Implementing Next-Generation Sequencing Process Changes to Increase Capacity and Improve Timeliness of Molecular Biomarker Profiling for Lung Cancer Patients.

BACKGROUND: Faced with expansion of molecular tumor biomarker profiling, the molecular genetics laboratory at Kingston Health Science Centre experienced significant pressures to maintain the provincially mandated 2-week turnaround time (TAT) for lung cancer (LC) patients. We used quality improvement methodology to identify opportunities for improved efficiencies and report the impact of the initiative. METHODS: We set a target of reducing average TAT from accessioning to clinical molecular lab report for LC patients. Process measures included percentage of cases reaching TAT within target and number of cases. We developed a value stream map and used lean methodology to identify baseline inefficiencies. Plan-Do-Study-Act cycles were implemented to streamline, standardize, and automate laboratory workflows. Statistical process control (SPC) charts assessed for significance by special cause variation. RESULTS: A total of 257 LC cases were included (39 baseline January-May 2021; 218 post-expansion of testing June 2021). The average time for baseline TAT was 12.8 days, peaking at 23.4 days after expansion of testing, and improved to 13.9 days following improvement interventions, demonstrating statistical significance by special cause variation (nonrandom variation) on SPC charts. CONCLUSIONS: The implementation of standardized manual and automated laboratory processes improved timeliness of biomarker reporting despite the increasing volume of testing at our center.

Prevalence of NTRK Fusions in Canadian Solid Tumour Cancer Patients.

INTRODUCTION: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions occur in ~ 0.3% of all solid tumours but are enriched in some rare tumour types. Tropomyosin receptor kinase (TRK) inhibitors larotrectinib and entrectinib are approved as tumour-agnostic therapies for solid tumours harbouring NTRK fusions. METHODS: This study investigated the prevalence of NTRK fusions in Canadian patients and also aimed to help guide NTRK testing paradigms through analysis of data reported from a national clinical diagnostic testing program between September 2019 and July 2021. RESULTS: Of 1,687 patients included in the final analysis, NTRK fusions were detected in 0.71% (n = 12) of patients representing salivary gland carcinoma (n = 3), soft tissue sarcoma (n = 3), CNS (n = 3), and one in each of melanoma, lung, and colorectal cancer. All three salivary gland carcinomas contained ETV6-NTRK3 fusions. Thirteen (0.77%) clinically actionable incidental findings were also detected. Two of the 13 samples containing incidental findings were NTRK fusion-positive (GFOD1-NTRK2, FGFR3-TACC3 in a glioblastoma and AFAP1-NTRK2, BRAF c.1799T>A in a glioma). The testing algorithm screened most patient samples via pan-TRK immunohistochemistry (IHC), whereas samples from the central nervous system (CNS), pathognomonic cancers, and confirmed/ putative NTRK fusion-positive samples identified under research protocols were reflexed straight to next-generation sequencing (NGS). CONCLUSION: These findings highlight the benefit and practicality of a diagnostic testing program to identify patients suitable for tumour-agnostic TRK inhibitor therapies, as well as other targeted therapies, due to clinically actionable incidental findings identified. Collectively, these findings may inform future guidance on selecting the appropriate testing approach per tumour type and on optimal NTRK testing algorithms.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Implementation of an NGS-based sequencing and gene fusion panel for clinical screening of patients with suspected hematologic malignancies.

OBJECTIVES: The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen. METHODS: Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results. RESULTS: The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%. CONCLUSIONS: Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories.