High resolution multi‐color detection of oncogenic rearrangements in single cells –molecular staging of tumors based on their ‘oncogene rearrangement status’

Oncogenic rearrangements leading to abnormal expression of proto‐oncogenes are a common feature of human solid tumors. The detection of such rearrangement events is complicated by the small amount of tissue available for analysis after tumor biopsy, the fact that cells in metaphase stage are not available in most cases, and by time constraints. We are developing sensitive assays based on fluorescence in situ hybridization (FISH) for detection of gene rearrangements. We describe our multicolor approach to the detection of rearrangements and amplification of the epidermal growth factor receptor (EGFR) gene on chromosome 7p12. The simultaneous hybridization of differently labeled DNA probes or probe contigs for the EGFR gene as well as adjacent loci or genes of interest allows an accurate stage classification of single interphase cells with regard to their ‘oncogene rearrangement status’. By providing high resolution information about relative position and copy number of multiple linked loci, our analysis is far superior to the commonly used dual‐color FISH analysis of tumor cells using probes for just EGFR and the centromere of chromosome 8. Our analysis of a broad spectrum of cell types ranging from normal cells and tissue sections to primary cultures to neoplastic tumor cell lines provides a detailed overview of the technology and highlights its potential role in predicting a tumor’s response to therapy. While results presented here focus on a FISH‐based detection and characterization of EGFR gene rearrangements, similar approaches, many of which are presently under development in our laboratories, can be applied to the characterization of other sites of frequent amplification in tumors.