AIMS: 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 focussed on the question of clone equivalence and effect of hotspot 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.
METHODS AND RESULTS: 42 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 3 different hotspot sizes. In manual comparisons using single hotspot 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 hotspot from 500 to 2000 cells resulted in a decrease in Ki67 PI.
CONCLUSION: Different anti-Ki67 clones do not produce equivalent PIs in GEP NETs and clone selection may therefore affect patient care. Increasing the hotspot size decreases the Ki67 PI. Greater standardisation in terms of antibody clone selection and hotspot 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 hotspot.