Fluorescence staining of oral cancer using a topical application of 5-aminolevulinic acid: fluorescence microscopic studies

Introduction: Topical application of 5-aminolevulinic acid (5-ALA) by means of a rinsing solution has been shown to be a promising new procedure in the diagnosis of oral malignancies. However, for assessing the reliability of this method regarding fluorescence-guided tumor resections and photodynamic therapy, further information on the distribution and penetration depth of 5-ALA-induced protoporphyrin IX (PPIX) in the tissue is needed. Methods: 24 patients suffering from oral cancer were included in this investigation. Biopsies were taken immediately after fluorescence examination and either used as native sections for immediate fluorescence microscopic examination (n=3) or shock frozen in liquid nitrogen and prepared as frozen sections (n=46). Fluorescence imaging and digital image processing were utilized in order to determine the presence of PPIX in regions of various histologies as well as the penetration depth of PPIX into solid tumor. Results: PPIX fluorescence in the tissue was limited to the epithelium. Both normal and dysplastic epithelium showed PPIX fluorescence. In the stroma, no PPIX fluorescence was found. In some cases (n=3/4) invasive carcinomas did not show PPIX fluorescence, while the adjacent or overlying normal epithelium was strongly fluorescent. The penetration depth of PPIX after topical application of 5-ALA was found to be limited to less than 1 mm. Conclusion: PPIX fluorescence induced by topical application of 5-ALA can be very useful in the determination of superficial tumor margins. However, due to the limited penetration depth there is a risk of not accurately recognizing the infiltration depth of solid tumors. The aim of further investigations will be to assess the tissue distribution and depth of penetration of PPIX following systemic application of 5-ALA.