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chapter 3: Fluorescence in situ Hybridization
2012). Although the exact incidence and mechanism of such change are not yet clear, repeat testing should be considered when the tumor demonstrates acquired resistance following treatment with an ALK inhibitor, and a new therapeutic regimen must be selected. However, the data are still immature, and further research and validation are needed.
Chromogenic in situ Hybridization (CISH)
FISH is not without limitations, which include the need for highly specialized equipment, inevitable signal fading after long-term storage of tissue, and dark-field examination that may obscure tissue architecture and cytomorphology. The latter factor can be particularly problematic because lung cancers often show complex growth patterns intimately admixed with nontumor cells; differentiating tumor cells from nontumor elements may be difficult without architectural or cytoplasmic information. CISH has been developed to overcome these disadvantages of FISH. With CISH, each hybridization probe is visualized by chromogens rather than fluorochromes (Figure 14). CISH allows for detection of specific genetic alterations while preserving tumor architecture and cytomorphology under the routine bright-field microscope. The utility of ALK break-apart CISH for lung cancer diagnosis has been evaluated, and all studies have demonstrated excellent concordance with the results of FISH and/or RT-PCR (Kim 2011, Yoshida 2011a, Nitta 2013, Schildhaus 2013). CISH still must be validated for use in detecting ALK rearrangement in lung cancers, and different cutoffs (15% and 20%) and scoring criteria (1-diameter gap versus 2-diameter gap) have been used in the few reported studies.
Substrate Enzyme Secondary antibody Probe
Chromogenic reaction
Primary antibody
Target DNA
Figure 14. Principles of CISH; left. Individual probes are labeled with different haptens that are visualized by antibody reactions similar to that in IHC. ALK break-apart CISH is applied to ALK-positive tumors (right). Black dots represents normal ALK gene, and rearranged ALK genes are seen as split single red or blue signals.
Conclusion
Break-apart FISH is a reliable technique for the diagnosis of ALK-rearranged NSCLCs and has been accepted as the criterion standard to select patients for treatment with an ALK inhibitor. However, FISH testing heavily depends on careful preparation and interpretation with strict adherence to