Journal of Molecular Diagnostics最新文献

The Idylla GeneFusion Assay detects gene fusions with fusion-specific and expression imbalance methods. The purpose of this study was to evaluate the diagnostic utility of detecting fusions with expression imbalance alone in non-small-cell lung cancer. Results of ALK, ROS1, and RET fusion detection with expression imbalance were compared with results of orthogonal testing. Of 1982 cases reviewed from October 2022 through August 2024, 63 (3.2%) had fusions detected with the expression imbalance method alone, including 47 ALK, 10 ROS1, and 7 RET fusions. One case had ALK and RET fusions. Fluorescence in situ hybridization (FISH) confirmation for 51 cases revealed 8 positive (15.7%), 3 equivocal (5.9%), and 40 negative (78.4%) results. Anaplastic lymphoma kinase (ALK) immunohistochemistry performed for 22 ALK-detected cases revealed 3 positive (13.6%) and 19 negative (86.4%) results that were concordant with ALK FISH results. The positive predictive value of expression imbalance detection alone varied by gene (12.5% for ALK, 16.7% for ROS1, and 33.3% for RET). RNA next-generation sequencing results for seven select cases (three ALK, two ROS1, and two RET) showed six novel fusions (eg, STRN::ALK, SQSTM1::ROS1, and ERC1::RET) and had 100% concordance with FISH results. One case showed an NOL10::ALK out-of-frame fusion with negative ALK immunohistochemistry and equivocal ALK FISH results. Because the expression imbalance method can detect novel fusions, its implementation with confirmation testing is recommended.

Previous work has shown that circulating miR-371a-3p has higher sensitivity and specificity than current biomarkers for malignant germ cell tumors (MGCTs). Herein, the performance of two methods commonly used to measure miR-371a-3p levels was compared: quantitative RT-PCR (RT-qPCR) and droplet digital PCR (ddPCR). Samples from the University of Texas Southwestern Medical Center (Dallas, TX) and the University of Cambridge (Cambridge, UK) were evaluated using both RT-qPCR and ddPCR, as per current protocols (RT-qPCR) or standard manufacturer's recommendations (ddPCR). Data were available for 70 individuals: 36 patients with MGCT and 34 control participants (nonmalignant GCT, non-GCT cancer, and noncancer). The performance of the two assays was compared using receiver operating characteristic curves and the area under the curve. Raw miR-371a-3p Cq values (RT-qPCR) were generally lower (ie, miR-371a-3p was more abundant) and the number of positive droplets (ddPCR) higher for patients with MGCT compared with control participants. The area under the curve (95% CI) was 0.94 (0.90-0.99) and 0.82 (0.70-0.93) when using RT-qPCR and ddPCR, respectively. Thus, RT-qPCR had better classification performance compared with ddPCR in the present cohort, supporting the continued use of RT-qPCR in standard clinical practice. Further investigation is required to optimize ddPCR before it should be considered for clinical adoption.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder with extensive allelic heterogeneity. Although RNA-based assays can increase sensitivity, their cost and complexity limit their routine use. A DNA-only tiered diagnostic approach was evaluated in 1917 unrelated Korean individuals with clinically suspected NF1. The process began with targeted sequencing of blood-derived genomic DNA, followed by reflex multiplex ligation-dependent probe amplification for copy number variants and a lesional tissue test for suspected mosaicism. Initial targeted sequencing of the NF1 gene established a diagnostic yield of 74.0%. The addition of reflex multiplex ligation-dependent probe amplification and tissue testing increased the cumulative yield to 79.2%. Subsequent post-report variant reclassification and whole-genome sequencing further increased the overall diagnostic yield to 81.6%. Among 901 distinct pathogenic variants identified-81.4% of which were private-truncating variants were predominant (79.0%). Notably, several variants enriched in European cohorts and with established genotype-phenotype correlations (eg, p.Arg1809Cys, p.Met992del, and p.Arg1276Gln) were rare in this cohort, highlighting population-specific differences. Individuals with large deletions were referred at younger ages, suggesting potential genotype-phenotype associations. These data demonstrate that a stepwise, DNA-only strategy delivers high yield and scalability for routine NF1 diagnostics, while delineating a Korean-specific mutational landscape. This practical workflow offers a robust alternative to RNA-based approaches in real-world clinical settings.

