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Diagnostics Genetics and epigenetics, Precision medicine, Oncology

On NTRK and Other Fusion Biomarkers

sponsored by Thermo Fisher Scientific

NTRK fusions are rapidly gaining attention in precision oncology as therapies that target these rearrangements are becoming available. Not only are these treatments evincing marked clinical responses – described as “amazing” – but they are doing so in tumor types and clinical situations that constitute a significant unmet patient need. But it also brings new challenge for pathology laboratories. Dr. Lopez-Rios tells us more…

Is NTRK fusion detection part of the routine testing algorithm today?

Yes and no… Nowadays, NTRK is included in NGS panels used routinely in some laboratories, but it’s rarely tested as an individual biomarker. This needs to change in the near future, and many more patients should have access to NTRK testing. One possibility proposed by the recently released ESMO guidelines (1) is to use immunohistochemistry to screen for NTRK rearrangements and then confirm all positive results with NGS. This is a sensible approach to take until NGS technology reaches the point where we can use it for every patient.

How will NGS testing fit into existing workflows?

You start with a question – is the histological tumor type known to harbor a highly recurrent NTRK arrangement? If the answer is yes, then there are several things that you can use: fluorescence in situ hybridization (FISH), real-time PCR (RT-PCR), or NGS to confirm it. But if the answer is no – and you have a sequencing platform available – you should consider using NGS up-front. If you don’t, then you start with IHC and confirming positive cases with NGS.

Laboratories that still perform individual biomarker tests will likely continue to do so with the addition of NTRK IHC. Those already performing NGS won’t need to change anything; they’ll just need to ensure that they are targeting all three genes.

What about fusion biomarkers in general?

Overall, the role of gene fusions in precision oncology is increasing. There are either clinical trials or  stablished treatment options available for patients with ALK, ROS1, NTRK, or RET translocations, so having sequencing information is clearly invaluable.

Is there enough evidence to argue for the use of NGS in all non-small cell lung cancer patients?

I think there’s no question that NGS makes a lot of sense for NSCLC because there are multiple clinically relevant biomarkers and several drugs per biomarker. In other tumors, NGS is still sometimes seen as a “last resort” because it’s used to identify patients for clinical trials, so we may have a harder time convincing our clinical colleagues to use first-line NGS in those instances.

With the arrival of RET, NTRK, and other druggable gene rearrangements in lung cancers, I think the balance will shift toward up-front NGS in increasing numbers of patients.

With the arrival of RET, NTRK, and other druggable gene rearrangements in lung cancers, I think the balance will shift toward up-front NGS in increasing numbers of patients. At the moment in our laboratory, the split is about 50/50 – but, every year, I estimate that NGS increases by 10 percent. So I think the question is not, “How will this fit into the current lung cancer workflow?” It’s, “How will this change the current lung cancer workflow?”

What challenges does NGS fusion testing still face?

The biggest challenge we have at the moment is obtaining high-quality RNA. I’m not worried about the quantity, because everyone in the cancer care pipeline – from surgeon to oncologist – is now aware of the need for larger sample sizes, but the quality of the RNA is still a problem. Our current failure rate is in the 3–5 percent range; it’s not huge, and I’m optimistic that we can improve on it.

We cannot report an advanced lung cancer without fusion testing results, so in cases where NGS testing fails due to sample quality, we rely on FISH or IHC to give us the answers we need. I think we also need to make changes to the workflow to reduce turnaround times and eliminate the need to batch patients. This last remains a challenge because, with the way existing products are presented and the way chips are designed, it’s difficult to overcome this barrier.

Much has been written about what NGS technology is best for fusion detection. What are your thoughts?

There are several lines of evidence demonstrating that RNA-based NGS is preferable. DNA-based NGS is associated with a significant risk of false-negatives.

What does the future of NGS look like?

I am convinced that, in the future, we will have “real-time NGS.” By that I mean two things: one, that we’ll have the flexibility to start running a patient sample at any point. Currently, with IHC, we have machines with as many as 30 different chambers, each one of which can run separately so that each sample doesn’t have to wait for the previous one to finish – and I think we’ll have something similar in the future for NGS (i.e., a continuous workflow).

Overall, the role of gene fusions in precision oncology is increasing.

And two, that we’ll have a short turnaround time. For me, the dream turnaround time would be 72 hours, which is doable, but at a significant cost. In the future, I think we’ll be able to bring the cost of rapid sequencing down. Given the number of patients who can benefit from comprehensive profiling, NGS becomes an economy of scale.

Right now, we can take a blood draw or a biopsy and return an RT-PCR result in a few hours. Some predictive IHC assays take very little time. Who knows? Perhaps one day, NGS will provide results within a single working day as well.

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  1. C Marchiò et al., “ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research”, Ann Oncol, 30, 1417 (2019). PMID: 31268127.
  2. R Benayed et al., “High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden”, Clin Cancer Res, 25, 4712 (2019). PMID: 31028088.
About the Author
Fernando Lopez-Rios

Head of Pathology and Targeted Therapies Laboratory, Hospital Universitario HM Sanchinarro, Madrid, Spain

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