Quantitative histopathological assessment of ocular surface squamous neoplasia using digital image analysis

Abstract

The aim of this retrospective pilot study was to evaluate the Aperio nuclear V9 algorithm as an image analysis tool to observe the histopathological changes of ocular surface squamous neoplasia (OSSN). A histopathological assessment, including the Ki-67 proliferative index (PI) of immunohistochemically-stained tumor conjunctiva (TC) and healthy conjunctiva (HC) tissues, was performed in six cases of OSSN. The Aperio V9 algorithm was applied to digital images of the tissue specimens to count the Ki-67 PI and to measure the nuclear area indices. This digital algorithm was validated using stereological and visual analysis methods. The visual scoring of Ki-67 PI ranged from 22 to 60% (mean, 38.5%), and from 5 to 20% (mean 9.5%) in TC and HC tissue, respectively. The computer-aided analysis, using the Aperio nuclear V9 algorithm, revealed that the Ki-67 PI ranged from 21.5 to 43.5% (mean, 33.6%), and from 1.9 to 21.0% (mean, 11.8%) in the TC and HC tissue, respectively. The stereological method demonstrated that the Ki-67 PI ranged from 30.1 to 51.5% (mean, 41.0%), and from 3.2 to 30.1% (mean, 15.1%) in the TC and HC tissues, respectively. The strongest association in the collinearity of regression analysis was observed between the Aperio nuclear V9 algorithm/stereological models in the TC tissue (r²=0.7; P=0.04) and the HC tissue (r²=0.7; P=0.03), and the visual/stereological models in the TC tissue (r²=0.7; P=0.04) and the visual/Aperio nuclear V9 algorithm models in the HC tissue (r²=0.7; P=0.04). A weak and statistically insignificant association was identified between the visual/Aperio nuclear V9 algorithm analysis in the TC tissue (r²=0.4; P=0.2) and the visual/stereological models in the HC tissue (r²=0.5; P=0.13). No significant difference was observed between the nuclear area of the TC (mean, 36.5 μm²) and HC (mean, 35.7 μm²; P=0.88) tissues. It was concluded that the Aperio nuclear V9 algorithm is a useful tool for the reliable analysis of histopathological changes of OSSN. The results of this computer-aided algorithm correlate strongly with the stereological method when assessing the Ki-67 PI.

Keywords: Ki-67; digital image analysis; ocular surface squamous neoplasia.

Figure 1

(A) Image of conjunctival squamous cell carcinoma in patient 3 showing a nodular mass with foci of leukoplakia on the surface of the lesion. (B) Digital image of the excisional biopsy (stain, Hematoxylin and Eosin). (C) Ki-67 immunohistochemically-stained slide shows areas of tumor conjunctiva (green) and healthy conjunctiva (red). (B and C: Magnification, ×20). (D) Aperio nuclear v9 algorithm indicates positive nuclei as brown, orange and yellow, and negative nuclei as blue. A grid of the stereology model has been superimposed over the digital Aperio image.