Uveal melanoma: mean of the longest nucleoli measured on silver-stained sections

To summarize and compare the various histologic methodologies for using nucleoli to assess the malignant potential of uveal melanoma.

This was an observational series of 100 samples of uveal melanoma in which histologic sections were studied. The cases were selected so that approximately half (n = 49) of the tumors were from patients who had died of metastatic malignant melanoma. The 51 remaining tumors were from patients who had survived at least 9 years without development of metastasis. Central sections from the uveal melanomas were stained using the colloidal silver nitrate stain for nucleolar organizing regions (AgNOR). These were compared with an adjacent hematoxylin and eosin (H&E)-stained section. A light microscope with a micrometer inset into the eyepiece (x10) was used at a final magnification of x1000 under oil immersion to measure the length of the nucleolus along the longest axis and the width perpendicular to that axis. From at least twenty cells selected from random fields throughout the tumor, the mean of the 10 longest and widest nucleoli (MLN) was calculated. Seven samples had to be discarded because the nucleoli were unmeasurable.

T-tests and Cox proportional hazard regression analysis indicated that the MLN of nucleolar length as measured on AgNOR-stained slides was as significant as cell type but was more significant than other histopathologic prognosticating variables measured and evaluated in this study. These prognosticators included tumor size, calculated as the largest tumor dimension; MLN width; and MLN length, as measured on H&E-stained sections.

It has previously been demonstrated that AgNOR-stained nucleoli, unlike H&E-stained nucleoli, can be captured and measured by an automated image analyzer with prognostically significant results. This new method of simple oil-immersion measurements of the longest AgNOR-stained nucleoli length in microscopic sections of uveal melanoma provides an inexpensive and highly significant method for predicting outcome in patients with uveal melanoma. Because of the high contrast with the background, the silver-stained nucleoli clearly define the nucleolar boundaries, rendering them readily discernible and allowing greater ease and speed of measurement when compared with H&E-stained nucleoli. The method of random sampling that was used was comparable with linear sampling in predicting outcome. Highly necrotic tumors, however, had to be excluded from the study because of loss of nucleolar morphology.