FDA Approves a Laboratory Immunohistochemical Test That Identifies Whether the ALK Protein is Present in a NSCLC Tissue Sample

If the test result indicates that the ALK protein is present, then the patient with NSCLC may be eligible for treatment with crizotinib

The US Food and Drug Administration (FDA) approved on 12 June 2015, the VENTANA ALK (D5F3) CDx Assay, a laboratory immunohistochemical (IHC) test intended for the qualitative detection of the ALK protein in formalin-fixed, paraffin-embedded (FFPE) non-small-cell lung cancer (NSCLC) tissue stained with a BenchMark XT automated staining instrument. It is indicated as an aid in identifying patients eligible for treatment with crizotinib (XALKORI®). There are no known contraindications for use for this test.

VENTANA ALK (D5F3) CDx Assay is a rabbit monoclonal primary antibody for use with the VENTANA OptiView DAB IHC Detection Kit and OptiView Amplification Kit on the automated BenchMark XT platform. The OptiView DAB IHC Detection Kit, OptiView Amplification Kit, Rabbit Monoclonal Negative Control Ig and the BenchMark XT instrument are all Ventana products and are procured by the end user commercially. The ALK (D5F3) antibody is a recombinant rabbit monoclonal antibody targeted for recognition of the C-terminal peptide region of the human ALK protein, and is purified from culture supernatant.

The ALK (D5F3) antibody binds to ALK in FFPE NSCLC tissue sections, which is followed by a sequential application of a secondary (linker) antibody that binds to the primary antibody, a tertiary antibody enzyme complex (multimer) that binds to the secondary antibody, and a chromogenic substrate. Interposed washing steps stop the reaction after appropriate incubation times between each of the steps. The enzymatic activation of the chromogen results in a visible reaction product at the antigen site, which is DAB (3,3'-diaminobenzidine) with amplification of the signal via tyramide. The specimen is counterstained and cover slipped.

Results are interpreted and visualised using light microscopy. The final slide staining results are evaluated by a pathologist.

NSCLC cases that are positive with the VENTANA ALK (D5F3) CDx Assay demonstrate strong granular cytoplasmic staining in any percentage of tumour cells.

NSCLC cases determined to be negative with the VENTANA ALK (D5F3) CDx Assay demonstrate an absence of strong granular staining.

The VENTANA ALK (D5F3) CDx Assay scoring algorithm is specified in the product labelling, which includes the Interpretation Guide and Quick Reference Guide.

One 5 mL dispenser of VENTANA ALK (D5F3) CDx Assay contains approximately 70 μg of the rabbit monoclonal antibody targeted for the ALK protein, which is sufficient reagent for 50 tests. The antibody is diluted in 0.08 M PBS with 3% carrier protein and 0.05% ProClin 300, a preservative. Total protein concentration of the reagent is approximately 10 mg/mL. Specific antibody concentration is approximately 14 μg/mL. Ancillary reagents that are compatible with the BenchMark XT instrument system are required. The VENTANA ALK (D5F3) CDx Assay Interpretation Guide for NSCLC (containing colour images of representative staining patterns and known artifacts) is available to the end user to assist in the interpretation of assay results

Instruction in the product labelling states that “This product should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls”. This product is intended for in vitro diagnostic (IVD) use.

Instrumentation and software

VENTANA ALK (D5F3) CDx Assay was developed for use on a VENTANA BenchMark XT automated slide stainer in combination with Rabbit Monoclonal Negative Control Ig, OptiView DAB IHC Detection Kit, OptiView Amplification Kit and ancillary reagents. An assay specific staining procedure must be used with the VENTANA ALK (D5F3) CDx Assay.

The assay specific staining procedure software is named “XT VENTANA ALK(D5F3) CDx”. The following steps in the staining procedure software are locked: deparaffinization, cell conditioning, pre-primary peroxidase inhibitor, antibody incubation, OptiView HRP Multimer, OptiView Amplification, OptiView AMP, OptiView H2O2 and OptiView AMP Multimer. The OptiView HQ Universal Linker, Counterstain and Post Counterstain steps remain selectable by users. For the OptiView HQ Universal Linker, the user can chose between only 2 options: 8 minutes or 12 minutes. The default option is 12 minutes, as specified in the product labeling. Similarly, for both the Counterstain and Post Counterstain options, users can select any time from 4 minutes to 32 minutes. The manufacturer’s recommended counterstain times are 4 minutes for each counterstain step.

Test controls and calibrator

Ventana recommends the use of human NSCLC or appendix tissue as run controls to establish run validity of this assay. These tissue controls are not provided by Ventana and must be sourced by end users. Instructions are included in the product labelling on appropriate staining, qualification and interpretation of these end user provided tissue controls to assess run validity. Each run must have a system-level control and each case requires a corresponding slide stained with the negative reagent control.

Negative reagent control

The Rabbit Monoclonal Negative Control Ig is a pre-diluted, ready-to-use antibody product for use with Ventana detection chemistries on Ventana IHC instrument systems. The Rabbit Monoclonal Negative Control Ig should be run for each case to be assessed with the VENTANA ALK (D5F3) CDx Assay. The Rabbit Monoclonal Negative Control Ig is directed against DNP (Dinitrophenyl), which does not occur naturally, and should demonstrate appropriate negative staining results in human tissue. It is purified from culture supernatant.

System level controls

End user provided tissue controls (appendix or NSCLC) are required in each run with patient specimens. A major advantage of using end user provided tissue controls is that they monitor all aspects of pre-analytical variables from fixation to processing, to embedding and sectioning.

Alternative practices and procedures

Currently, there is one FDA-approved alternative for detection of ALK status in FFPE NSCLC tissues, for the selection of patients who are eligible for crizotinib treatment. Namely, the Vysis ALK Break Apart FISH Probe Kit was approved under PMA P110012. This FDA-approved test is a fluorescence in situ hybridization (FISH)-based assay that detects rearrangements involving the ALK gene in human NSCLC tissue specimens. To date, there are no FDA-approved anti-ALK antibodies commercially available for use in immunohistochemistry applications.

Marketing history

Prior to this approval, the VENTANA ALK (D5F3) CDx Assay has not been marketed in the United States. It is marketed in the following countries under the VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody product name: Austria, France, Germany, Italy, Middle East, Belgium, Denmark, Finland, Netherlands, Norway, Portugal, Sweden, Slovenia, Baltics, Croatia, Czech Republic, Estonia, Greece, Hungary, Poland, Romania, Russia, Slovakia, Spain, Switzerland, Turkey, United Kingdom, Argentina, Brazil, Chile, Colombia, Ecuador, Mexico, Peru, Venezuela, Uruguay, New Zealand, India, Indonesia, Malaysia, Singapore, Taiwan, Vietnam, China, Hong Kong, Thailand, Israel, and South Africa.

The device has not been withdrawn to date from the market in any country for reasons relating to safety and effectiveness of the device.