NTRK fusion testing: ups, downs of four methods

For proper sensitivity, partner agnostic methods are important. The only way to do a partner agnostic fusion detection and learn the identity of a partner gene is with a next-generation sequencing approach, Dr. Segal said. DNA or RNA can be used as a starting material, and each has pros and cons. “If you go the DNA route, then you’re in the position of always needing to pay careful attention to introns, within which a fusion event—breakage and recombination—will take place. But even if we know which intron is involved, we don’t know where in the intron this is going to occur. So we’re stuck.” The only thing that’s abnormal about that fusion gene, he said, is the exact breakpoint—“the place where you go from being a part of one gene to being a part of another gene.” The gene sequence on either side is normal. To detect the fusion, that exact breakpoint must be found, and that means evaluating and sequencing the entire intron, he said. For that, his laboratory uses a hybrid capture sequencing approach. “We try to sequence all possible fragments from the intron by pulling down everything we can from the intron using specially designed hybrid capture probes.” Decisions are still being made about which introns need to be covered for the NTRKs. “But for NTRK1, probably the bare minimum area you need to cover is intron seven through 11.”

The problem of looking at introns gets worse as more introns are looked at, Dr. Segal said. “And that’s unfortunate for NTRK2 and NTRK3 in particular, because the regions that need to be picked up are huge. For NTRK2, it’s over 100,000 base pairs of intron coverage. For NTRK3, it’s almost 200,000 base pairs, and these introns are littered with areas of bad, repetitive regions and bad homology. So a lot of labs don’t even try to do NTRK3 [intron] tiling, even if they can do NTRK2.”

Many laboratories choose instead to focus on introns four and five of ETV6, hoping to pick up ETV6-NTRK3, he said. “But if you do that, you’re going to miss all of the other fusion partners of NTRK3.” Thus, sensitivity for detection via DNA-based approaches “may be a little questionable,” he said.

With an RNA-based approach, all of the intron-associated problems are gone. “It’s been spliced out.” The gene will be transcribed into a pre-spliced RNA transcript, and that is going to be spliced down into a final spliced mRNA. Evaluation of post-spliced mRNA allows for straightforward partner agnostic NGS fusion detection, “and all we have to worry about now are direct exon to exon connections.”

There are two main ways to engineer the library prep and sequencing to look for this properly. One is anchored multiplex PCR, for which the panels are designed for anchor exons of target genes (Fig. 3) and which is designed for low input of RNA. “You can design them for a variety of different genes, and it’s easy to add additional primers to them.”

His laboratory uses hybrid capture RNA-seq, which takes a little more RNA but is easily scalable to large gene numbers because any number of probes can be added. “Because we’re using exon capture probes, we can use the same capture probes we use for our DNA mutation panel.” The RNA-based NGS methods are much easier to multiplex compared with DNA-based NGS, he said, because there’s no need to cover “intron after intron after intron” for a lot of genes, which can become costly.

The published sensitivity range for RNA-based fusion detection systems is 93 percent to 100 percent. “For our laboratory, we’ve tested a variety of fusions, in many different genes, and our overall sensitivity is about 98 percent, including 100 percent for all the NTRKs that we’ve tested,” he said.

But there are limitations, the largest of which is that the RNA quality from pathology tissue samples is poor. But quality control can be performed up front, “so at least you will figure out which samples won’t work, and maybe that’s around 10 percent of them. For the ones that do work, you can be quite confident about the data from them.” The other limitation: The expression levels of fusion gene and normal transcripts may affect the sensitivity.

Overall, he said, the RNA-based approach is good (though it’s the lesser used of the two) with the caveat that the lab will have some samples that don’t work properly. “And you’ll need to figure those out up front and what you’re going to do with them.”

If a laboratory is not already doing these types of RNA and DNA analyses routinely, adding one could mean a steep extra cost and use of tissue. “But if you’re already doing this type of analysis in the lab, then it’s trivial and cost free to add a new marker,” Dr. Segal said. “So the big question is what are you already doing, and how do we move the field over time so that we’re doing these analyses and getting reimbursed for them on a more routine basis.”

More near term, he said, is the challenge of figuring out whom and how to test. For now, “it comes down to institutional-specific factors.” At the University of Chicago Medical Center, RNA and DNA assessments are performed on every lung cancer case—but not for other tumors. “We need to figure out how we expand this over time and in a reasonably cost-affordable way,” Dr. Segal said.

“We would all like to expand the screening that we do for these NTRK fusions,” he added, “because if you can find the patients who have them, we now have a great intervention.” 

Sherrie Rice is editor of CAP TODAY. The full webinar is at www.captodayonline.com.