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J Pathol Clin Res. 2019 Jan;5(1):63-78. doi: 10.1002/cjp2.119. Epub 2018 Nov 29.

Combined epithelial marker analysis of tumour budding in stage II colorectal cancer.

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Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland.
Department of Pathology, Misurata Cancer Center, Misurata, Libya.
Department of Basic Sciences, Dentistry Faculty, University of Misurata, Misurata, Libya.
Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland.
Auria Biobank, University of Turku and Turku University Hospital, Turku, Finland.
Department of Surgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland.
Department of Pathology, University of Helsinki and HUSLAB, Helsinki University Hospital, Helsinki, Finland.
Department of Oncology and Pathology, Science for Life Laboratory, Karolinska Institutet, Solna, Sweden.
Department of Oncology, Turku University Hospital, Hämeentie 11, Turku, Finland.


Tumour budding predicts survival of stage II colorectal cancer (CRC) and has been suggested to be associated with epithelial-to-mesenchymal transition (EMT). However, the underlying molecular changes of tumour budding remain poorly understood. Here, we performed multiplex immunohistochemistry (mIHC) to phenotypically profile tumours using known EMT-associated markers: E-cadherin (adherence junctions), integrin β4 (ITGB4; basement membrane), ZO-1 (tight junctions), and pan-cytokeratin. A subpopulation of patients showed high ITGB4 expression in tumour buds, and this coincided with a switch of ITGB4 localisation from the basal membrane of intact epithelium to the cytoplasm of budding cells. Digital image analysis demonstrated that tumour budding with high ITGB4 expression in tissue microarray (TMA) cores correlated with tumour budding assessed from haematoxylin and eosin (H&E) whole sections and independently predicted poor disease-specific survival in two independent stage II CRC cohorts (hazard ratio [HR] = 4.50 (95% confidence interval [CI] = 1.50-13.5), n = 232; HR = 3.52 (95% CI = 1.30-9.53), n = 72). Furthermore, digitally obtained ITGB4-high bud count in random TMA cores was better associated with survival outcome than visual tumour bud count in corresponding H&E-stained samples. In summary, the mIHC-based phenotypic profiling of human tumour tissue shows strong potential for the molecular characterisation of tumour biology and for the discovery of novel prognostic biomarkers.


colorectal cancer; digital pathology; epithelial-to-mesenchymal transition; integrin β4; multiplex immunohistochemistry; prognostics; tumour budding

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