title: Development and Validation of a Histological Method to Measure Microvessel Density in Whole-Slide Images of Cancer Tissue.
abstract: Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.
authors: Division of Physiopharmacology, University of Antwerp, Antwerp, Belgium.|HistoGeneX NV, Antwerp, Belgium.
authors: Koen M
authors: http://orcid.org/0000-0001-7212-7682
authors: KM
authors: Marien
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Valerie
authors: V
authors: Croons
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Yannick
authors: Y
authors: Waumans
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Ellen
authors: E
authors: Sluydts
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Stefanie
authors: S
authors: De Schepper
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Luc
authors: L
authors: Andries
authors: Department of Pathology, University Hospital Brussels (UZ Brussel), Brussels, Belgium.
authors: Wim
authors: W
authors: Waelput
authors: StatUa Center for Statistics, University of Antwerp, Antwerp, Belgium.
authors: Erik
authors: E
authors: Fransen
authors: CORE (Translational Cancer Research Unit, GZA Hospitals), Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.
authors: Peter B
authors: PB
authors: Vermeulen
authors: HistoGeneX NV, Antwerp, Belgium.
authors: Mark M
authors: MM
authors: Kockx
authors: Division of Physiopharmacology, University of Antwerp, Antwerp, Belgium.
authors: Guido R Y
authors: GR
authors: De Meyer
mesh_terms: D001808:Blood Vessels / Q000276:immunology / Q000473:pathology*; D006801:Humans; D009369:Neoplasms / Q000098:blood supply*; D009389:Neovascularization, Pathologic / Q000473:pathology*; D015588:Observer Variation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D015203:Reproducibility of Results
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1