Long-read whole-transcriptome sequencing (WTS) has the potential to precisely characterize fusion oncogenes that drive leukemia and other cancers. Although there is a variety of general-purpose fusion detection algorithms that use modern long-read sequencing data, they show poor sensitivity for precision diagnostics in B-cell acute lymphoblastic leukemia (B-ALL) and do not robustly assess technical and analytical parameters (eg, sequencing depth) to reliably detect fusion transcripts. FUSILLI (FUSions In Leukemia Long-read sequencing Investigator) is a novel long-read fusion detection algorithm, with a focus on targeted genomic subtyping in B-ALL. FUSILLI was evaluated against extant methods using nanopore WTS from 51 pediatric B-ALL samples sequenced at high depth and 68 at low depth (mean of 11.2 and 1.4 million reads, respectively). In the high-depth cohort, FUSILLI demonstrated increased sensitivity (0.81) compared with FusionSeeker, JAFFAL, and LongGF (0.63, 0.76, and 0.70, respectively), while maintaining high specificity (0.92). At lower sequencing depth, FUSILLI showed correspondingly lower sensitivity (0.27) but still outperformed the other fusion callers (sensitivities ranging from 0.09 to 0.16). Computational down sampling suggests that 10 million reads is sufficient to sensitively detect B-ALL-relevant fusions using this approach. FUSILLI detects B-ALL fusions with high sensitivity at modest sequencing depth, supporting implementation of nanopore WTS as a low-cost and globally accessible sequencing-based molecular diagnostic platform for pediatric B-ALL and other fusion-driven cancers.

Molecular profiling of solid tumors is increasingly essential in oncology practice, guiding diagnosis-prognosis and providing patients with access to molecularly matched therapies that can improve outcomes. In this study, 554 patients with advanced solid tumors were evaluated through the POWER (Precision Oncology at Western University) study, a first of its kind Canadian study, designed to prospectively assess the clinical impact of expanded pan-cancer next-generation sequencing (NGS) panel testing on patient management, in real-world oncology practice and evaluate the overall health system impact. The findings reveal that 79% of patients had clinically relevant variants, and nearly 28% experienced changes in treatment eligibility because of the identification of novel druggable mutations. Additionally, the analysis shows that a pan-cancer NGS panel significantly impacted patient management, with 18% of patients receiving access to clinical trials and off-label therapy with expected better outcomes and 19% (31/162) patients, previously tested by tumor-specific panels, experiencing management changes when tested through POWER. This study also highlights the broader health system impact: access to safer treatment options (14.5%), change in management (17.6%), treatment sequence changed (17.3%), and Ministry of Health formulary treatment saved (12.5%). These results underline the benefits of expanded NGS testing over tumor-specific panels in guiding personalized treatment decisions, optimizing patient care, and enhancing health care delivery in oncology.

Acquired TERT promoter (TERTp) variants are found in the blood of patients with telomere biology disorders and carry diagnostic and prognostic significance. Detection of these variants is challenging because of low variant allele frequencies (VAFs) and high GC content. The sensitivity of long-read amplicon sequencing with deepSNV analysis, referred to as LR-deep AmpSeq, for TERTp variant detection was tested. Among 47 patients with telomere biology disorder features, an average depth of coverage of 7943× was achieved, and seven TERTp variants were detected in six individuals (13%) with VAFs ranging from 0.006 to 0.33. These results demonstrate that LR-deep AmpSeq is a sensitive, cost-effective method to detect low VAF TERTp variants.