Quantitative evaluation of in vitro transformation by analysis of morphological and biochemical markers and statistical descriptors

The EC directive REACH (EC1907/2006) sets, amongst others, the need for all the chemicals to be tested for their carcinogenic potential. In vitro assays can provide a fast and reliable tool for screening purposes. The Cell Transformation Assay (CTA) is one of the in vitro assays in the most advanced phase of the validation process and the only one able to evaluate both the genotoxic and the non-genotoxic potentials. The evaluation of results of the CTA is based on the scoring of transformed colonies (foci) by a trained expert on the basis of their morphological features. Levels of cell packing and multilayered growth, as well as fibroblastic shape of cells, criss-crossing and invasion of the surrounding monolayer features are evaluated for classification. While the decision making process is based on standard criteria, their interpretation is potentially biased, especially in borderline cases, due to a certain degree of subjectivism inherent in the evaluation of qualitative features. This aspect is critical towards the international validation of the CTA assay: subjectivity driven error might in fact result in under or over estimation of the carcinogenic potential of tested compounds. In this work, different approaches were used to develop an objective method to give decisional support to the operator in the classification procedure. Biological markers related to the transformation process (p53, cx43), and to a general cell stress (Hsp70) were analyzed. A novel technique for the in focus localization of biological markers of transformation was developed. RNA whole genome screening was used to set the conditions for future molecular characterization of foci-derived cell lines. A novel, Quantitative Index of Dissimilarity has been obtained by statistical descriptors capturing morphological features and employing an unsupervised image analysis approach, in order to help the operator in the decisional process of scoring the borderline cases.