Relentless clinical research has led to advances in precision cancer medicine and targeted treatments, resulting in better patient outcomes. At the same time, clinicians have shifted away from only classifying cancers according to histology and the site of origin (e.g., breast, lung, etc.) to classifying them based on molecular biomarkers and the genetics of the cancer.
For many clinicians, this poses a challenge as there are often so many targeted drugs, each with different molecular targets, that it can be expensive and cumbersome to identify which patients would benefit from which treatment. Coupled with the limitations of small biopsy samples, it no longer makes sense to run single-gene tests on samples. As a result, pathologists are increasingly turning to next-generation sequencing (NGS) and multigene panels.
The challenges of accessing NGS testing
For community hospitals that have to send out their samples for sequencing, it can take weeks, or even months, to receive results—time that many patients don’t have.
Until recently, this was a reality for Dr. Brandon Sheffield, a pathologist at William Osler Health System, a community hospital in Brampton and Etobicoke, Canada: “The majority of patients in Canada are treated in a community setting. Typically, it's very difficult for these patients to get access to comprehensive NGS because those services are usually only available in academic centers.”
Difficulty accessing NGS testing has been a problem for patients with aggressive or refractory cancers, who are often the ones that novel and highly targeted cancer treatments have been designed to treat.
For example, in 2018, the FDA approved larotrectinib, a drug approved for use in cancers with neurotrophic tyrosine receptor kinase (NTRK) gene fusions—known oncogenic drivers. Although relatively rare across all types of solid tumors (~0.3 percent), NTRK fusions are frequently (>90 percent) found in rare tumor types.1 There are now several effective TRK inhibitors approved in Canada and the US, providing patients with NTRK fusion-positive cancers with effective targeted treatment options: “The difference is night and day for the patients because these are very difficult to treat cancers and there’s really no other effective therapy,” says Sheffield.
However, identifying these patients requires access to high-quality testing.
Bringing NGS testing to community settings
Because there is only one FDA-approved companion diagnostic for NTRK fusion detection—FoundationOne CDx (2020)—clinical labs often rely on laboratory-developed NGS assays to detect these rare gene fusions. Specifically, the European Society for Medical Oncology guidelines on NTRK fusion detection recommends RNA-based sequencing,2 as RNA-based NGS assays allow clinicians to detect exon–exon junctions, a tell-tale sign of gene fusions.
Sheffield’s team at William Osler Health System has been one of the first to set up comprehensive NGS testing in a community setting so that patients can be tested, diagnosed, and treated in their community.
To help other community pathologists make informed choices regarding gene fusion testing, in a recent study3 supported by Genentech, Inc., Sheffield and colleagues compared the ability of four commercially available RNA-based NGS assays to detect NTRK fusions:
- Archer’s FusionPlex Lung Panel
- Illumina’s TruSight Oncology 500
- Thermo Fisher Scientific’s Oncomine Precision Assay (+automated Genexus System sequencer)
- Thermo Fisher Scientific’s Oncomine Focus Assay
Because NTRK fusion-positive cancers are relatively rare, Sheffield and colleagues purchased commercial formalin-fixed, paraffin-embedded (FFPE) cell lines to use as a testing standard, and also collaborated with other hospital laboratories to acquire 30 NTRK fusion-negative control samples and 14 NTRK fusion-positive samples.
Comparing commercial NGS assays
In their study, all four assays demonstrated high specificity for NTRK fusion detection, with no false-positive results in the clinical control samples. Across the assays, each had different strengths and weaknesses (see Table 1).
