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 |