TSA in the evaluation of the HPV status on formalin-fixed paraffinembedded tissue sections from early stage invasive cervical carcinoma.




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