Four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. In total 2779 patients identified with either Alpha (n = 1153), Gamma (n = 122), Delta (n = 808), or Omicron variants (n = 696) were selected for analysis. No difference in frequency of hospitalization, intensive care unit (ICU) admission, and death were found among Alpha, Gamma, and Delta variants. However, patients with Omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (χ² = 12.8, P < .001; χ² = 21.6, P < .002; χ² = 9.6, P = .01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with Delta or Omicron variants (218/808 [26.9%] and 513/696 [73.7%], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between Omicron subvariants BA.1 and BA.2. Disease severity associated with Alpha, Gamma, and Delta variants is comparable while Omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with Omicron infection.

BACKGROUND: The detection of mutated epidermal growth factor receptor (EGFR) in non–small cell lung cancer (NSCLC) with residual cell pellets derived from liquid‐based cytology (LBC) samples (eg, endoscopic ultrasound–guided fine‐needle aspiration) has been validated with allele‐specific polymerase chain reaction. The aim of this study was to validate next‐generation sequencing (NGS) technology for detecting gene mutations with residual cell pellets from LBC.METHODS: Archived DNA extracted from LBC samples of adenocarcinoma stored in PreservCyt with a known EGFR mutation status was retrieved. Genomic DNA was multiplex‐amplified and enriched with Ion AmpliSeq Cancer Hotspot Panel v2 chemistry and the OneTouch 2 instrument; this was followed by semiconductor sequencing on the Ion Personal Genome Machine platform. The mutation hotspots of 6 NSCLC‐related genes (BRAF, EGFR, ERBB2, KRAS, MET, and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit α [PIK3CA]) were analyzed with NextGENe and Torrent Suite bioinformatics tools.RESULTS: The commonly identified EGFR sequence changes, including 4 L858R mutations, 3 exon 19 deletions, and 1 exon 20 insertion, were in 100% concordance between the assay platforms. Less common NSCLC variants were also found in the mutation hotspots of ERBB2, KRAS, MET, and PIK3CA genes.CONCLUSIONS: NSCLC mutation analysis using NGS can be successfully performed on residual cell pellets derived from LBC samples. This approach allows the simultaneous examination of multiple mutation hotspots in a timely manner to improve patient care. Cancer Cytopathol 2017;125:178–187. © 2016 American Cancer Society.

Background: Somatic mosaicism is to date an uncommon finding in genetic autoinflammatory syndromes such as Cryopyrin‐associated periodic syndrome, Blau syndrome, and TNF receptor‐associated periodic syndrome (TRAPS). However, somatic mosaicism may be particularly important in adult‐onset or atypical phenotypes of these conditions. Herein, we report a unique adult‐onset TRAPS patient presenting with intermittent daily fever for 3 weeks, rash, peritonitis, and lymphadenopathy, who was found with hematopoietic mosaicism involving different white blood cell populations.Methods: Patient's lymphocyte genomic DNA was initially analyzed by periodic fever seven‐gene next‐generation sequencing panel. Genomic DNAs extracted from patient's hair roots, buccal swab, and subpopulations of white blood cells were subsequently examined on the identified TNFRSF1A variant using Sanger sequencing.Results: A de novo mosaic missense variant, c.265 T>C(p.Phe89Leu), in the TNFRSF1A gene was found in the patient's buccal swab, B cells, neutrophils, and NK cells but not detected in monocytes, T cells, and hair roots.Conclusion: These data provide additional information about somatic mosaicism in autoinflammatory conditions and provide new insights regarding cellular players that are indispensable for the phenotypic expression of TRAPS.

Allele specific real-time PCR and next-generation sequencing (NGS) are widely used to detect somatic mutation in non-small cell lung cancer (NSCLC). Both methods commonly use formalin-fixed paraffin-embedded (FFPE) tissues as diagnostic materials. Real-time PCR has the advantage of being easy to use and more tolerant of variable DNA quality, but has limited multiplex capability. NGS, in contrast, allows simultaneous analysis of many genomic loci while revealing the exact sequence changes; it is, however, more technically demanding and more expensive to employed. A challenge for both platforms is the varied limit of detection (LoD) for target genomic loci, even within the same gene. The variability of detection sensitivity may be problematic if well-known actionable somatic mutations are missed. We compared LoDs between real-time PCR and targeted NGS tests for some commonly observed EGFR mutations in NSCLC specimens. The FDA-approved real-time PCR test was superior to the NGS in detecting low level EGFR exon 19 deletion (near 1% variant allele fraction (VAF)). The cancer hotspot NGS detects low level EGFR c.2369C > T, p.T790M (2-5% VAF) better than the FDA-approved real-time PCR method. We conclude that the real-time PCR and hotspot NGS methods have complementary strengths in accurately determining clinically important EGFR mutations in NSCLC.

Methods for identifying gene fusion events, such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and transcriptome analysis, are either single gene approaches or require bioinformatics expertise not generally available in clinical laboratories. We analytically validated a customized next-generation sequencing (NGS) panel targeting fusion events in 34 genes involving soft-tissue sarcomas. Specimens included 87 formalin-fixed paraffin-embedded (FFPE) tissues with known gene fusion status. Isolated total nucleic acid was used to identify fusion events at the RNA level. The potential fusions were targeted by gene-specific primers, followed by primer extension and nested PCR to enrich for fusion candidates with subsequent bioinformatics analysis. The study generated results using the following quality metrics for fusion detection: (a) ≥100 ng total nucleic acid, (b) RNA average unique start sites per gene-specific primer control ≥10, (c) quantitative PCR assessing input RNA quality had a crossing point <30, (d) total RNA percentage ≥30%, and (e) total sequencing fragments ≥500 000. The test validation study demonstrated analytical sensitivity of 98.7% and analytical specificity of 90.0%. The NGS-based panel generated highly concordant results compared to alternative testing methods.