Analytical Validation of a 37-Gene Next-Generation Sequencing Panel for Myeloid Malignancies and Review of Initial Findings Incorporating Updated 2022 Diagnostic and Prognostic Guidelines.

Myeloid neoplasms are clonal disorders that arise via acquisition of genetic mutations leading to excessive proliferation and defective differentiation. Mutational profiling is vital as it has implications for diagnosis, prognosis, and therapeutic decision-making. Next-generation sequencing (NGS) has become a mainstay in the evaluation of myeloid malignancies, as it enables efficient characterization of multiple genetic changes. Herein, the analytical validation of the 37-gene Archer VariantPlex Core Myeloid panel is reported, using 58 DNA specimens with 87 single-nucleotide variants and 23 insertions/deletions. The panel achieved good depth of coverage, 100% analytical sensitivity and specificity for single-nucleotide variants and insertions/deletions ≤21 bp, and 100% reproducibility, with a reportable limit of detection determined as 5%. The Archer NGS panel can accurately and reproducibly detect variants of clinical significance in myeloid neoplasms. A retrospective analysis of 535 clinical specimens tested with the Archer NGS panel showed a frequency and pattern of mutations across myeloid malignancies that were similar to other published studies. A review of the diagnostic classification of patients with acute myeloid leukemia and myelodysplastic syndrome using the World Health Organization 2017/2022 and International Consensus Classification 2022 guidelines, in addition to European LeukemiaNet 2017/2022 risk stratification of patients with acute myeloid leukemia, was also performed to assess the utility of the molecular information provided by the Archer NGS panel.

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Shariant platform: Enabling evidence sharing across Australian clinical genetic-testing laboratories to support variant interpretation.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

A Multi-Disciplinary Team Approach to Genomic Testing for Drug-Resistant Epilepsy Patients-The GENIE Study.

BACKGROUND: The genomic era has led to enormous progress in clinical care and a multi-disciplinary team (MDT) approach is imperative for integration of genomics into epilepsy patient care. METHODS: The MDT approach involved patient selection, genomic testing choice, variant discussions and return of results. Genomics analysis included cytogenomic testing and whole exome sequencing (WES). Neurologist surveys were undertaken at baseline and after genomic testing to determine if genomic diagnoses would alter their management, and if there was a change in confidence in genomic testing and neurologist perceptions of the MDT approach. RESULTS: The total diagnostic yield from all genomic testing was 17% (11/66), with four diagnoses from cytogenomic analyses. All chromosomal microarray (CMA) diagnoses were in patients seen by adult neurologists. Diagnostic yield for WES was 11% (7/62). The most common gene with pathogenic variants was DCX, reported in three patients, of which two were mosaic. The genomic diagnosis impacted management in 82% (9/11). There was increased confidence with integrating genomics into clinical care (Pearson chi square = 83, p = 0.004) and qualitative comments were highly supportive of the MDT approach. CONCLUSIONS: We demonstrated diagnostic yield from genomic testing, and the impact on management in a cohort with drug-resistant epilepsy. The MDT approach increased confidence in genomic testing and neurologists valued the input from this approach. The utility of CMA was demonstrated in epilepsy patients seen by adult neurologists as was the importance of considering mosaicism for previously undiagnosed patients.

Giant axonal neuropathy diagnosed on skin biopsy.

Evaluation of hereditary axonal neuropathy in childhood is complex. Often, the child has to be subjected to general anaesthesia for a nerve biopsy to guide further genetic testing, which may or may not be readily available. We describe a toddler with clinical features suggesting giant axonal neuropathy (GAN), whose diagnosis was confirmed by minimally invasive skin biopsy and corroborated by the finding of compound heterozygous mutations involving the GAN gene, including a novel interstitial microdeletion at 16q23.2 detected by microarray and a point mutation detected by direct sequencing.

Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction.

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.