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.
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.
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.
| 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 |
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| 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 |
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| 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 |
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| 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 |
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| 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 |
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| 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 |
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| 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 |
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| 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 |
References:
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Hsiao, Susan J., et al. (2019) Jour Molec Diag.