Standardized demonstration of silver-stained nucleolar organizer regions-associated proteins in archival oral squamous cell carcinomas and adjacent non-neoplastic mucosa.

Standardized silver staining and quantitation of nucleolar organizer regions-associated proteins (AgNORs) on routinely formalin-fixed and paraffin-embedded tissues has been recently established. The present study was designed to assess its applicability and reproducibility on archival oral squamous cell carcinoma specimens, with special emphasis on the invasive tumor front. Standardized image cytometric AgNOR analysis was performed at the invasive zone and central parts of the tumors as well as in adjacent dysplastic and normal oral mucosa by applying a modified silver staining on routinely processed archival tissues after wet autoclave pretreatment for protein retrieval. A statistically highly significant and reproducible difference of all of the four standardized AgNOR parameters evaluated (mean AgNOR number, mean AgNOR area, coefficient of variation (CV) of AgNOR number, and CV of AgNOR area per nucleus) was observed between the different areas assessed; there was a statistically significant gradual decrease in the CV of the AgNOR number from the invasive tumor front to central parts of the carcinomas, from dysplastic mucosa to normal epithelium. The observed increase of AgNORs at the invasive front of oral squamous cell carcinomas seems to indicate a subgroup of tumor cells with increased biosynthetic activity and malignant potential. Wet autoclave pretreatment before silver staining of AgNORs is necessary for a reliable visualization of single AgNOR dots. Second-order statistics of AgNOR features, e.g., CV values, offer a potent and highly reproducible parameter for AgNOR quantitation.