Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis.

Abstract

Ki67 proliferative index (PI) is essential for grading gastroenteric and pancreatic neuroendocrine tumours (GEP NETs). Analytical and preanalytical variables can affect Ki67 PI. In contrast to counting methodology, until now little attention has focused on the question of clone equivalence and the effect of hot-spot size on Ki67 PI in GEP NETs. Using manual counting and image analysis, this study compared the Ki67 PI achieved using MM1, K2 and 30-9 to MIB1, a clone which has been validated for, and is referenced in, guidelines relating to assessment of Ki67 PI in GEP NETs. Forty-two pancreatic NETs were each immunohistochemically stained for the anti-Ki67 clones MIB1, MM1, K2 and 30-9. Ki67 PI was calculated manually and by image analysis, the latter using three different hot-spot sizes. In manual comparisons using single hot-spot high-power fields, non-MIB1 clones overestimated Ki67 PI compared to MIB1, resulting in grading discordances. Image analysis shows good agreement with manual Ki67 PI but a tendency to overestimate absolute Ki67 PI. Increasing the size of tumour hot-spot from 500 to 2000 cells resulted in a decrease in Ki67 PI. Different anti-Ki67 clones do not produce equivalent PIs in GEP NETs, and clone selection may therefore affect patient care. Increasing the hot-spot size decreases the Ki67 PI. Greater standardisation in terms of antibody clone selection and hot-spot size is required for grading GEP NETs. Image analysis is an effective tool for assisting Ki67 assessment and allows easier standardisation of the size of the tumour hot-spot.