Table 1. A summary of the comparison of the four commercially available assays and their ability to detect NTRK fusions in cell lines and clinical samples.3
|FusionPlex Lung Panel||TruSight Oncology 500||Oncomine Precision Assay||Oncomine Focus Assay|
|Technology||Anchored multiplex PCR||Hybrid capture||Amplicon-based enrichment||Amplicon-based enrichment|
|RNA sample input (ng)||20–250||40||10||10|
|Turnaround time (days)||~5||~5||~1||~5|
|Cell lines||Detected all NTRK fusions||Detected all NTRK fusions||Detected all NTRK fusions||Detected all NTRK fusions|
|NTRK fusion-negative control samples||No false-positive results||No false-positive results||No false-positive results||No false-positive results|
|Sensitivity:Total fusion copies at estimated limit of detection||30 to 620 fusion copies||~30 to 290 copies||~1 to 28 copies||~1 to 28 copies|
|Specificity: Specificity (% samples pass)||100 (43)||100 (77)||100 (internal validation, data not shown)||100 (83)|
|Clinical performance: Sensitivity (% passing success rate)||100 (83)||100 (75)||Fusion Caller: 100 (100)|
Imbalance Caller: 67 (100)
According to Sheffield and colleagues, it’s not clear whether the low-quality control pass rate of the FusionPlex Lung Panel assay (43 percent) was due to the age of the FFPE samples, resulting in poor quality RNA extracts, or whether it may have been the RNA extraction kit or the FusionPlex Lung Panel assay itself. Nonetheless, the FusionPlex Lung Panel reliably detected NTRK fusions even when the samples failed to meet the manufacturer’s quality control standards (see Table 1). The researchers also noted that the limit of detection for the Oncomine Focus and Precision Assays was approximately 10 times lower than the limit of detection provided by Thermo Fisher Scientific. The researchers suggested that this difference could be because the manufacturer used a different gene fusion (EML4:ALK) to set the limit of detection.
Setting up in-house NGS testing
According to Sheffield, it was difficult to set up in-house NGS testing: “There isn’t a lot of funding available for new molecular services, the validation of NGS tests is very difficult, and then there’s technical challenges, such as finding space to set up the testing lab and hiring the right staff to perform and interpret the tests.”
Though NGS is currently difficult to implement in community settings, it has many benefits. Fully automated NGS panels can enable community pathologists to order tests and receive actionable results within days instead of weeks or even months. Fusion panels can be multiplexed as part of larger panels to detect all classes of genomic driver alterations. “That's really the power behind that approach,” says Sheffield. With a highly automated NGS machine, such as the Genexus System, technical staff trained in histopathology can use the instrument, he says.
“With this study, we characterized [commercially available] NTRK reference samples, which are commonly available online. And these are resources that labs can find and use to evaluate their own NGS setup,” says Sheffield. “But we also exchanged samples with other sites around the world to ensure we were getting the same results as labs in France, South Korea, Italy, and in the US.”
If your lab wants to validate NTRK tests, Sheffield recommends you form a network with other labs, as it's not possible for any one hospital to acquire enough samples to fulfill the validation criteria for the assays.
“What we've set up at William Osler Health can essentially be set up in any other community site,” he says. “In the next few years, we're hoping to see other community hospitals follow in our footsteps.”
Improving community patient care
“Many labs might still be using IHC [immunohistochemistry] as a means for cost saving,” says Sheffield, “but the most important conclusion from this comparison is that, regardless of what method you choose, NGS is a very effective tool for identifying NTRK fusion cancers.”
In addition, he says that many of the study conclusions around NTRK testing may be true for other gene fusions like RET and ROS1. Further evaluating the ability of commercially available NGS assays to detect these types of gene fusions across different tumor types will help support broader access to testing and accordingly, broader access to targeted cancer treatments.
“This research demonstrates that currently available tools can help community hospitals bring NGS in house and match patients to new, cutting-edge therapies,” says Sheffield. “Every patient should have access to comprehensive NGS to guide their systemic therapy.”
1. Rolfo C. NTRK gene fusions: a rough diamond ready to sparkle. Lancet Oncol. 2020;21(4):472–474.
2. Marchiò C, Scaltriti M, Ladanyi M, et al. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research. Ann Oncol. 2019;30(9):1417–1427.
3. Chung CB, Lee J, Barritault, et al. Evaluating targeted next-generation sequencing assays and reference materials for NTRK fusion detection. J Mol Diagn. 2022;24(1):18–32.