Comparison of SARS-CoV-2 detection from nasopharyngeal swab samples by the Roche cobas 6800 SARS-CoV-2 test and a laboratory-developed real-time RT-PCR test.

The urgent need to implement and rapidly expand testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has led to the development of multiple assays. How these tests perform relative to one another is poorly understood. We evaluated the concordance between the Roche Diagnostics cobas 6800 SARS-CoV-2 test and a laboratory-developed test (LDT) real-time reverse transcription-polymerase chain reaction based on a modified Centers for Disease Control and Prevention protocol, for the detection of SARS-CoV-2 in samples submitted to the Clinical Laboratories of the Mount Sinai Health System. A total of 1006 nasopharyngeal swabs in universal transport medium from persons under investigation were tested for SARS-CoV-2 as part of routine clinical care using the cobas SARS-CoV-2 test with subsequent evaluation by the LDT. Cycle threshold values were analyzed and interpreted as either positive ("detected" or "presumptive positive"), negative (not detected), inconclusive, or invalid. Statistical analysis was performed using GraphPad Prism 8. The cobas SARS-CoV-2 test reported 706 positive and 300 negative results. The LDT reported 640 positive, 323 negative, 34 inconclusive, and 9 invalid results. When excluding inconclusive and invalid results, the overall percent agreement between the two platforms was 95.8%. Cohen's κ coefficient was 0.904 (95% confidence interval, 0.875-0.933), suggesting almost perfect agreement between both platforms. An overall discordance rate of 4.2% between the two systems may reflect differences in primer sequences, assay limit of detection, or other factors, highlighting the importance of comparing the performance of different testing platforms.

Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples.

Glioblastoma is a malignant brain tumor with dismal prognosis. Oncogenic mutations in glioblastoma frequently affect receptor tyrosine kinase pathway components that are challenging to quantify because of heterogeneous expression. EGFRvIII, a common oncogenic receptor tyrosine kinase mutant protein in glioblastoma, potentiates tumor malignancy and is an emerging tumor-specific immunotarget, underlining the need for its more accessible and quantitative detection. We used normalized next-generation sequencing data from 117 brain and 371 reference clinical tumor samples to detect focal gene amplifications across the commercial Ion AmpliSeq Cancer Hotspot Panel version 2 and infer EGFRvIII status based on relative coverage dropout of the gene's truncated region within EGFR. In glioblastomas (n = 45), amplification of EGFR [18 (40%)], PDGFRA [3 (7%)], KIT [2 (4%)], MET [1 (2%)], and AKT1 [1 (2%)] was detected. With respect to EGFR and PDGFRA amplification, there was near-complete agreement between next-generation sequencing and in situ hybridization. Consistent with previous reports, this method detected EGFRvIII exclusively in EGFR-amplified glioblastomas [8 (44%)], which was confirmed using long-range PCR. Our study offers a practical method for detecting oncogene amplifications and large intragenic mutations in a clinically implemented hotspot panel that can be quantified using z scores. The validated detection of EGFRvIII using DNA sequencing eliminates problems with transcript degradation, and the provided script facilitates efficient incorporation into a laboratory's bioinformatic pipeline.

Cancer gene profiling in non-small cell lung cancers reveals activating mutations in JAK2 and JAK3 with therapeutic implications.

BACKGROUND: Next-generation sequencing (NGS) of cancer gene panels are widely applied to enable personalized cancer therapy and to identify novel oncogenic mutations. METHODS: We performed targeted NGS on 932 clinical cases of non-small-cell lung cancers (NSCLCs) using the Ion AmpliSeq™ Cancer Hotspot panel v2 assay. RESULTS: Actionable mutations were identified in 65% of the cases with available targeted therapeutic options, including 26% of the patients with mutations in National Comprehensive Cancer Network (NCCN) guideline genes. Most notably, we discovered JAK2 p.V617F somatic mutation, a hallmark of myeloproliferative neoplasms, in 1% (9/932) of the NSCLCs. Analysis of cancer cell line pharmacogenomic data showed that a high level of JAK2 expression in a panel of NSCLC cell lines is correlated with increased sensitivity to a selective JAK2 inhibitor. Further analysis of TCGA genomic data revealed JAK2 gain or loss due to genetic alterations in NSCLC clinical samples are associated with significantly elevated or reduced PD-L1 expression, suggesting that the activating JAK2 p.V617F mutation could confer sensitivity to both JAK inhibitors and anti-PD1 immunotherapy. We also detected JAK3 germline activating mutations in 6.7% (62/932) of the patients who may benefit from anti-PD1 treatment, in light of recent findings that JAK3 mutations upregulate PD-L1 expression. CONCLUSION: Taken together, this study demonstrated the clinical utility of targeted NGS with a focused hotspot cancer gene panel in NSCLCs and identified activating mutations in JAK2 and JAK3 with clinical implications inferred through integrative analysis of cancer genetic, genomic, and pharmacogenomic data. The potential of JAK2 and JAK3 mutations as response markers for the targeted therapy against JAK kinases or anti-PD1 immunotherapy warrants further investigation.