Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as ''interphase cytogenetics'', can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed. Most of the currently applied methods, e.g., conventional cytogenetics, analysis of restriction fragment length polymorphisms, or the various modifications of the polymerase chain reaction (PCR), require in vitro cultivation of the tumor cells or tissue disaggregation and cell lysis. A close correlation with morphology, however, is necessary to elucidate the significance of molecular or cytogenetic findings associated with malignancy. In situ hybridization using appropriate DNA probes allows for the simultaneous assessment of chromosomal aberrations and morphological features in tumor specimens (Gray et al. 1994; Joos et al. 1994). With this technique, that is also referred to as ''interphase cytogenetics'', karyotypic changes can be analyzed at a single cell level. The major advantage of interphase cytogenetics is the possibility of examining chromosomal aberrations among morphologically or immunophenotypically different cells or cell groups that may be evident in an individual tumor sample. Therefore, interphase cytogenetics is especially useful for correlating molecular or cytogenetic alterations to morphological details in cytological preparations and tissue sections. The method of choice for interphase cytogenetics is fluorescence in situ hybridization (FISH) which is performed with DNA probes that are labeled with fluorochromes. In this technical overview, we discuss the principles, applications, and limitations of FISH and provide elaborated protocols.