Quantitation of preinvasive neoplastic progression in animal models of chemical carcinogenesis

Abstract

An assay method that precisely quantitates the cellular and tissue changes associated with early, preinvasive neoplasia is much needed as a surrogate endpoint biomarker (SEB) in clinical trials to predict the potential efficacy of chemopreventive agents in bringing about cancer incidence reduction. Quantification of histological changes at the tissue level are potentially powerful SEB's since these visually apparent changes are common in all neoplastic development, regardless of tissue type or neoplastic cause. Currently, subjective inspection of the histological appearance of sectioned and stained material, or "grading," by experienced pathologists is used to evaluate neoplastic progression. This has well-known limitations of reproducibility, accuracy, and resolution of grading scale. Since neoplastic changes are visually apparent and morphologic in nature, quantification by image analysis is a measurement modality of choice. Image analysis was implemented through the use of high-resolution "tiled" images of complete tissue sections. A histological grading system, or "scale," was developed that could be expressed in terms of normal deviate units of multiple and different morphometric descriptors. Neoplastic growth was characterized quantitatively with multiple measurements on each tissue image tile, which were combined into a single number for each tile, i.e., a histologic grade per tile, and parameters from the distributions of these measurements were used to represent the histologic grade for the entire region considered. This concept provided a uniform final scale in similar units of measurement, regardless of which tissues were graded. Also, the grading scale automatically adjusted measurement variance for different tissues by using normal tissue for each different type to obtain the normalization to standard deviation (z) units. This further defined a uniform final scale and maintained standard references. Using this method, results from two well-known animal models of carcinogenesis, squamous cell carcinoma of SENCAR mouse skin induced by benzo(a)pyrene (B[a]P), and squamous cell carcinoma of the rat esophagus induced by N-nitrosomethylbenzylamine (NMBA), were compared to each other. Image analysis was performed on skin tissue sections from a total of 64 SENCAR mice, and esophagus tissue sections from 96 Fischer-344 rats. In both cases, a quantitative expression of the preinvasive neoplastic response to the carcinogen as a function of time of exposure was expressed along a continuous grading scale in standard deviation units (z). In the SENCAR mouse skin animal model, similar cohorts of 4 mice at 20 weeks showed significant modulation of B[a]P-induced neoplasia by treatment with the antiproliferative agent difluoromethylornithine, P < .05. In the rat esophagus animal model, similar cohorts of 6 rats at 10 and 15 weeks showed significant modulation of NMBA-induced neoplasia by treatment with the antimutagen phenethyl isothiocyanate, P < .05.