c-FOS Expression Analysis in Pterygia Cell Spot Arrays

Abstract

Background/aim: Mechanisms of c-FOS activation in the onset and progression of pterygia remain under investigation. This study aimed to comparatively analyze c-FOS proto-oncogene expression levels in neoplastic pterygia and normal epithelia.

Materials and methods: We used a liquid-based cytology assay on thirty (n=30) pterygia cell populations and normal epithelia (n=10) extracted by a smooth scraping of conjunctiva epithelia. Applying a cell spot-based technique, we constructed five (n=5) slides, each containing eight (n=8) cell spots. A modified immune-cytochemistry (ICC) assay for c-FOS protein was used. Additionally, digital image analysis was implemented to calculate c-FOS immunostaining intensity levels.

Results: High staining intensity levels of c-FOS were detected in 12/30 (40%), whereas the rest 18/30 (60%) demonstrated moderate expression. c-FOS levels were statistically significantly higher in the pterygia compared to control tissues (p=0.001). c-FOS levels in the pterygia were not associated with the sex of patients (p=0.678), the presence of recurrent lesion (p=0.390) or the location of the lesion (p=0.158). The levels of c-FOS, however, were marginally significantly affected by the morphology of the pterygia (p=0.005). More precisely, the c-FOS levels were significantly higher in pterygia with a fleshy morphology.

Conclusion: c-FOS over-expression is observed frequently in pterygia. It seems to be critically involved in the molecular mechanism of the lesion by its over-expression affecting partially their morphological features. Cell spot liquid - based array analysis is an innovative, easy to implement technique for simultaneously evaluating a broad spectrum of molecules in multiple specimens on the same slide.

Keywords: Pterygia; c-fos; cell; image analysis; ocular; oncogenes.

Figure 1. c-FOS protein expression levels in pterygia fibro-endothelial cells A) Cell spot array. Note 8 spots of liquid-based cytological specimens obtained by scrapping, B) c-FOS dense expression [dark brown nuclear mainly, cytoplasmic staining pattern produced using 3-3,diaminobenzidinetetrahydrocloride (DAB) chromogen, original magnification 400×], C) Digital image analysis on a pterygia cell spot stained using a c-FOS antibody. In this progressive accurate and objective measurement process, reddish areas demonstrate different expression values, whereas green lines encircle specific areas of interest (range=0-255 continuous grey scale immunostaining intensity levels).