Automated quantification of MART1-verified Ki67 indices by digital image analysis in melanocytic lesions

The proliferation marker Ki67 is an important diagnostic and prognostic aid in surgical pathology. However, manual quantification in a counting frame to accurately establish the proliferation rate (Ki67 index) is cumbersome and time-consuming. Instead, digital image analysis of Ki67/MART1 double stains may provide fast and novel index computations for entire tumor sections.

To design and compare image analysis protocols that compute Ki67 indices of Ki67/MART1 double stains, to compare automated indices with previously published manual indices, and to compare the total number of proliferating cells (mimicking a Ki67 single stain) with the number of MART1-verified proliferating cells.

Whole slide images were captured from 48 melanomas and 77 nevi stained with an immunohistochemical cocktail against Ki67 and MART1. Ki67 indices were determined by digital image analysis and different equations based on number or area.

The differences between mean indices of melanomas and nevi were significant (P < .001) in all index computations. Number-based image analysis of lesions with more than 250 melanocytic cells misclassified 1 of 42 melanomas and 4 of 53 nevi, numbers comparable with manual counting. Automated indices were significantly higher than manual indices, as were indices of mimicked Ki67 single stains compared with MART1-verified Ki67 indices (P < .001).

Ki67 indices established by digital image analysis of Ki67/MART1 double stains demonstrated excellent abilities to discriminate melanomas from nevi with diagnostic performances equal to manually performed indices. Testing different definitions of the automated MART1-verified Ki67 index, no single definition stood out; thus, a variety of definitions may be used.