The U.S. Food and Drug Administration (FDA)-approved VENTANA ALK (D5F3) CDx Assay more accurately selected patients eligible to receive anakinase lymphoma kinase (ALK) tyrosine kinase inhibitor treatment compared with another commonly used immunohistochemistry (IHC) assay, the 5A4 IHC assay, a study published in the Journal of Thoracic Oncology has shown.1

For the study, researchers sought to compare the 2 widely used IHC assays in lung adenocarcinoma samples. Fluorescence in situ hybridization (FISH) and IHC with the VENTANA ALK (D5F3) CDx Assay with the OptiView Amplification Kit and a standard IHC test with the clone 5A4 were performed on 1031 lung adenocarcinomas. Next-generation sequencing was performed in samples with inconsistent results between FISH and IHC.

Results showed that the FDA-approved IHC assay had a sensitivity of 90.9% ± 2.6%, specificity of 99.8% ± 0.6%, positive predictive values of 93.8% ± 2.1%, and negative predictive values of 99.7% ±0.6%, while the 5A4 IHC assay had a sensitivity of 90.9% ± 2.6%), specificity of 98.3% ± 1.3%, positive predictive value of 63.8% ± 4.2%, and negative predictive value of 97.7% ± 0.6%. These findings suggest a high degree of false positives with the 5A4 IHC assay.

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Next-generation sequencing was performed in 5 samples in which FISH and IHC data differed. Next-generation sequencing confirmed the FISH data, suggesting that there were a very low proportion of patients that had ALK rearrangements that were negative at the ALK protein expression level, and a low percentage of patients without detectable ALK rearrangements that are ALK protein expression-positive.

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The study also demonstrated a 100% response rate to ALK inhibitors in FISH-negative/IHC analysis-positive cases and a 46% response rate in FISH-positive/IHC analysis-negative cases.


  1. Marchetti A, Di Lorito A, Pace MV, et al. ALK protein analysis by IHC staining after recent regulatory changes: a comparison of two widely used approaches, revision of the literature, and a new testing algorithm [published online ahead of print February 22, 2016]. J Thorac Oncol. doi: 10.1016/j.jtho.2015.12.111.