A framework for quantitative assessment of Ki67 distribution in preneoplastic bronchial epithelial lesions.

OBJECTIVE: Deregulated cell proliferation is a hallmark of cancer, and Ki67 immunostaining can be used to identify proliferating cells. Evaluation of cell proliferation may have utility as a biomarker of epithelial malignant transformation risk. To date, most analyses of Ki67 staining have been restricted to semiquantitative estimations of the degree of staining or the measurement of the fraction of Ki67-positive cells within the epithelium. We sought to develop a robust, objective means of quantitatively evaluating Ki67 immunostaining for lung precancerous lesions.

STUDY DESIGN: We quantified the spatial distribution of Ki67-expressing cells within the epithelium by means of (1) a cell-based Voronoi tessellation and (2) a basement membrane-referenced distance transform. This was undertaken in a large cohort of 613 lung biopsy sections representing normal, hyperplasia, squamous metaplasia and mild, moderate and severe dysplasia. For each section 21 features quantifying different aspects of the Ki67 staining were calculated. Intraobserver and inter-observer variation were recorded for a subset of the biopsy sections. We examined the behavior of each feature with respect to histopathological grade.

RESULTS: These measures demonstrated that proliferation is generally limited to layers 2, 3 and 4 of the epithelium (layer 1 being the basal layer). The proliferation in the basal layer is limited and does not increase with increasing grade of dysplasia. Interobserver and intraobserver effects on these features were assessed, and several were more robust with respect to measuring Ki67 expression pattern than the commonly used fraction of Ki67-positive cells.

CONCLUSION: Many of these quantitative features showed associations with histological grade that were as strong as the association that exists based on the fraction of Ki67-positive cells while being much more robust to interobserver- and intraobserver-associated variations. The measured spatial distribution of proliferating cells statistically demonstrated asymmetric cell division behavior in cells in the basal layer, a pattern attributed to stem cells giving rise to transient amplifying cells.