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Several challenges exist when testing for cancers for the presence of NTRK gene fusion including, testing for the particular gene defect or protein, as well as which patients or tumour types would be most appropriate to test.

Each test methodology has advantages and disadvantages in terms of resource efficiency, turnaround time, sensitivity, and specificity which must be balanced with the individual case in mind. For example, NTRK3 FISH would be a good choice in patients where there is high suspicion of ETV6-NTRK3 fusion while RNA testing would be ideal to clarify cryptic NTRK gene rearrangements detected via DNA sequencing. In addition, considering the rarity of NTRK gene fusions, there is some uncertainty regarding which patients should undergo testing for NTRK gene fusions.

To address these challenges, target enrichment strategies can be used to enhance the study population being tested for NTRK gene fusions [1-3]. Target enrichment involves the selection of patients from a larger patient population, of particular tumour histologies or molecular subgroups, or with the use of a rapid screening assay. Then test these patients using a highly sensitive assay in order to identify the small percentage of those who have NTRK gene fusions. IHC may be used as a target enrichment technique in cases where NTRK fusions are somewhat common [4].

To further enrich for or identify patients with NTRK fusions triaging both, histology-based and genomic-based approaches may be useful [5-6]. For example, FISH or RNA-based testing should be performed if IHC testing is negative in tumour histologies with a high likelihood of NTRK fusions. Likewise, confirmatory testing with IHC should be performed to detect functional fusion protein expression in patients with positive NGS-based fusion testing.

The selection of the appropriate assay for NTRK fusion detection depends on the tumour type concerned and genes involved. Other factors should also be considered including, available tumour/ tissue material for testing, availability of various assays in the particular clinical setting, and whether comprehensive genomic testing is needed concurrently [5-7].

Depending on the source of the material for testing for NTRK gene fusions, assays may be categorized as a) tissue-based (tumour) or b) liquid-based (blood). Techniques routinely used in clinical practice at present all require tumour tissue samples. Details of the assay methodologies used, their advantages, disadvantages and applications in NTRK gene fusion detection can be found by clicking on each method below. 

An algorithm to support decision on testing for NTRK gene fusion in cancer patients can be found here


  1. Hechtman JF, Benayed R, Hyman DM et al. Pan-Trk Immunohistochemistry Is an Efficient and Reliable Screen for the Detection of NTRK Fusions. Am J Surg Pathol 2017; 41: 1547-1551.
  2. Murphy DA, Ely HA, Shoemaker R et al. Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing. Appl Immunohistochem Mol Morphol 2017; 25: 513-523.
  3. Zheng Z, Liebers M, Zhelyazkova B et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med 2014; 20: 1479-1484.
  4. De Winne K, Sorber L, Lambin S, Siozopoulou V, Beniuga G, Dedeurwaerdere F, D'Haene N, Habran L, Libbrecht L, Van Huysse J, Weynand B, Wouters K, Pauwels P, Zwaenepoel K. Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial. Virchows Arch. 2021; 478(2):283-291.
  5. Hechtman JF. NTRK insights: best practices for pathologists. Mod Pathol. 2022; 35(3):298-305.
  6. Conde E, Hernandez S, Sanchez E, Regojo RM, Camacho C, Alonso M, Martinez R, Lopez-Rios F. Pan-TRK Immunohistochemistry: An Example-Based Practical Approach to Efficiently Identify Patients With NTRK Fusion Cancer. Arch Pathol Lab Med. 2021; 145(8):1031-1040.
  7. Solomon JP, Linkov I, Rosado A, Mullaney K, Rosen EY, Frosina D, Jungbluth AA, Zehir A, Benayed R, Drilon A, Hyman DM, Ladanyi M, Sireci AN, Hechtman JF. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 2020; 33(1):38-46.

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