High-depth next-generation sequencing panel testing in the evaluation of arteriovenous malformations.

Arteriovenous malformations (AVMs) are vascular lesions in which an overgrowth of blood vessels of varying sizes develops with one or more direct connections between the arterial and venous circulation. We performed a retrospective review of a cohort of 54 patients with AVMs referred to our clinical genomic laboratory for high-depth next-generation sequencing (NGS) panel of Disorders of Somatic Mosaicism (DoSM). Thirty-seven of 54 patients were female (68.5%). Among the 54 cases, 37 (68.5%) cases had pathogenic and/or likely pathogenic (P/LP) variants identified, two cases (3.7%) had variants of uncertain clinical significance, and the remaining 15 cases (27.8%) had negative results. MAP2K1 variants were found in 12 cases, followed by eight cases with KRAS variants and seven with TEK variants, and the remainder being identified in several other genes on the panel. Among the 37 positive cases, 32 cases had somatic alterations only; the remaining five cases had at least one germline P/LP variant, including four cases with PTEN and one with RASA1. Of note, two cases had the unexpected co-existence of two P/LP variants. In summary, this study illustrated the molecular diagnostic yield (68.5%) of this cohort of patients with a clinical indication of AVMs by our high-depth DoSM NGS panel.

A comparative analysis of RAS variants in patients with disorders of somatic mosaicism.

PURPOSE: RAS genes (HRAS, KRAS, and NRAS) are commonly found to be mutated in cancers, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed. METHODS: A total of 938 individuals with suspected DoSM underwent high-sensitivity clinical next-generation sequencing-based testing. We investigated the mutational spectrum and genotype-phenotype associations of mosaic RAS variants. RESULTS: In this article, we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Furthermore, we present 12 individuals with HRAS and KRAS in-frame duplication/insertion (dup/ins) variants in the switch II domain. Among the 18.3% individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations of these. The median age at testing was higher and the variant allelic fraction was lower in individuals with in-frame dup/ins variants than those in individuals with mosaic RAS hotspots. CONCLUSION: Our work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in nonmalignant conditions.

Comparison of gene fusion detection methods in salivary gland tumors.

Salivary gland neoplasms may pose diagnostic difficulties due to overlapping morphologic features. Recently, specific gene fusions have been discovered that correspond to particular tumor types, and can aid in accurate diagnosis. Gene rearrangements are commonly assessed by fluorescence in situ hybridization (FISH), although use of next-generation sequencing is increasing. However, there is no "gold standard" for fusion detection. We determined the concordance between FISH and a targeted RNA sequencing panel in gene fusion detection across twenty-two salivary gland tumors, including five mucoepidermoid carcinomas, four acinic cell carcinomas, four pleomorphic adenomas, two adenoid cystic carcinomas, two NUT carcinomas, and one each of basal cell adenoma, salivary duct carcinoma ex-pleomorphic adenoma, salivary duct carcinoma, clear cell carcinoma, and secretory carcinoma. Directed FISH testing based on the diagnosis was performed on cases that did not already have FISH conducted during clinical workup. Targeted RNA sequencing of 507 genes and their partners (using the Illumina TruSight Fusion Panel) was completed. Six of twenty-two (27.3%) cases had discordant results. In three cases, FISH results were negative while RNA sequencing results found fusion transcripts, which were all confirmed with RT-PCR and Sanger sequencing. In three cases, RNA sequencing results were negative while FISH results were positive for a gene rearrangement. Thus, if fusion analysis results are conflicting with the morphologic impression, a second mode of fusion detection may be warranted. Although both methods have advantages and drawbacks, RNA sequencing provides additional information about novel fusion partners and fusions that may not have been originally considered.

Clinical Implications of a Targeted RNA-Sequencing Panel in the Detection of Gene Fusions in Solid Tumors.

