Fluorescence in situ hybridization was performed on unstained 4 μm formalin-fixed paraffin embedded tumor tissue sections with the use of an ALK break-apart probe set (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular, Rungis, France) using a paraffin pretreatment reagent kit (Vysis, Abbott Molecular). Assays were performed following the manufacturer’s instructions. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole/Vectashield (Vektor Laboratories, AbCys, Paris, France).
Sections were analyzed with a Metafer slide scanning system (Metasystems, Altlussheim, Germany) under a 63 × oil immersion objective with a fluorescence microscope (M1, Zeiss, Stuttgart, Germany) equipped with appropriate filters, a charge-coupled device camera, and the FISH imaging and capturing software Metafer 4 (Metasystems). Signals were enumerated with the ISIS imaging system (Metasystems). Non-rearranged ALK showed fusion (orange signals) or very close apposition of the probes adjacent to the 3′ (red) and 5′ (green) ends of the gene. Rearranged ALK appeared as split 3′ and 5′ signals. Tumor tissues were considered ALK-FISH positive (ALK rearranged) if >15% of tumor cells showed split red and green signals and/or single red signals, according to previous publications.3 (link),7 (link),10 (link),11 (link) Otherwise the samples were considered ALK-FISH negative.
Sections were analyzed with a Metafer slide scanning system (Metasystems, Altlussheim, Germany) under a 63 × oil immersion objective with a fluorescence microscope (M1, Zeiss, Stuttgart, Germany) equipped with appropriate filters, a charge-coupled device camera, and the FISH imaging and capturing software Metafer 4 (Metasystems). Signals were enumerated with the ISIS imaging system (Metasystems). Non-rearranged ALK showed fusion (orange signals) or very close apposition of the probes adjacent to the 3′ (red) and 5′ (green) ends of the gene. Rearranged ALK appeared as split 3′ and 5′ signals. Tumor tissues were considered ALK-FISH positive (ALK rearranged) if >15% of tumor cells showed split red and green signals and/or single red signals, according to previous publications.3 (link),7 (link),10 (link),11 (link) Otherwise the samples were considered ALK-FISH negative.
