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Physiologically, ALK-RTK expression is limited to rare embryonic and adult brain tissue cells, suggesting
[8,9]
a role in neural development and function . However, in a small subset of NSCLC affected patients (4%-6%),
typically following an inversion rearrangement in the short arm of chromosome 2 between the ALK gene
and the Echinoderm microtubule-associated protein-like 4 (EML4) gene [inv2(p21;p23)], the resulting
chimeric fusion protein (EML4-ALK) becomes aberrantly expressed, resulting in enhanced tyrosine kinase
activity and constitutive activation of the signaling pathways mediated by mitogen-activated protein kinase,
Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Phosphatidylinositol-
4,5-bisphosphate 3-kinase-RAC-alpha serine/threonine-protein kinase, leading to cell proliferation and
anti-apoptosis and eventually to tumorigenesis [10-12] ; on a side note, ALK rearrangements with other genes
(fusion partners) other than EML4 have also been reported (e.g., TFG, KLC1, PTPN3). However, the eventual
[13]
outcome does not vary .

Moreover, based on the EML4 breakpoint, we can further identify several EML4-ALK fusion variants (15 to
date), expressing different amounts of EML tandem atypical β-propeller in EML protein (TAPE) domain, or
no TAPE domain at all; among all EML4-ALK fusion variants, variants 1 (partial TAPE), 2 (partial TAPE)
and 3a/b (no TAPE) are the most common ones, accounting for approximately 90% of all cases. According
to the most recent studies, variants lacking TAPE domain show earlier TKI-treatment failure and worse
outcome, when compared to TAPE-harboring ones [14,15] .

DIAGNOSING ALK + NSCLC
Currently, we can use four different methods to detect ALK rearrangements: fluorescent in situ hybridization
(FISH), immunohistochemistry (IHC), reverse transcriptase polymerase chain reaction (RT-PCR) and
next generation sequencing (NGS); FISH and IHC are the only US Food and Drug Administration (FDA)
approved techniques [16,17] .

FISH
To date, FISH is considered the gold standard assay for diagnosing ALK rearrangements. The rationale
behind this method is represented by the fact that using two differently colored break-apart probes (green
and red) specific to the inversion breakpoints of the ALK gene, we will obtain a single yellow signal in
non rearranged cells, while two split signals (green and red) will be obtained in rearranged cells; however,
although very reliable, this technique cannot be automated and requires trained personnel and equipment
(fluorescence microscope and the devices for the hybridization probes) for results interpretation [16,17] .

IHC
Whereas ALK rearrangements are followed by EML4-ALK expression, IHC relies on highly ALK-specific
monoclonal antibodies to detect the products of this inversion on formalin fixed tissue sections; while low-
cost and easy to perform, IHC requires laboratory validation and standardization [16,17] .

RT-PCR
The use of this method is currently not recommended, in fact, it does not provide a good quality of RNA
from the formalin-fixed paraffin embedded tissue and furthermore can only detect known fusion partners.
However, with this approach subjectivity in assessment of the analysis can be completely ruled out, unlike
IHC and FISH [16,17] .

NGS
Even though NGS is still not FDA-approved, it is an undoubtedly promising technique, in fact, it grants
screening of both known and novel ALK gene rearrangements, alongside with screening for other NSCLC-
related gene mutations; nevertheless, trained personnel is required for results interpretation and costs are
still prohibitive [16,18] .