PCR BASED ASSAY OF MITOCHONDRIAL DNA MUTATIONS IN ORAL LEUKOPLAKIA: CORRELATION WITH COX-2 EXPRESSION

Abstract

The global cancer burden is growing at an alarming pace.Head and neck cancersare amongst the most aggressive cancers, with OSCC representing the vast majority.OSCCis usually preceded by visible clinical changes in the oral mucosa (two-step process of cancer development) with varying types of dysplasia. Oral leukoplakia is by far the most common symbol of such clinical changes, representing 85% of these changes. The early diagnosis of such oral lesions is the key to preventing the progression to advanced-stage disease. Many molecular markers have been recognized to overcome the limitations inherent in the histologic grading and clinical assessmentto predict the malignant potential of lesions at risk. Cyclo-oxygenase-2 (COX-2) is considered one of the early molecular markers to detect the malignant potentiality of premalignant lesions. COX-2 enzymes contribute to cancerization via synthesis of mutagens, decreasing apoptosis and increasing immunosuppression and angiogenesis. Along years, many studies suggested a pivotal role of nuclear DNA mutations in the process of carcinogenesis. Recently, more and more authors claim that disruption of mtDNA should not be excluded from this analysis. Mutational screening of mtDNA revealed that mitochondrial D-loop (control region) is considered a hot spot for mutation in many cancers including oral squamous cell carcinoma. This study aimedto investigate COX-2 immunoexpression in both normal oral mucosa and oral leukoplakias whether dysplastic or not. Along with this molecular marker (COX-2), considered of a nuclear DNA origin, another molecular marker, but of a mitochondrial DNA origin, was investigated in this study on the same lesions. Correlation between these two markers was another aim in order to evaluate mtDNA as a marker for carcinogenesis. Mutational screening in the hot spot region of mtDNA (D-loop) was carried out after PCR and semiquantitation of this region. The results of this study showed early gradual up-regulation of COX-2 immunoreactivity starting from non dysplastic leukoplakias to severely dysplastic ones. In parallel with this nuclear marker results, both qualitative and quantitative alterations in mitochondrial D-loop were observed also as early as non dysplastic leukoplakia and intensified with dysplasia gradient.Even in non dysplastic cases showing COX-2 immunonegativity, these mtDNA alterations were observed indicating early occurrence of mtDNA changes, whether qualitative or quantitative, during the early stages of carcinogenesis which favor the leading role of mtDNA in the process of carcinogenesis rendering it as a promising molecular marker for early detection and progression of malignancy.