Homologous recombination pathway gene variants identified by tumor-only sequencing assays in lung carcinoma patients.

Background: The homologous recombination (HR) repair pathway plays a key role in double-stranded DNA break repair, and germline HR pathway gene variants are associated with increased risk of several cancers, including breast and ovarian cancer. HR deficiency is also a therapeutically targetable phenotype. Methods: Somatic (tumour-only) sequencing was performed on 1,109 cases of lung tumors, and the pathological data were reviewed to filter for lung primary carcinomas. Cases were filtered for variants (disease-associated or of uncertain significance) in 14 HR pathway genes, including BRCA1, BRCA2, and ATM. The clinical, pathological and molecular data were reviewed. Results: Sixty-one HR pathway gene variants in 56 patients with primary lung cancer were identified. Further filtering by variant allele fraction (VAF) of ≥30% identified 17 HR pathway gene variants in 17 patients. ATM gene variants were most the commonly identified (9/17), including two patients with c.7271T>G (p.V2424G), a variant in the germline that is associated with increased familial cancer risk. Four (4/17) patients had a family history of lung cancer, among which three patients had ATM gene variants suspected to be germline in origin. In three other patients with BRCA1/2 or PALB2 gene variants who had undergone germline testing, the variants were confirmed to be germline; lung cancer was the sentinel cancer in two of these patients with a BRCA1 or PALB2 variant. Conclusions: Genomic variants in the HR repair pathway identified in tumor-only sequencing and occurring at higher VAFs (i.e., ≥30%) may suggest a germline origin. Correlating with personal and family history, a subset of these variants is also suggested to be associated with familial cancer risks. Patient age, smoking history and driver mutation status are expected to be a poor screening tool in identifying these patients. Finally, the relative enrichment for ATM variants in our cohort suggests a possible association between ATM mutation and lung cancer risk.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms.

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

CNViz: An R/Shiny Application for Interactive Copy Number Variant Visualization in Cancer.

Copy number variants (CNVs) comprise a class of mutation which includes deletion, duplication, or amplification events that range in size from smaller than a single-gene or exon, to the size of a full chromosome. These changes can affect gene expression levels and are thus implicated in disease, including cancer. Although a variety of tools and methodologies exist to detect CNVs using data from massively parallel sequencing (also referred to as next-generation sequencing), it can be difficult to appreciate the copy number profile in a list format or as a static image. CNViz is a freely accessible R/Bioconductor package that launches an interactive R/Shiny visualization tool to facilitate review of copy number data. As inputs, it requires genomic locations and corresponding copy number ratios for probe, gene, and/or segment-level data. If supplied, loss of heterozygosity (LOH), focal variant data [single nucleotide variants (SNVs) and small insertions and deletions (indels)], and metadata (e.g., specimen purity and ploidy) can also be incorporated into the visualization. The CNViz R/Bioconductor package is an easy-to-use tool built with the intent of encouraging visualization and exploration of copy number variation. CNViz can be used in a clinical setting as well as for research to study patterns in human cancers more broadly. The intuitive interface allows users to visualize the copy number profile of a specimen, dynamically change resolution to explore gene and probe-level copy number changes, and simultaneously integrate LOH, SNV, and indel findings. CNViz is available for download as an R package via Bioconductor. An example of the application is available at rebeccagreenblatt.shinyapps.io/cnviz_example.

TERT gene rearrangement in chordomas and comparison to other TERT-rearranged solid tumors.

Chordomas are rare, slow-growing neoplasms thought to arise from the foetal notochord remnant. A limited number of studies that examined the mutational profiles in chordomas identified potential driver mutations, including duplication in the TBXT gene (encoding brachyury), mutations in the PI3K/AKT signaling pathway, and loss of the CDKN2A gene. Most chordomas remain without clear driver mutations, and no fusion genes have been identified thus far. We discovered a novel TERT in-frame fusion involving RPH3AL (exon 5) and TERT (exon 2) in the index chordoma case. We screened a discovery cohort of 18 additional chordoma cases for TERT gene rearrangement by FISH, in which TERT rearrangement was identified in one additional case. In our independent, validation cohort of 36 chordomas, no TERT rearrangement was observed by FISH. Immunohistochemistry optimized for nuclear TERT expression showed at least focal TERT expression in 40/55 (72.7%) chordomas. Selected cases underwent molecular genetic profiling, which showed low tumor mutational burdens (TMBs) without obvious driver oncogenic mutations. We next examined a cohort of 1,913 solid tumor patients for TERT rearrangements, and TERT fusions involving exon 2 were observed in 7/1,913 (0.4%) cases. The seven tumors comprised five glial tumors, and two poorly differentiated carcinomas. In contrast to chordomas, the other TERT-rearranged tumors were notable for higher TMBs, frequent TP53 mutations (6/7) and presence of other driver oncogenic mutations, including a concurrent fusion (TRIM24-MET). In conclusion, TERT gene rearrangements are seen in a small subset (2/55, 3.6%) of chordomas. In contrast to other TERT-rearranged tumors, where the TERT rearrangements are likely passenger events, the possibility that TERT protein overexpression representing a key event in chordoma tumorigenesis is left open.

Recombination rate plasticity: revealing mechanisms by design.

For over a century, scientists have known that meiotic recombination rates can vary considerably among individuals, and that environmental conditions can modify recombination rates relative to the background. A variety of external and intrinsic factors such as temperature, age, sex and starvation can elicit 'plastic' responses in recombination rate. The influence of recombination rate plasticity on genetic diversity of the next generation has interesting and important implications for how populations evolve. Further, many questions remain regarding the mechanisms and molecular processes that contribute to recombination rate plasticity. Here, we review 100 years of experimental work on recombination rate plasticity conducted in Drosophila melanogaster We categorize this work into four major classes of experimental designs, which we describe via classic studies in D. melanogaster Based on these studies, we highlight molecular mechanisms that are supported by experimental results and relate these findings to studies in other systems. We synthesize lessons learned from this model system into experimental guidelines for using recent advances in genotyping technologies, to study recombination rate plasticity in non-model organisms. Specifically, we recommend (1) using fine-scale genome-wide markers, (2) collecting time-course data, (3) including crossover distribution measurements, and (4) using mixed effects models to analyse results. To illustrate this approach, we present an application adhering to these guidelines from empirical work we conducted in Drosophila pseudoobscuraThis article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'.