Despite the absence of clear guidelines or firmly established best practices, next-generation sequencing (NGS) assays are becoming the method of choice for gene fusion detection.
This is significant because, although some of the cancers that contain fusion RNAs are rare, they’re now treatable thanks to new targeted therapies. If your assay can detect fusion RNAs, it can help profile tumors for important diagnostic, prognostic, and therapeutic targets, which can lead to improved patient outcomes.
The old FISH method limited you to one type of fusion variant at a time; it was effective, but also slow and cumbersome. With the latest NGS techniques, detecting fusion RNAs is more efficient than ever. It’s more sensitive and can detect multiple fusions in the same assay.
Nevertheless, it’s still challenging because of the complex workflows and the need to rigorously ensure performance across all fusion variants. From extraction, to library prep, to sequencing, to the bioinformatics pipeline, there are countless points where something could go wrong.
What Are You Missing?
Take, for example, the case of Dr. Greg Tsongalis, Director, Clinical Genomics and Advanced Technology (CGAT) at Dartmouth-Hitchcock Medical Center.
At this year’s Precision Medicine World Conference (read our summary and analysis here), Dr. Tsongalis described how his lab used a 50-gene cancer hotspot panel on a lung adenocarcinoma sample and found no mutations. The problem was, another lab had definitively identified an 18 bp EGFR exon 19 deletion at 15% alleic frequency in the sample.
As Dr. Tsongalis explained, his lab went back and updated its analysis pipeline to make sure the mutation would now be detected.
“We were missing this mutation not because of the chemistry, but because of the software we had to detect the mutations,” he said.
Because he was using biosynthetic NGS reference material and QC management software, Tsongalis could track and tweak his pipeline to quickly determine what he was missing.
The Time/Cost Equation
One of the most revealing quotes from Dr. Tsongalis’ presentation was this:
“It takes time and money, but we have to make the effort to re-validate (on an ongoing basis), otherwise we will miss mutations and not even know about it.”
Although RNA-seq techniques have vastly sped up the time it takes to detect cancer-causing mutations, assay development remains a laborious and expensive process.
Compounding the problem is how hard it is to track down rare variants to use as reference material. Fusion RNA variants such as the NTRK family of fusions can be involved in many types of cancers but generally have low rates of occurrence. It’s unlikely your lab has samples on hand, especially in the quantities needed to sufficiently test your assay across the range of allelic frequencies.
As an assay developer, this doesn’t leave you with many good options. You can dedicate your limited time, money, and staff to tracking down patient samples from healthcare providers and biobanks. Or you can forego running the rare variants altogether and hope for the best.
The first option is wasteful. As we’ll see, the second option can jeopardize the clinical utility of your assay.
How Many Variants Should You Look At?
The answer is, as many as you possibly can.
In Dr. Tsongalis’ case, had he not known the sample he was working with contained a rare variant, he wouldn’t have known his assay was missing it. Without proper reference material, your lab can put an assay through validation and go live without addressing unknown limitations.
This matters because you want to be sure your assay detects any and all variants that are critical to patient care. While fusion RNA variants like TRK may be uncommon, they have prognostic and therapeutic implications.
If you neglect to test your assay’s ability to detect these rare variants, you would have to disclose to patients and physicians that certain variants in the assay had not been appropriately validated. If they are detected, they cannot be reported.
Where to Find Rare Variants
Fortunately, locating material with rare variants like TRK fusions - and ensuring your assay detects them - is easier than you might think. As we noted recently, biosynthetic alternatives have many advantages over remnant patient samples.
First, they’re readily available. With biosynthetic NGS reference materials from companies like SeraCare you can order exactly the variants and quantities you need. If your needs change, or you need additional materials for re-validation or routine monitoring, you can be assured of their lot-to-lot consistency.
Second, biosynthetic NGS reference materials can be highly multiplexed and blended with many other rare and challenging variants at precise allele frequencies. They can also be customized easily and quickly.
In their recently released guidelines, the Association for Molecular Pathology and the College of American Pathologists wrote:
“[S]ynthetic DNA fragments...have particular advantages because they can be designed to incorporate specific sequence variants at known positions. They, likewise, can be mixed in known allelic ratios, to simultaneously evaluate many aspects of platform performance, library preparation, and bioinformatics analysis.”
Because biosynthetic NGS reference materials are consistent from lot-to-lot and precisely quantitated by digital PCR (unlike remnant patient specimens), they serve as an unchanging truth-set that can help you identify sources of variability within your assay, workflow, and bioinformatics pipeline.
Learn More About Using Biosynthetic NGS Reference Materials
The right biosynthetic NGS reference materials can help your lab improve the robustness and reliability of your NGS assays while spending less time and money developing and validating them. Learn how in our new white paper, “How to Develop a Clinical NGS Assay Without Losing Your Mind or Your Shirt.” Click on the link below to access your free copy.