Automated assessment of Ki-67 in breast cancer: the utility of digital image analysis using virtual triple staining and whole slide imaging.

AIMS Precise evaluation of proliferative activity is essential for the stratified treatment of luminal-type breast cancer (BC). Immunohistochemical staining of Ki-67 has been widely used to determine proliferative activity and is recognized to be a useful prognostic marker. However, there remains discussion about the methodology. We aimed to develop an automated and reliable Ki-67 assessment approach for invasive BC. MATERIALS AND RESULTS A retrospective study was designed to include two cohorts consisting of 152 and 261 consecutive patients with luminal-type BC. Representative tissue blocks upon surgery were collected, and three serial sections were stained automatically with Ki-67, pan-cytokeratin, and p63. The whole slides were scanned digitally and aligned using VirtualTripleStaining - an extension to the VirtualDoubleStainingTM technique provided by Visiopharm software. The aligned files underwent automated invasive cancer detection, hot spot identification, and Ki-67 counting. The automated scores showed a significant positive correlation with the pathologists' scores (r=0.82, P<0.0001). The digitally assessed low Ki-67 group (<20%) demonstrated a significant better prognosis (breast cancer-specific survival, P=0.030; hazard ratio: 0.038) than high Ki-67 group among curative patients with standard therapies (n=130). CONCLUSIONS Digital image analysis yielded similar results to the scores determined by experienced pathologists. The prognostic utility was verified in our cohort, and an automated process is expected to have high reproducibility. Although some pitfalls were confirmed and thus need to be monitored by laboratory staff, the application could be utilized for the assessment of BC. This virtual technique on whole slide might give us new insights into the future cancer research.