Combined Use of ALK Immunohistochemistry and FISH for Optimal Detection of ALK-Rearranged Lung Adenocarcinomas

INTRODUCTION ALK gene rearrangements occur in ~5% of lung adenocarcinomas (ACA), leading to ALK overexpression and predicting response to targeted therapy. Fluorescence in situ hybridization (FISH) is the gold standard for detection of ALK rearrangements in lung ACA but requires specialized equipment and expertise. Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with reports of newer antibodies showing excellent sensitivity and specificity for ALK-rearranged lung ACA. METHODS In this study, we analyze ALK IHC (5A4 clone) in 186 cases from our clinical service and compare with ALK FISH and EGFR and KRAS mutation status. RESULTS Twelve cases had concordant ALK protein overexpression and ALK rearrangement by FISH. Three ALK-rearranged cases lacked ALK protein expression. Of these discrepant cases, one had a coexisting EGFR mutation and a subtle “atypical” ALK rearrangement with a break in the 5’ centromeric portion of the FISH probe. One case had a concurrent BRAF mutation; followup testing on a metastasis revealed absence of the ALK-rearrangement with persistent BRAF mutation. In one ALK-rearranged, protein negative case, very limited tissue remained for ALK IHC, raising the possibility of false negativity due to protein expression heterogeneity. Importantly, ALK protein expression was detected in one case initially thought not to have an ALK rearrangement. In this case, FISH was falsely negative due to interference by benign reactive nuclei. After correcting for these cases, ALK IHC was 93% sensitive and 100% specific as compared to FISH. CONCLUSIONS ALK IHC improves the detection of ALK rearrangements when used together with FISH, and its use in lung adenocarcinoma genetic testing algorithms should be considered