Table 2.

Overview of testing methods currently available for NTRK gene fusions

Assay	Advantages	Disadvantages
IHC	Low cost52 53 Readily available34 Detects TRKA, B and C18 Turnaround time 1–2 days53	May not be specific for NTRK gene fusion as it detects both wild-type and fusion proteins18 Possible false positives34 Possible false negatives for fusions involving TRKC60 There is no standardisation of scoring algorithms52
FISH	The location of the target within the cell is visible54 55 Several targets can be detected in one sample using several fluorophores54 Requires knowledge of only one of the two fusion partners when using break-apart probes NTRK gene fusions with unknown partners can be detected using break-apart FISH FISH is readily available in most laboratories and institutes	The target sequence must be known for conventional FISH otherwise three separate tests are required for NTRK1, NTRK2 and NTRK3 56 Complex chromosomal translocations can result in false positive signals56 False negative results may be above 30%63
RT-PCR	High sensitivity and specificity34 Low cost per assay52	Target sequences must be known (i.e., cannot readily detect novel fusion partners)32 52 A comprehensive multiplex RT-PCR assay might be challenging because of the potentially large number of possible 5’ fusion partners52 57
NGS	May detect novel fusion partners (depending on the assay used)32 Can be used to evaluate multiple actionable targets simultaneously while preserving limited tissue32 Currently used for NTRK testing10 RNA-NGS is preferred over DNA-NGS as sequencing for RNA-based testing is focused on coding sequences not introns56	Commercially available DNA-based NGS platforms may not be capable of identifying all NTRK gene fusions, especially those involving NTRK2 and NTRK3, which have large intronic regions58 DNA-NGS is limited by intron size56 RNA-NGS is limited by RNA quality56