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Digital image analysis (DIA) is becoming central to the quantitative evaluation of tissue biomarkers for discovery, diagnosis and therapeutic selection for the delivery of precision medicine. In this study, automated DIA using a new purpose-built software platform (QuPath) is applied to a cohort of 293 breast cancer patients to score five biomarkers in tissue microarrays (TMAs): ER, PR, HER2, Ki67 and p53. This software is able to measure IHC expression following fully automated tumor recognition in the same immunohistochemical (IHC)-stained tissue section, as part of a rapid workflow to ensure objectivity and accelerate biomarker analysis. The digital scores produced by QuPath were compared with manual scores by a pathologist and shown to have a good level of concordance in all cases (Cohen's κ>0.6), and almost perfect agreement for the clinically relevant biomarkers ER, PR and HER2 (κ>0.86). To assess prognostic value, cutoff thresholds could be applied to both manual and automated scores using the QuPath software, and survival analysis performed for 5-year overall survival. DIA was shown to be capable of replicating the statistically significant stratification of patients achieved using manual scoring across all biomarkers (P

Digital pathology and the adoption of image analysis have grown rapidly in the last few years. This is largely due to the implementation of whole slide scanning, advances in software and computer processing capacity and the increasing importance of tissue-based research for biomarker discovery and stratified medicine. This review sets out the key application areas for digital pathology and image analysis, with a particular focus on research and biomarker discovery. A variety of image analysis applications are reviewed including nuclear morphometry and tissue architecture analysis, but with emphasis on immunohistochemistry and fluorescence analysis of tissue biomarkers. Digital pathology and image analysis have important roles across the drug/companion diagnostic development pipeline including biobanking, molecular pathology, tissue microarray analysis, molecular profiling of tissue and these important developments are reviewed. Underpinning all of these important developments is the need for high quality tissue samples and the impact of pre-analytical variables on tissue research is discussed. This requirement is combined with practical advice on setting up and running a digital pathology laboratory. Finally, we discuss the need to integrate digital image analysis data with epidemiological, clinical and genomic data in order to fully understand the relationship between genotype and phenotype and to drive discovery and the delivery of personalized medicine.

The involvement of Wnt signalling in cancers including ovarian, gastric, and breast cancer is well characterised. Wntless (Wls) (also known as GPR177, Evi, and Srt) is a key modulator of Wnt protein secretion, and was recently found to be highly overexpressed in malignant astrocytomas, promoting proliferation, survival, and migration of glioma cells. It was hypothesised that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signalling. Using immunohistochemical analysis of ovarian, gastric, and breast cancer tissue microarrays, we found that Wls was overexpressed in a subset of tumours from each cancer subtype. Wls overexpression was associated with poorer clinical outcomes in gastric cancer. Interestingly, a strong correlation was observed between Wls expression and human epidermal growth factor receptor 2 (HER2) expression. Eight of eight (100%) HER2-positive intestinal gastric carcinomas, five of five (100%) HER2-positive serous ovarian carcinomas, and 41 of 64 (64%) HER2-positive breast carcinomas expressed Wls. This finding has clear biological and clinical implications. HER2 is an important predictive biomarker in breast and gastric cancer for selection of patients likely to respond to trastuzimab. However, a substantial proportion of HER2-positive patients do not respond to this therapy. Recent literature suggested a possible pathway in which Wls and HER2 may be involved via the known mediator of trastuzimab and chemotherapy resistance NFkB, which may explain the low response rate of patients with HER2-positive breast, ovarian, and gastric cancers to agents that target HER2. An in-vitro study has been commenced to try to confirm the existence of this pathway. This study should provide significant insights into the mechanistic association between these important signalling molecules. Funding Queens University Belfast.