Immunohistochemistry (IHC) may be considered a standard method for determination of ALK tumor status to select treatment-naive patients with advanced non-small cell lung cancer (NSCLC) for alectinib therapy, according to results of an exploratory analysis published in the Journal of Thoracic Oncology.

An accurate assessment of ALK tumor status has been previously identified as an essential step in the selection of patients with advanced NSCLC for treatment with ALK receptor tyrosine kinases (TKIs), including crizotinib and alectinib. Both IHC and fluorescence in situ hybridization (FISH) have been used to evaluate whether NSCLC tumor specimens are characterized by ALK gene fusions. The former test evaluates the level of ALK protein expression on tumors, whereas the FISH test provides a more direct measure of ALK gene rearrangement.

In order to assess the concordance of ALK IHC and ALK FISH testing within the context of the efficacy outcomes of patients treated with ALK TKIs, an exploratory, retrospective analysis was performed based on data from a randomized, phase 3 trial (ALEX study; Identifier: NCT02075840) that evaluated first-line alectinib vs crizotinib in advanced ALK-positive NSCLC as determined centrally by the Ventana IHC assay.

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Retrospective central FISH testing (Vysis assay) was performed on tumor specimens for all 303 patients enrolled in the study who had been randomly assigned in a 1:1 ratio to receive alectinib or crizotinib. Of the 303 tumor specimens, 242, 61, and 39 were classified as IHC-positive/FISH-positive, IHC-positive/FISH indeterminate, and IHC-positive/FISH-negative, respectively.

At a median follow-up of approximately 2 years, a comparison of the primary study outcome measure, investigator-assessed progression-free survival (PFS), for patients receiving alectinib vs crizotinib in each of these tumor status subgroups, IHC-positive/FISH-positive (106 receiving alectinib; 92 receiving  crizotinib), IHC-positive/FISH indeterminate (25 receiving  alectinib; 36 receiving  crizotinib), and IHC-positive/FISH-negative (21 receiving alectinib; 18 receiving  crizotinib), revealed the following:

  • Median PFS for IHC-positive/FISH-positive subgroup: 34.8 months (alectinib) vs 12.6 months (crizotinib); (hazard ratio [HR], 0.37; 95% CI, 0.25-0.56)
  • Median PFS for IHC-positive/FISH-indeterminate subgroup: 22.8 months (alectinib) vs 9.8 months (crizotinib); (HR, 39; 95% CI, 0.20–0.78)
  • Median PFS for IHC-positive/FISH-negative subgroup (39 individuals): 3.8 months (alectinib) vs 7.4 months (crizotinib); (HR, 1.33; 95% CI, 0.6–3.2)
  • Other efficacy endpoints, such as objective response rate and duration of response, reflected the trends observed for PFS in these 3 subgroups.

Based on these results, the study authors concluded that “an uninformative ALK FISH test result should not prevent physicians from treating patients with alectinib if the patient’s tumor is determined to be ALK positive by another approved method, such as [the Ventana ALK IHC assay].”

However, they noted that “the question is how best to identify patients who will derive benefit from ALK inhibitors in the small group of patients with discordant ALK IHC/FISH results.”

They further noted that in this subgroup “nonresponders may either be truly ALK fusion-negative, have high ALK protein expression or ALK gene copy number in the absence of ALK rearrangements, or may not respond for other reasons than ALK fusion negativity.”

To address this question in this analysis, targeted next-generation sequencing (NGS) was performed on the tumor and/or plasma samples classified as IHC-positive/FISH-negative. Of these samples, no ALK fusion and an EML4-ALK fusion was found in 57.1% 42.9% of cases, respectively.

For those cases of IHC-positive/FISH-negative tumors where tissue-based NGS does not reveal the underlying cause of the discrepancy, the study authors commented that “careful consideration should be taken to identify the optimal treatment for patients with discordant IHC/FISH results. RNA-based or plasma [circulating tumor DNA] ALK fusion detection methods should be considered.”


Mok T, Peters S, Camidge DR, et al. Outcomes according to ALK status determined by central IHC or FISH in patients with ALK-positive NSCLC enrolled in the phase III ALEX study. J Thorac Oncol. Published online October 23, 2020. doi:10.1016/j.jtho.2020.10.007