Only sensitive and specific tests can reliably detect NTRK and ROS1 gene fusions1–3

  • There are multiple molecular tests available for detecting gene fusions, but some are more specific than others1,2,4–11
  • Next-generation sequencing (NGS) has the high sensitivity, accuracy and throughput necessary to test for all gene fusions9–16

Technologies available for gene fusion detection


Next-generation sequencing (NGS)

Ability to detect different fusions 

Detects fusions in all three NTRK and ROS1 genes4,9

NGS is a sensitive and specific way to detect all the possible variations of NTRK gene fusions and also identify other biomarkers in one comprehensive molecular test.
RNA-NGS can detect ROS1 fusion partners and the position of gene rearrangements.4,5,9


Reliability varies depending on the assay used17

Different NGS panels target different regions of the sequence and depth of coverage varies between assays. Requirements for tumour content can also vary between assays.17


Can detect novel fusion partners and evaluates multiple actionable targets18

Depending on the assay used, NGS can evaluate multiple and novel fusion partners while preserving limited tissue. RNA sequencing is focused on coding sequences rather than introns, and is suited to gene fusion detection.5,18


May not identify all NTRK/ROS1 gene fusions18

DNA-NGS is limited by intron size. RNA-NGS is suited to gene fusion detection but may be limited by RNA quality.5,18


When you order an NGS test, be certain to test for all three NTRK gene fusions; NTRK1, NTRK2, and NTRK319

Test results will help identify which samples have NTRK fusion+ solid tumours.

The specific content in your patient’s report will depend on the testing method and the laboratory.

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Immunohistochemistry (IHC)

Ability to detect different fusions

May detect proteins encoded by NTRK and ROS1 genes but requires confirmation by other tests2,5,9,13

To be used as a screening tool only – other methodologies are required to confirm gene fusions. Pan-TRK antibodies may detect presence of protein encoded by any of the three NTRK genes but can’t differentiate between wild-type and fusion proteins.2,5,13


Detects TRK and ROS1 proteins with high specificity and sensitivity2,5

Pan-TRK antibodies have been reported to have 95–100% sensitivity and up to
100% specificity for TRK proteins. ROS1 antibodies have been reported to have
100% sensitivity and a specificity ranging from 70% to 100%, depending on the threshold used to define positivity.2,9,10  


Low cost and readily available2,9,18

Pan-TRK antibodies detect TRK A, B and C proteins with a turnaround time of
1–2 days.18


Cannot reliably differentiate between normal protein expression and proteins resulting from gene rearrangements9,18

IHC can detect both wild-type and fusion TRK and ROS1 proteins leading to possible false positives. There is also a risk of possible false negatives for fusions involving TRKC. Therefore, orthogonal techniques are required to confirm the presence of gene rearrangements.9,18


Pan-TRK assay staining of EML4-NTRK1 fusion in colorectal cancer (left) and ETV6-NTRK3 fusion in secretory carcinoma (right). Fusion status is based upon next-generation sequencing.20 

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Fluorescence in-situ hybridisation (FISH)

Ability to detect different fusions

Can detect both NTRK and ROS1 fusions, depending on the probes used18,19

Different probes are required for each NTRK gene.21 For ROS1 fusion detection dual colour ‘break-apart’ probes are used which label the 3′ (centromeric) part of the fusion breakpoint with one colour fluorochrome and the 5′ (telomeric) part with another. Rearrangements are determined by looking at the pattern of fluorochrome expression.9,21 


False positives or negatives might occur9,18

Reliability depends on the probes used. There is a risk of false-positive results due to complex chromosomal translocations and detection of non-functional fusion proteins.9,18 Variant or complex rearrangements may be missed.1,2,9,18 False negative results may be above 30% in some cases.18


Readily available, break-apart FISH is the gold-standard for detecting ROS1 gene fusions10,21

Allows visualisation of the target within the cell and enables several targets to be detected in one sample using multiple fluorophores.10,18 The use of break-apart probes allows fusions with unknown partners to be detected.18


Conventional FISH may require multiple tests to detect NTRK gene fusions

The target sequence must be known for conventional FISH and three separate tests are required for NTRK1, NTRK2 and NTRK3.18 Depending on the type of translocation there is also a risk of false positive or false negative results.9,18

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Polymerase chain reaction (PCR)

Ability to detect different fusions

Requires multiple primer sets to detect gene fusions18,22

As the location of gene rearrangements is not known at the time of testing, multiple primer sets are required, one for each fusion variant.18,21,22


Reliable for known gene fusions, requires FISH confirmation when testing for ROS1 fusions10

Each variant requires a specific primer set. Therefore, PCR may miss unknown or untested variants.7,9,21,22 ROS1 expression is detectable in normal lung tissue, PCR testing is not specific enough to detect ROS1+ lung cancer without FISH confirmation.11,22


Low cost per assay with high sensitivity and specificity18


Can only detect known target sequences18

Target sequences must be known and cannot readily detect novel fusion partners.
A comprehensive multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay might be challenging because of the potentially large number of possible 5’ fusion partners.10,18

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Appropriate molecular testing can help uncover gene fusions.4,5,18 Your pathologist can help you decide what is the best testing option for each individual patient.

FISH, DNA fluorescence in-situ hybridisation; IHC, immunohistochemistry; NGS; next-generation sequencing; PCR, polymerase chain reaction; RT-PCR, reverse-transcriptase polymerase chain reaction.


Su D, et al. J Exp Clin Cancer Res 2017;36:1–12.


Hechtman JF, et al. Am J Surg Pathol 2016;41:1547–1551.


Kumar-Sinha C, et al. Genome Med 2015;7:1–18.


Vaishnavi A, et al. Cancer Discov 2015;5:25–34.


Murphy DA, et al. Appl Immunohistochem Mol Morphol 2017;25:513–523.


Stack EC, et al. Methods 2014;70:46–58.


Knezevich SR, et al. Nat Genet 1998;18:184–187.


Naidoo J, Drilon A. Am J Hematol Oncol 2014;10:4–11.


International Association for the Study of Lung Cancer. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. Available at: (Accessed November 2020).


Shan L, et al. PLoS One 2015;10:e0120422.


Bubendorf L, et al. Virchows Arch 2016;469:489–503.


Cao B, et al. Onco Targets Ther 2016;31:131–138.


National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Non-Small Cell Lung Cancer. V.6.2020, 2020. Available at: (Accessed November 2020).


Zheng Z, et al. Nat Med 2014;20:1479–1484.


Drilon A, et al. Clin Cancer Res 2015;21:3631–3639.


Grada A, Weinbrecht K. J Invest Dermatol 2013;133:e11.


Horak P, et al. ESMO Open 2016;1:e000094.  


Penault-Llorca F, et al. J Clin Pathol 2019;72:460–467.


Kummar S, Lassen UN. Target Oncol 2018;13:545–556.


VENTANA pan-TRK (EPR17341) Assay: Package Insert 1017533EN Rev A.


Rossi G, et al. Lung Cancer (Auckl) 2017;8:45–55.


Ali G, et al. Arch Pathol Lab Med 2018;142:480–489.