Clinical Implications

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 inhibitors.

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).

Volume

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).

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.

Limitations

Fixatives other than buffered formalin, prolonged fixation time or exposure to severe heat or chemicals may give rise to inadequate results.
Concordance between the ALK IHC and ALK FISH assay was determined at CellCarta. Out of the 86 samples tested for validation, the concordance between FISH and IHC was 92% (79/86) according to 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.
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.

Turn-Around Time

Five to seven business days for slides and paraffin blocks respectively. If confirmation by FISH is required: additional 5 to 7 business days.