ALK Status (Lung Cancer) Methodology

To determine the ALK status in non-small-cell lung cancer (NSCLC), a validated ALK IHC test is used as a first screening method. Patient samples that show ALK IHC positivity are subsequently subjected to ALK FISH analysis for confirmation of a positive ALK status (1).

ALK IHC testing is performed using the Ventana anti-ALK (D5F3) Rabbit monoclonal primary antibody (Roche, CE-IVD) used in combination with the OptiView DAB IHC Detection kit and OptiView Amplification Kit as a fully automated immunohistochemistry assay on the Ventana BenchMark XT automated slide stainer.

ALK FISH testing is performed using the Vysis ALK Break Apart Probe kit (2p23/ALK translocation detection, Abbott, CE-IVD).

Clinical Implication

ALK is considered a key oncogenic driver in non-small cell lung cancer (NSCLC). Rearrangement of the ALK locus on 2p23 has been implicated in the development of NSCLC. The ALK gene codes for a transmembrane glycoprotein with tyrosine kinase activity. In-frame rearrangements with the known fusion partners place the ALK kinase domain under the control of a different gene promoter. This fusion results in a chimeric protein (like EML4-ALK) with constitutive tyrosine kinase activity that has been demonstrated to play a key role in controlling cell proliferation. ALK rearrangements are seen in about 5% of NSCLC patients. Importantly, ALK rearrangement-positive patients have been shown to respond to treatment with ALK inhibitor crizotinib (2).

Specimen Requirements

Acceptable specimens for the assay are formalin (10% buffered or Zinc formalin)-fixed (6-48 hours), paraffin-embedded NSCLC tissue specimens. Sectioned slides must be analyzed within 3 months after sectioning date (required for ALK IHC testing). 


1 representative paraffin block is preferred. Alternatively, 5 unstained freshly sectioned tissue sections are accepted (1 slide of 4 or 5 µm thickness for H&E staining , a minimum of 2 slides of 4-5 µm thickness for ALK IHC testing and a minimum of 2 slides of 3 µm thickness for ALK FISH testing).

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.


Fixatives other than buffered formalin, prolonged fixation time or exposure to severe heath or chemicals may give rise to inadequate results.

Concordance between the ALK IHC and ALK FISH assay was determined at HistoGeneX. Outof the 86 samples tested for validation, the concordance between FISH and IHC was 92% (79/86) according the criteria used in the initial crizotenib study (cut-off: ≥ 15%). Yet, if a cutoff of > 15% is used (as defined by the FDA) concordance between FISH and IHC in our sample set was 97% (83/86; 4 samples had 15% of cells with ALK rearrangement).

Initial data suggested that a cutoff point of “≥15 % of cells exhibiting an ALK rearrangement by break-apart FISH” for classifying a tumor as ALK positive would generate few borderline cases. The ≥15 % cutoff point used in the initial crizotinib studies appeared to develop in a naturally occurring gap in the continuum of the assay. As more samples have been tested using both methods considerable proportions of cases closely approach the established cutoff point (3).

In the “Summary of safety and effectiveness data of the DNA FISH Probe Assay for the Vysis ALK Break Apart Probe Kit” (PMA P110012) the FDA defines the normal cutoff as the maximum amount of scoreable interphase nuclei with a specific abnormal signal pattern at which a specimen is considered negative for that signal pattern. The normal cutoff value is expressed in terms of a percentage, or the actual number of nuclear FISH patterns positive for rearrangement per the standard number of nuclei tested. The normal cutoff for the ALK assay was established as 15% using NSCLC FFPE specimens.

Special Requirements


Turn-Around Time

Five to 7 business days for slides and paraffin blocks respectively. If confirmation by FISH is required: 10 to 15 business days. 


  1. Lindeman NI et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Mol Diagn. 2013,15(4):415-453. 
  2. Kwak EL et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010,363:1693-1703.
  3. Camidge DR et al. Native and Rearranged ALK Copy Number and Rearranged Cell Count in Non-Small Cell Lung Cancer. Cancer. 2013,119(22):3968-75.