Conditional JAG1 Mutation Shows the Developing Heart Is More Sensitive Than Developing Liver to JAG1 Dosage

Mutations of Jagged 1 (JAG1), a ligand in the Notch signaling pathway, cause Alagille syndrome (AGS). AGS is an autosomal dominant, multisystem disorder with variable expressivity, characterized by bile duct paucity and resultant liver disease in combination with cardiac, ocular, skeletal, and facial findings. JAG1 mutations in AGS include gene deletions and protein truncating, splicing, and missense mutations, suggesting that haploinsufficiency is the mechanism of disease causation. With limited exceptions, there is no genotype-phenotype correlation. We have studied a JAG1 missense mutation (JAG1-G274D) that was previously identified in 13 individuals from an extended family with cardiac defects of the type seen in patients with AGS (e.g., peripheral pulmonic stenosis and tetralogy of Fallot) in the absence of liver dysfunction. Our data indicate that this mutation is “leaky.” Two populations of proteins are produced from this allele. One population is abnormally glycosylated and is retained intracellularly rather than being transported to the cell surface. A second population is normally glycosylated and is transported to the cell surface, where it is able to signal to the Notch receptor. The JAG1-G274D protein is temperature sensitive, with more abnormally glycosylated (and nonfunctional) molecules produced at higher temperatures. Carriers of this mutation therefore have >50% but <100% of the normal concentration of JAG1 molecules on the cell surface. The cardiac-specific phenotype associated with this mutation suggests that the developing heart is more sensitive than the developing liver to decreased dosage of JAG1

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Context.-Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK.Objective.-To validate an anchored multiplex polymerase chain reaction-based panel for the detection of fusion transcripts in a university hospital-based clinical molecular diagnostics laboratory.Design.-We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffinembedded specimens with as little as 10% tumor nuclei.Results.-Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant Ill (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific.Conclusions.-This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing

<div><p>Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (<i>EGFR</i>, <i>KRAS</i>, <i>BRAF</i>, <i>NPM1</i>, <i>FLT3</i>, and <i>JAK2</i>). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two <i>FLT3</i> internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.</p></div

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes

Two Specimens with Low-Allele Frequency <i>FLT3</i> Internal Tandem Duplications.

<p>In one specimen (A and B), a 24 bp internal tandem duplication (ITD) was seen in 7 out of 529 reads for an allele frequency of 1.3%. (A) Four of the reads containing insertions (purple bars) are shown using the Integrative Genomics Viewer. This specimen additionally harbored a <i>FLT3</i> D839G mutation in 45% of reads (B). A second specimen (C) harbored a 33 bp <i>FLT3</i> ITD in 12 out of 739 reads, for an allele frequency of 1.6%. Nine of the reads carrying an ITD are pictured.</p

Next-generation sequencing data analysis pipeline.

<p>Data analysis occurs in three sequential stages, pre-processing of NGS reads, variant calling, and variant annotation. Of note, large indels are detected by an examination of reads that failed to map to target regions of the reference genome and are recovered from a pool of rejected reads (“Trash”). SNVs, single nucleotide variants. CNVs, copy number variation.</p

Concordance Analysis of Solid Tumor Specimens.

<p>The specimens shown were submitted for both NGS and targeted tests for <i>EGFR</i> (A), <i>KRAS</i> (B), and <i>BRAF</i> (C) mutations. Note that all mutations seen by targeted testing were also found by NGS when specimens with inadequate DNA quantity and/or quality are excluded.</p

Tumor Percentage of Specimens Analyzed by Targeted <i>EGFR</i> Tests.

<p>Cases were placed into 5 bins (<5%, 5–10%, 11–25%, 26–50%, >50% tumor cells in specimen).</p