Proliferation assessment in breast carcinomas using digital image analysis based on virtual Ki67/cytokeratin double staining
- 496 Downloads
Manual estimation of Ki67 Proliferation Index (PI) in breast carcinoma classification is labor intensive and prone to intra- and interobserver variation. Standard Digital Image Analysis (DIA) has limitations due to issues with tumor cell identification. Recently, a computer algorithm, DIA based on Virtual Double Staining (VDS), segmenting Ki67-positive and -negative tumor cells using digitally fused parallel cytokeratin (CK) and Ki67-stained slides has been introduced. In this study, we compare VDS with manual stereological counting of Ki67-positive and -negative cells and examine the impact of the physical distance of the parallel slides on the alignment of slides. TMAs, containing 140 cores of consecutively obtained breast carcinomas, were stained for CK and Ki67 using optimized staining protocols. By means of stereological principles, Ki67-positive and -negative cell profiles were counted in sampled areas and used for the estimation of PIs of the whole tissue core. The VDS principle was applied to both the same sampled areas and the whole tissue core. Additionally, five neighboring slides were stained for CK in order to examine the alignment algorithm. Correlation between manual counting and VDS in both sampled areas and whole core was almost perfect (correlation coefficients above 0.97). Bland–Altman plots did not reveal any skewness in any data ranges. There was a good agreement in alignment (>85 %) in neighboring slides, whereas agreement decreased in non-neighboring slides. VDS gave similar results compared with manual counting using stereological principles. Introduction of this method in clinical and research practice may improve accuracy and reproducibility of Ki67 PI.
KeywordsKi67 Breast carcinoma Immunohistochemistry Digital image analysis Virtual double staining Standardization
Compliance with ethical standards
All experiments in this article were performed in accordance to Danish law.
Conflicts of interest
RR and MV have collaborated with Visiopharm in the development of the software. MV is member of the Visiopharm Scientific Advisory Board. The remaining authors declare that they have no conflict of interest.
- 1.Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311–322. doi: 10.1002/(SICI)1097-4652(200003)182:3<311:AID-JCP1>3.0.CO;2-9 CrossRefPubMedGoogle Scholar
- 3.Bosman FT, Carneiro F, Hruban R, Theise N (2010) Classification of tumours of the digestive system. WHO, LyonGoogle Scholar
- 5.Goldhirsch A, Wood WC, Coates AS (2011) Strategies for subtypes–dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol 22:1736–1747. doi: 10.1093/annonc/mdr304 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Goldhirsch A, Winer EP, Coates AS (2013) Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 24:2206–2223. doi: 10.1093/annonc/mdt303 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Gudlaugsson E, Klos J, Skaland I et al (2013) Prognostic comparison of the proliferation markers (mitotic activity index, phosphohistone H3, Ki67), steroid receptors, HER2, high molecular weight cytokeratins and classical prognostic factors in T1−2N0M0 breast cancer. Pol J Pathol 64:1–8CrossRefPubMedGoogle Scholar