The detection of recurrent gene fusions can help confirm diagnoses in solid tumors, particularly when the morphology and staining are unusual or nonspecific, and can guide therapeutic decisions. Although fluorescence in situ hybridization and PCR are often used to identify fusions, the rearrangement must be suspected, with only a few prioritized probes run. It was hypothesized that the Illumina TruSight RNA Fusion Panel, which detects fusions of 507 genes and their partners, would uncover fusions with greater sensitivity than other approaches, leading to changes in diagnosis, prognosis, or therapy. Targeted RNA sequencing was performed on formalin-fixed, paraffin-embedded sarcoma and carcinoma cases in which fluorescence in situ hybridization, RT-PCR, or DNA-based sequencing was conducted during the diagnostic workup. Of the 153 cases, 138 (90%) were sequenced with adequate quality control metrics. A total of 101 of 138 (73%) cases were concordant by RNA sequencing and the prior test method. RNA sequencing identified an additional 30 cases (22%) with fusions that were not detected by conventional methods. In seven cases (5%), the additional fusion information provided by RNA sequencing would have altered diagnosis and management. A total of 19 novel fusion pairs (not previously described in the literature) were discovered (14%). Overall, the findings show that a targeted RNA-sequencing method can detect gene fusions in formalin-fixed, paraffin-embedded specimens with high sensitivity.

Somatic PIK3R1 variation as a cause of vascular malformations and overgrowth.

PURPOSE: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1. METHODS: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity. RESULTS: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone. CONCLUSION: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

A Next-Generation Sequencing Test for Severe Congenital Neutropenia: Utility in a Broader Clinicopathologic Spectrum of Disease.

Severe congenital neutropenia (SCN) is a collection of diverse disorders characterized by chronically low absolute neutrophil count in the peripheral blood, increased susceptibility to infection, and a significant predisposition to the development of myeloid malignancies. SCN can be acquired or inherited. Inherited forms have been linked to variants in a group of diverse genes involved in the neutrophil-differentiation process. Variants that promote resistance to treatment have also been identified. Thus, genetic testing is important for the diagnosis, prognosis, and management of SCN. Herein we describe clinically validated assay developed for assessing patients with suspected SCN. The assay is performed from a whole-exome backbone. Variants are called across all coding exons, and results are filtered to focus on 48 genes that are clinically relevant to SCN. Validation results indicated 100% analytical sensitivity and specificity for the detection of constitutional variants among the 48 reportable genes. To date, 34 individuals have been referred for testing (age range: birth to 67 years). Several pathogenic and likely pathogenic variants have been identified, including one in a patient with late-onset disease. The pattern of cases referred for testing suggests that this assay has clinical utility in a broader spectrum of patients beyond those in the pediatric population who have classic early-onset symptoms characteristic of SCN.

Diagnostic Utility of Next-Generation Sequencing for Disorders of Somatic Mosaicism: A Five-Year Cumulative Cohort.

Disorders of somatic mosaicism (DoSM) are a diverse group of syndromic and non-syndromic conditions caused by mosaic variants in genes that regulate cell survival and proliferation. Despite overlap in gene space and technical requirements, few clinical labs specialize in DoSM compared to oncology. We adapted a high-sensitivity next-generation sequencing cancer assay for DoSM in 2014. Some 343 individuals have been tested over the past 5 years, 58% of which had pathogenic and likely pathogenic (P/LP) findings, for a total of 206 P/LP variants in 22 genes. Parameters associated with the high diagnostic yield were: (1) deep sequencing (∼2,000× coverage), (2) a broad gene set, and (3) testing affected tissues. Fresh and formalin-fixed paraffin embedded tissues performed equivalently for identification of P/LP variants (62% and 71% of individuals, respectively). Comparing cultured fibroblasts to skin biopsies suggested that culturing might boost the allelic fraction of variants that confer a growth advantage, specifically gain-of-function variants in PIK3CA. Buccal swabs showed high diagnostic sensitivity in case subjects where disease phenotypes manifested in the head or brain. Peripheral blood was useful as an unaffected comparator tissue to determine somatic versus constitutional origin but had poor diagnostic sensitivity. Descriptions of all tested individuals, specimens, and P/LP variants included in this cohort are available to further the study of the DoSM population.