Validation of mitosis counting by automated phosphohistone H3 (PHH3) digital image analysis in a breast carcinoma tissue microarray.

Mitosis counting in H&E stained sections is the most informative constituent of the Nottingham histological grade in breast carcinoma prognosis. Phosphohistone H3 (PHH3) immunohistochemistry (IHC) is a highly specific marker of mitoses, with practical application in identifying mitoses in poorly fixed or distorted tissue and is of prognostic significance in breast carcinoma. Our aim was to assess methods of PHH3 IHC mitosis counting in a tissue microarray (TMA) of 2 mm cores from 36 resected breast carcinomas. Mitoses in H&E and PHH3 stained slides were manually scored by pathologist consensus and expressed as counts/2 mm. PHH3 stained cores were also evaluated by automated digital image analysis (DIA). Results were compared using Spearman correlation. A strong and significant correlation was observed between manual PHH3 and manual H&E mitotic counts (correlation = 0.81; p < 0.0001) and between automated PHH3 DIA and manual H&E mitotic counts (correlation = 0.79; p < 0.0001). More mitoses were identified with PHH3 IHC than with H&E. Manual and DIA PHH3 counts were strongly and significantly correlated (correlation = 0.83; p < 0.0001) and of similar absolute values. PHH3 DIA is a valid alternative to manual counting with potential application in breast cancer reporting and prognostication.