Automated image analysis tools for Ki67 breast cancer digital pathology images would have significant value if integrated into diagnostic pathology workflows. Such tools would reduce the workload of pathologists, while improving efficiency and accuracy. Developing tools that are robust and reliable to multicentre data is challenging, however, differences in staining protocols, digitization equipment, staining compounds and slide preparation can create variabilities in image quality and colour across digital pathology datasets. In this work, a novel unsupervised colour separation framework based on the IHC colour histogram (IHCCH) is proposed for the robust analysis of Ki67 and hematoxylin stained images in multicentre datasets. An “overstaining” threshold is implemented to adjust for background overstaining, and an automated nuclei radius estimator is designed to improve nuclei detection. Proliferation index and F1 scores were compared between the proposed method and manually labeled ground truth data for 30 TMA cores that have ground truths for Ki67+ and Ki67- nuclei. The method accurately quantified the PI over the dataset, with an average proliferation index difference of 3.25%. To ensure the method generalizes to new, diverse datasets, 50 Ki67 TMAs from the Protein Atlas were used to test the validated approach. As the ground truth for this dataset is PI ranges, the automated result was compared to the PI range. The proposed method correctly classified 74 out of 80 TMA images, resulting in a 92.5% accuracy. In addition to these validations experiments, performance was compared to two colour-deconvolution based methods, and to six machine learning classifiers. In all cases, the proposed work maintained more consistent (reproducible) results, and higher PI quantification accuracy.

In this work, a novel proliferation index (PI) calculator for Ki67 images called piNET is proposed. It is successfully tested on four datasets, from three scanners comprised of patches, tissue microarrays (TMAs) and whole slide images (WSI), representing a diverse multi-centre dataset for evaluating Ki67 quantification. Compared to state-of-the-art methods, piNET consistently performs the best over all datasets with an average PI difference of 5.603%, PI accuracy rate of 86% and correlation coefficient R = 0.927. The success of the system can be attributed to several innovations. Firstly, this tool is built based on deep learning, which can adapt to wide variability of medical images—and it was posed as a detection problem to mimic pathologists’ workflow which improves accuracy and efficiency. Secondly, the system is trained purely on tumor cells, which reduces false positives from non-tumor cells without needing the usual pre-requisite tumor segmentation step for Ki67 quantification. Thirdly, the concept of learning background regions through weak supervision is introduced, by providing the system with ideal and non-ideal (artifact) patches that further reduces false positives. Lastly, a novel hotspot analysis is proposed to allow automated methods to score patches from WSI that contain “significant” activity.

In this work, a novel proliferation index (PI) calculator for Ki67 images called piNET is proposed. It is successfully tested on four datasets, from three scanners comprised of patches, tissue microarrays (TMAs) and wholeslide images (WSI), representing a diverse multicentre dataset for evaluating Ki67 quantification. Compared to state-of-the-art methods, piNET consistently performs the best over all datasets with an average PI difference of 5.603%, PI accuracy rate of 86% and correlation coefficient R = 0.927. The success of the system can be attributed to a number of innovations. Firstly, this tool is built based on deep learning, which can adapt to wide variability of medical images – and it was posed as a detection problem to mimic pathologists’ workflow which improves accuracy and efficiency. Secondly, the system is trained purely on tumour cells, which reduces false positives from non-tumour cells without needing the usual pre-requisite tumour segmentation step for Ki67 quantification. Thirdly, the concept of learning background regions through weak supervision is introduced, by providing the system with ideal and non-ideal (artifact) patches that further reduces false positives. Lastly, a novel hotspot analysis is proposed to allow automated methods to score patches from WSI that contain “significant” activity.