About oncogenetic drivers

Only sensitive and specific tests can reliably detect gene alterations

  • 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 most gene fusions9–16

Technologies available for gene alterations


Next-generation sequencing (NGS)

Ability to detect different alterations 

Comprehensive detection of fusions and mutations in oncogenes4,9

NGS is a sensitive and specific way to detect fusions and mutations and can also identify other biomarkers in one comprehensive molecular test.
RNA-NGS can also detect fusion partners and the position of gene rearrangements.
Liquid biopsy is emerging as a valuable option to test patients but should always be confirmed with a tissue test if negative


Reliability varies depending on the assay used17

Different  NGS panels target different regions of genome 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 simultaneously evaluate multiple and novel gene fusion partners and mutations while requiring limited tissue. RNA sequencing is focused on coding sequences rather than introns, and is suited for gene fusion detection.5,18


May not identify all gene fusions and targetable mutations18

DNA-NGS is limited for fusion detection by intron size. RNA-NGS is suited for gene fusion detection but may be limited by RNA quality.5,18
Depending on assay, not all genes or alterations might be covered, and certain assays can only detect known fusions due to their design.

Assay Design

When ordering an NGS test, be certain it detects the most common gene fusions and mutations in actionable genes19

Ensure that driver mutations in EGFR, KRAS and fusions in NTRK, ROS1, ALK and RET are included to help identify the best treatment options for patients.



Immunohistochemistry (IHC)


Ability to detect different fusions

May detect abnormal expression of proteins encoded by fusion genes indirectly but sometimes requires confirmation by other tests2,9,10

May be used as a screening tool or diagnostic depending on the biomarker.  Screening assays require confirmation by other methodologies.


Depending on the biomarker, it can detect aberrant protein expression with high sensitivity and specificity2,5,13

IHC antibodies have been reported to have 95-100% sensitivity and range from 70-100% specificity depending on the threshold used to define positivity.


Low cost and readily available2,9,18

Lower cost than NGS with a turnaround time of 1-2 days18.


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

IHC can detect both wild-type and fusion proteins leading to possible false positives.
IHC cannot identify fusion partners and there is also a risk of false negative for fusions in some biomarkers. Therefore, orthogonal techniques are required to confirm the presence of gene rearrangements.



Fluorescence in-situ hybridisation (FISH)

Ability to detect different fusions

Can detect most common fusions, depending on probes used

Different probes are required for each NTRK gene.21 For ROS1 fusion detection by FISH relies on dual color ‘break-apart’ probes which label the fusion breakpoints. Rearrangements are determined by looking at the pattern of fluorochrome labeling9,21


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 a common method for detecting 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 confirmatory testing depending on the biomarker9,18

Conventional FISH may require multiple tests to detect gene fusions and does not detect novel fusions as compared to NGS.
FISH is primarily DNA based and will not provide information about the expression of the fusion protein and cannot detect mutations. FISH requires technical expertise to interpret and score.



Polymerase chain reaction (PCR)

Ability to detect different alterations

Requires multiple primer sets to detect gene fusions18,22

Different gene alterations can be detected by RT-PCR with multiple primer sets required for each fusion variant.18,21,22


Reliable for known gene fusions and mutations, some biomarkers require confirmatory testing

Each variant requires a specific primer set. Therefore, PCR may miss unknown or untested variants. Low specificity for some biomarkers in certain indications will require confirmation using FISH or NGS11,22.


Low cost per assay with high sensitivity and specificity18

RT-PCR can also provide information on the expression level of oncogene being tested.


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



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.



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

2. Hechtman JF, et alAm J Surg Pathol 2016;41:1547–1551.

3. Kumar-Sinha C, et alGenome Med 2015;7:1–18.

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

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

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

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

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

9. International Association for the Study of Lung Cancer. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. Available at: https://www.iaslc.org/research-education/publications-resources-guidelines/iaslc-atlas-alk-and-ros1-testing-lung-cancer (Accessed November 2020).

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

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

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

13. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Non-Small Cell Lung Cancer. V.6.2020, 2020. Available at: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf (Accessed November 2020).

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

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

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

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

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

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

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

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

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

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