reconCNV: interactive visualization of copy number data from high-throughput sequencing.

SUMMARY: Copy number variation (CNV) is an important category of unbalanced structural rearrangement. While methods for detecting CNV in high-throughput targeted sequencing have become increasingly sophisticated, dedicated tools for interactive and dynamic visualization of CNV from these data are still lacking. We describe reconCNV, a tool that produces an interactive and annotated web-based dashboard for viewing and summarizing CNVs detected in next-generation sequencing (NGS) data. reconCNV is designed to work with delimited result files from most NGS CNV callers with minor adjustments to the configuration file. The reconCNV output is an HTML file that is viewable on any modern web browser, requires no backend server, and can be readily appended to existing analysis pipelines. In addition to a standard CNV track for visualizing relative fold change and absolute copy number, the tool includes an auxiliary variant allele fraction track for visualizing underlying allelic imbalance and loss of heterozygosity. A feature to mask assay-specific technical artifacts and a direct HTML link out to the UCSC Genome Browser are also included to augment the reviewer experience. By providing a light-weight plugin for interactive visualization to existing NGS CNV pipelines, reconCNV can facilitate efficient NGS CNV visualization and interpretation in both research and clinical settings. AVAILABILITY AND IMPLEMENTATION: The source code and documentation including a tutorial can be accessed at https://github.com/rghu/reconCNV as well as a Docker image at https://hub.docker.com/repository/docker/raghuc1990/reconcnv. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrated DNA and RNA sequencing reveals targetable alterations in metastatic pediatric papillary thyroid carcinoma.

BACKGROUND: Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy. METHODS: Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq. RESULTS: Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonly BRAF p.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%): RET (n = 8), NTRK3 (n = 4), and BRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. Both BRAF V600E mutations and gene fusions were correlated with the presence of cervical metastases. CONCLUSIONS: Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease.

Alternative genetic mechanisms of BRAF activation in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207+ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in ∼75% of LCH cases, including recurrent BRAF-V600E and MAP2K1 mutations. To elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole-exome sequencing (WES, n = 6), targeted BRAF sequencing (n = 19), and/or whole-transcriptome sequencing (RNA-seq, n = 6) on 24 LCH patient samples lacking BRAF-V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the ß3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A-BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207+ cells from lesions. ERK activation was resistant to BRAF-V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for patients with LCH.