Diagnostic assays for identification of anaplastic lymphoma kinase-positive non-small cell lung cancer

Abstract

In series dominated by adenocarcinoma histology, approximately 5% of non-small cell lung cancers (NSCLCs) harbor an anaplastic lymphoma kinase (ALK) gene rearrangement. Crizotinib, a tyrosine kinase inhibitor with significant activity against ALK, has demonstrated high response rates and prolonged progression-free survival in ALK-positive patients enrolled in phase 1/2 clinical trials. In 2011, crizotinib received accelerated approval from the US Food and Drug Administration (FDA) for the treatment of proven ALK-positive NSCLC using an FDA-approved diagnostic test. Currently, only break-apart fluorescence in situ hybridization testing is FDA approved as a companion diagnostic for crizotinib; however, many other assays are available or in development. In the current review, the authors summarize the diagnostic tests available, or likely to become available, that could be used to identify patients with ALK-positive NSCLC, highlighting the pros and cons of each.

Figure 1

This is a schematic of major anaplastic lymphoma kinase (ALK) testing methodologies. (a) Note that reverse transcriptase-polymerase chain reaction (RT-PCR) focused solely on the ALK kinase domain, searching for a threshold signal in a quantitative assay, is not fusion partner-specific; whereas RT-PCR that spans the common breakpoint in rearrangements is fusion partner-specific and searches for the presence/absence of a generated amplicon. EML4 indicates echinoderm microtubule-associated protein like 4; EML4 v1 and EML4 v2 refer to two of the possible variants of EML4. Break-apart fluorescence in situ hybridization (FISH) testing is not fusion partner-specific. IHC is also not strictly fusion partner-dependent. However, because transcriptional activity is set by the promoter/enhancer of the 5′ fusion partner, and different fusion proteins may have both different intracellular locations (because of an association between the 5′ oligomerization domains in the fusion protein with the native form of the 5′ partner as well as with the rearranged form) and, along with different fusion variants, potentially different half-lives, the absolute protein expression levels and patterns of staining with IHC may vary, depending on the exact ALK fusion and/or variant present.^- Techniques not presented here include FISH testing using fusion probe sets, chromogenic in situ hybridization, and next-generation sequencing. (b) Different break-apart FISH testing patterns include (clockwise from top left) fused (negative), single green (negative), split (positive), and single red (positive).

Figure 2

Hypothetical comparisons are illustrated for different diagnostic techniques versus a biologically “true” anaplastic lymphoma kinase (ALK)-positive patient population. Definitive determination of the validity of techniques with higher or lower positivity rates will depend on head-to-head clinical comparisons entailing the expansion of access to ALK inhibitors beyond only patients who have positive results from break-apart fluorescence in situ hybridization (FISH) analysis.

Figure 3

This schematic illustrates a possible 2-tier testing strategy for detecting anaplastic lymphoma kinase (ALK) using immunohistochemistry (IHC) and confirmatory ALK fluorescence in situ hybridization (FISH) for IHC equivocal cases. For the purposes of this illustration, an IHC staining score of 3+ would be considered true-positive, and an IHC score of 0 would be considered true-negative. The resource and cost-effectiveness implications of this approach will depend on the ultimate cost of a validated IHC assay, the absolute number of cases falling within the equivocal category, and the proportion of cases that are FISH-positive residing within the equivocal category. These equivocal category variables will be affected both by the specific IHC assay used and by any aspects of patient enrichment applied to the tested population.