Chapter 3
Fluorescence in situ Hybridization (FISH)
By Akihiko Yoshida and Marileila Varella-Garcia
FISH with a break-apart probe set was originally developed for detecting gene fusions created by interchromosomal translocations. Break-apart FISH is a reliable diagnostic method in surgical pathology because it is easily applicable to FFPE specimens even when the exact fusion partners are not known. FISH with break-apart probes for ALK has been successfully incorporated into diagnostic practice for lymphomas and mesenchymal tumors, and the discovery of ALK rearrangement in a rare subset of NSCLCs broadened the application of break-apart FISH (Soda 2007). However, in the latter setting, FISH has been associated with unexpected challenges, primarily because the common fusion variants occur between ALK (2p23.2) and the closely situated gene EML4 (2p21) through intrachromosomal inversions; only rarely is ALK fused with other genes through intrachromosomal translocations. Thus, break-apart FISH for the diagnosis of lung cancers with ALK rearrangement must be performed with special attention to technical details and interpretation of the results.
FISH Probe Design
The ALK break-apart probe is typically designed by labeling the 3' (telomeric) part of the fusion breakpoint with one fluorochrome and the 5' (centromeric) part with another fluorochrome. Some variation exists among different commercial and custom-made reagents as to the specific genomic areas covered by the probes and the distinct fluorochromes used for labeling. In the kit developed by Abbott Molecular (Figure 1; Vysis LSI ALK Break Apart FISH Probe Kit), 300 kb 442 kb 3' 5' the 3' part (approximately 300 kb) 2p23.2 is represented by an orange signal ALK EML4 (Spectrum Orange, often referred to as red in the literature because the Normal signal is detected by the interference filter in the red wavelength), and the ~12.5 Mb inversion EML4-ALK fusion 5' part (approximately 442 kb) is repALK:EML4 resented by a green signal (Spectrum ALK:EML4 Green). This probe set was approved as a companion diagnostic assay by Figure 1. The Vysis LSI ALK Break Apart FISH Probe Kit (Abbott Molecular) the US FDA for an ALK inhibitor and is for testing for the presence of EML4-ALK fusion gene (ALK rearrangement) in lung cancers. commonly used worldwide.