Testing for NTRK
Gene Fusions

Classic and Novel Methods of Detection

NTRK fusions can be detected using a few different techniques. Fusions are now commonly screened for shortly after diagnosis to determine whether NTRK-specific therapy can be included in the treatment regimen. Clinicians may use one or a combination of the following testing methods to detect NTRK fusions.

Testing Methods

Fluorescent in situ Hybridization (FISH)

FISH is a cytogenetic technique that uses fluorescent tags attached to nucleotide sequences. These sequences are designed to complement specific genes or genomic regions, so that when added to a sample, they hybridize to and elucidate the presence, absence, rearrangement, or amplification of target genes. FISH is typically performed on formalin-fixed, paraffin-embedded tissue or peripheral blood. FISH has helped to characterize the particular genetic abnormalities at the root of countless diseases, especially cancers and prenatal conditions.


The identification of tumors harboring particular gene fusions by immunohistochemistry rests on the premise that the chromosomal rearrangements result in an up-regulation of fusion gene expression in the tumor cell. Regarding the identification of patients with TRK fusion cancer, recently published studies have shown that immunohistochemistry might be an effective diagnostic approach in certain indications that are not yet routinely subjected to molecular genomic profiling, and that have a low incidence of tumors harboring NTRK gene fusions, as a way of selecting patients for subsequent molecular testing.

Next Generation Sequencing (NGS)

NGS performs sequencing of millions of small DNA or RNA fragments simultaneously, which are arranged using bioinformatics analyses that map the fragments to the human reference genome. Both DNA and RNA based NGS can be used to detect NTRK fusions, but RNA-based NGS is preferred, as it detects solely in-frame, transcribed gene fusions, and doesn’t sequence large intronic regions resulting from NTRK fusions.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

RT-PCR quantifies RNA expression. It’s currently the most sensitive means of detecting and measuring mRNA. The technique uses the reverse transcriptase enzyme to produce complementary DNA (cDNA) from mRNA transcripts, then amplifies regions of interest with PCR.

Clinical Laboratory Techniques Used to Identify Tumors Harboring NTRK Gene Fusions
Analytical technique Sample requirements Preanalytical considerations Turnaround time Advantages Disadvantages
Pan-TRK IHC FFPE tissue Variability in fixation processes may impact the quality of staining 1–2 days Rapid and inexpensive process. Established approach, widely available within clinical laboratories Indication-specific specificity for NTRK gene fusion prediction not well characterized

Sensitivity with respect to TRKC fusion proteins may be low

Assay not easily multiplexed for other biomarkers
FISH FFPE tissue Must ensure adequate tumor cellularity 1–2 days Established approach, widely available within clinical laboratories. Probes are costly, but FISH is generally reimbursable Requires expert interpretation

Does not confirm detected fusion is expressed

Not easily multiplexed with other biomarkers and may require more than one FISH assay to adequately cover all possible NTRK1 to NTRK3 fusions

Limited scalability for high-volume testing
Fusion High specificity. Can detect alterations present in small subsets of cells Individual assay limited to detection of specific 5′ partner and NTRK gene pair
NTRK break apart Detects NTRK rearrangements without knowledge of 5′ partner Sensitivity and specificity variable, depending on assay design and parameters

Multiple or complex FISH assays may be required for complete coverage
RT-PCR FFPE, snap- frozen, or stabilized tissue With variable intronic breakpoints, RT-PCR assays can be dependent on high-quality RNA from frozen/stabilized tissue 5–10 days Rapid and inexpensive test. Well-established technique in molecular genetics laboratories Does not confirm that protein is generated

Might miss fusion because of breakpoint variability
Defined gene partners High specificity because of PCR design. Assays can be multiplexed, although limited PCR primer pairs must be designed and validated for each specific fusion

For FFPE tissue-based analyses, primers must closely flank breakpoints
3′/5′ NTRK ratio May be challenging to optimize assay, especially if RNA quality is variable Implies presence of NTRK gene fusion without knowledge of 5′ partner Sensitivity depends on expression difference between wild-type gene and fusion, which is currently unvalidated/unstudied

Comprehensive coverage may require different/complex primer designs to allow for variable alternative splicing
NGS Data acquisition may be affected by tumor heterogeneity. Sensitivity for fusions varies, according to enrichment method. Fixation conditions may affect DNA quality 2–3 weeks Ability to interrogate all clinically actionable genomic content. Most tissue-sparing approach for broad genomic analysis. Commercial kits available May require high level of infrastructure investment

Requires high-level bioinformatics capability

Evolving reimbursement landscape

Does not confirm that protein is generated
DNA-based NGS FFPE or frozen tissue For FFPE tissue, sample age might affect DNA quality and sequencing read quality Readily multiplexed across multiple biomarkers. Commercially available kits available Commercially available kits not configured to cover all NTRK introns involved in fusions

Detected fusions may not be expressed or in frame
Whole genome Covers most coding and noncoding regions, including large introns Lower analytical sensitivity

Slower to generate data, and requires more computational resources than targeted approaches
Hybridization capture Highly scalable. Theoretically capable of detecting all classes of actionable mutations, including fusions with unknown partners Requires more input DNA than amplicon methods

Complex library preparation processes

Large introns of NTRK2 and NTRK3 can prove problematic
Amplicon (target enrichment by PCR) Deep sequence coverage using low DNA input. Most suitable for analysis of SNVs, indels, and defined gene fusions Requires complex multiplex amplicon design

For gene fusions, 5′ and 3′ partners must be defined and potential breakpoint regions must be covered by amplicons
RNA-based NGS FFPE, snap- frozen, or stabilized tissue RNA is more labile than DNA Only transcriptionally active fusions detected. Allows in-frame vs out-of-frame confirmation for all fusions. Commercially available kits are designed to cover all potentially oncogenic actionable fusions, without knowledge of 5′ partners or breakpoints Detection of transcripts expressed at low levels may be challenging
Hybridization capture Highly scalable. Can detect unknown fusion partnersIs not impacted by large intronic regions of NTRK genes Requires more input RNA than amplicon methods

Complex library preparation processes
Amplicon (anchored multiplex RT-PCR) Highly scalable. Allows analysis of low-input and/or degraded RNA. Simple design based on use of unidirectional gene-specific primers, allowing for detection of unknown partners Limited published data on sensitivity
DNA plus RNA NGS FFPE, snap- frozen, or stabilized tissue For FFPE tissue, sample age might affect DNA quality and sequencing read quality Broad-based screen, allowing for the efficient detection of all classes of relevant genomic alterations in cancer, including gene fusions, SNVs, indels, and CNVs. Sample libraries can be prepared without the physical separation of DNA and RNA. Inclusion of RNA sequencing provides robust fusion detection