The Canadian NTRK (CANTRK) is a ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

Nearly 14% of disease-causing germline variants result from the disruption of mRNA splicing. Most (67%) DNA variants predicted in silico to disrupt splicing are classified as variants of uncertain significance. An analytic workflow-splice effect event resolver (SPEER)-was developed and validated to use mRNA sequencing to reveal significant deviations in splicing, pinpoint the DNA variants potentially involved, and measure the deleterious effects of the altered splicing on mRNA transcripts, providing evidence for assessing the pathogenicity of the variant. SPEER was used to analyze leukocyte RNA encoding 63 hereditary cancer syndrome-related genes in 20,317 patients. Among 3563 patients (17.5%) with at least one DNA variant predicted to affect splicing, 971 (4.8%) had altered splicing with a deleterious effect on the transcript, and 40 had altered splicing due to a DNA variant located outside of the reportable range of the test. Integrating SPEER results into the interpretation of variants allowed variants of uncertain significance to be reclassified as pathogenic or likely pathogenic in 0.4%, and as benign or likely benign in 5.9%, of the 20,317 patients. SPEER-based evidence was associated with a significantly greater effect on classifications of pathogenic or likely pathogenic and benign or likely benign in nonwhite versus non-Hispanic white patients, illustrating that evidence derived from RNA-splicing analysis may help to reduce ethnic/ancestral disparities in genetic testing.