Standardized Immunohistochemical and FISH Assays for ALK Detection

IHC analysis of ALK in formalin-fixed, paraffin-embedded tumor tissue sections was performed according to a clinically optimized and standardized assay using a highly sensitive intercalated antibody-enhanced polymer method based on 5A4-Histofine staining (ALK Detection kit; Nichirei Bioscience, Tokyo, Japan) [4 (link)]. Samples were prospectively analyzed with the FDA-approved Vysis ALK Break-Apart FISH Probe kit (Abbott Molecular, Abbott Park, IL, USA) using an ALK break-apart (i.e., split-signal) probe [20 (link)]. Samples were deemed FISH-positive if > 15% of scored tumor cells had split ALK 5' and 3' probe signals or had isolated 3' signals. CTCs detected by OBP-1101 were fixed and collected on glass slides to visualize ALK rearrangement in the nuclei of GFP-positive cells. FISH was also performed using the Vysis ALK Break-Apart FISH Probe kit along with the Vysis Paraffin & Post-Hybridization Wash Buffer kit (Abbott Molecular) as previously described [49 (link)].