Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

Abstract

BackgroundBased on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples.MethodsForty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system.ResultsAll positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively.ConclusionsThe specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

Therapeutic management of ALK+nonsmall cell lung cancer patients

“…While IHC is a reliable screening tool, FISH confirmation is required in the event of positive IHC and even in some cases for negative IHC in patients presenting predictive rearrangement markers, including younger age, light smokers (⩽10 pack-years) and testing negative for other mutations, notably EGFR and KRAS [34,37].…”

Lung Carcinoma Predictive Biomarker Testing by Immunoperoxidase Stains in Cytology and Small Biopsy Specimens: Advantages and Limitations

“…Some attempts have been made to standardize IPOX protocols, especially for biomarkers, 24,25 and the Food and Drug Administration (FDA) recently approved IPOX assays for the detection of anaplastic lymphoma kinase (ALK) rearrangement (D5F3 clone, Ventana Medical Systems, Oro Valley, Arizona) and programmed death ligand-1 (PD-L1) expression (Dako, Carpinteria, California) as companion diagnostic tests to identify patients eligible for targeted therapies. 26 However, even when a protocol is optimized, validated, and consistently applied, consistency of interpretation may be challenging to achieve. False-negative results deprive patients of potentially course-altering therapy, whereas false-positive results lead to the use of targeted therapies in mutation-negative tumors.…”

“…45,46 Although the first ALK antibody clones were unreliable, the subsequently developed clones D5F3 (Cell Signaling Technology) and 5A4 (Novocastra, Leica Biosystems, Buffalo Grove, Illinois) have a reported sensitivity of 100% and specificity of 99% in biopsy specimens, with excellent interobserver agreement and concordance with FISH results. 6,7,17,26,47,48 Clone 5A4, which is as reliable as D5F3 in the detection of ALK rearrangement, is used more commonly in Europe. 17,49 The performance of ALK IPOX in cytology specimens (formalin-fixed cell blocks) thus far is similar to that in surgical specimens.…”

“…[6][7][8]16 The recently FDA-approved IPOX assay for ALK rearrangement using the D5F3 clone qualitatively detects ALK rearrangement in NSCLC as a companion diagnostic test to select patients for crizotinib treatment. 26 The assay is FDA approved for use on formalin-fixed, paraffin-embedded NSCLC tissue using Ventana's rabbit monoclonal antibody (licensed from Cell Signaling Technology), OptiView Amplification Kit, OptiView DAB IHC Detection Kit, and BenchMark XT automated slide staining instrument.…”

“…9,16,17,46,50 In some studies a reaction of 1þ is considered positive, 9,46,50 whereas in others a 1þ reaction is considered inconclusive and is followed by FISH testing, 51,52 and in a recent study of the FDA-approved assay, 1þ was considered negative. 26 Reports of weak reactivity are more likely due to preanalytic factors such as fixation and antigen retrieval, as well as the clone and detection system used, rather than different levels of fusion protein expression. Therefore, each laboratory must validate its own test with strong internal positive and negative controls before implementing an IPOX screening algorithm for ALK rearrangement detection.…”