Utilisation of semiconductor sequencing for detection of actionable fusions in solid tumours.

Oncogenic fusions represent compelling druggable targets in solid tumours highlighted by the recent site agnostic FDA approval of larotrectinib for NTRK rearrangements. However screening for fusions in routinely processed tissue samples is constrained due to degradation of nucleic acid as a result of formalin fixation., To investigate the clinical utility of semiconductor sequencing optimised for detection of actionable fusion transcripts in formalin fixed samples, we have undertaken an analysis of test trending data generated by a clinically validated next generation sequencing platform designed to capture 867 of the most clinically relevant druggable driver-partner oncogenic fusions. Here we show across a real-life cohort of 1112 patients with solid tumours that actionable fusions occur at high frequency (7.4%) with linkage to a wide range of targeted therapy protocols including seven fusion-drug matches with FDA/EMA approval and/or NCCN/ESMO recommendations and 80 clinical trials. The more prevalent actionable fusions identified were independent of tumour type in keeping with signalling via evolutionary conserved RAS/RAF/MEK/ERK, PI3K/AKT/MTOR, PLCy/PKC and JAK/STAT pathways. Taken together our data indicates that semiconductor sequencing for detection of actionable fusions can be integrated into routine diagnostic pathology workflows enabling the identification of personalised treatment options that have potential to improve clinical cancer management across many tumour types.

Utilisation of semiconductor sequencing for the detection of predictive biomarkers in glioblastoma.

The standard treatment for glioblastoma involves a combination of surgery, radiation and chemotherapy but have limited impact on survival. The exponential increase in targeted agents directed at pivotal oncogenic pathways now provide new therapeutic opportunities for this tumour type. However, lack of comprehensive precision oncology testing at diagnosis means such therapeutic opportunities are potentially overlooked. To investigate the role of semiconductor sequencing for detection of predictive biomarkers in routine glioblastoma samples we have undertaken analysis of test trending data generated by a clinically validated next generation sequencing platform designed to capture actionable genomic variants distributed across 505 genes. Analysis was performed across a cohort of 55 glioblastoma patients. Analysis of trending data has revealed a complex and rich actionable mutational landscape in which 166 actionable mutations were detected across 36 genes linked to 17 off label targeted therapy protocols and 111 clinical trials. The majority of patients harboured three or more actionable mutations affecting key cancer related regulatory networks including the PI3K/AKT/MTOR and RAS/RAF/MEK/MAPK signalling pathways, DNA-damage repair pathways and cell cycle checkpoints. Linkage with immunotherapy and PARP inhibitors was identified in 44% of glioblastoma patients as a consequence of alterations in DNA-damage repair genes. Taken together our data indicates that precision oncology testing utilising semiconductor sequencing can be used to identify a broad therapeutic armamentarium of targeted therapies and immunotherapies that can be potentially employed for the improved clinical management of glioblastoma patients.

SARS-CoV-2 testing and sequencing for international arrivals reveals significant cross border transmission of high risk variants into the United Kingdom.

BACKGROUND: Mandatory Day 2 and Day 8 PCR testing and variant sequencing of international arrivals has been recently introduced by the UK Government to mitigate against cross-border transmission of high-risk SARS-CoV-2 variants. METHODS: SARS-CoV-2 testing and sequencing combines TaqPath CE-IVD COVID-19 RT-PCR with Ion AmpliSeq SARS-CoV-2 Next Generation Sequencing Assay. Retrospective analysis of test trending data was performed from initiation of testing on the 11th March through to the 14th April 2021. FINDINGS: During this time interval, 203,065 SARS-CoV-2 PCR tests were performed, with 3,855 samples testing positive, giving a prevalence of 1.9%. In total 1,913 SARS-CoV-2 genomes were sequenced from positive cases with Ct values < 30 and 1,635 (85.5%) sequences passed quality metrics for lineage analysis. A high diversity of 49 different SARS-CoV-2 variants were identified, including the VOCs B.1.1.7 (Kent; 80.6%), B.1.351 (South Africa; 4.2%), B.1.617.2 (India; 1.7%), P.1 (Brazil; 0.4%) and B.1.1.7 with E484K (Bristol; 0.2%). Vaccine effectiveness was age-related and dose-dependent, ranging from 5% in > 60 with a single dose to 83% in <60 with both doses of a vaccine. Viral load was variant dependent with the B.1.617.2 showing a 21 fold increase in viral copy number compared to the other variants. INTERPRETATION: The unexpectedly high prevalence of COVID-19 infection in UK arrivals is associated with a rich diversity of SARS-CoV-2 high risk variants entering the UK including the VOC B.1.617.2. Vaccination does not preclude infection and its effectiveness is significantly age-dependent and impacted by variant type. The rapid high-throughput test and sequence workflow we have adopted is particularly suited to the monitoring of cross border transmission and enables immediate public health interventions. FUNDING: Data analysis conducted in this study was limited to secondary use of information previously collected in the course of normal care.

A DNA-Binding Bromodomain-Containing Protein Interacts with and Reduces Rx1-Mediated Immune Response to Potato Virus X.

Plant NLR proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming. Some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato binds and distorts double-stranded DNA. However, the components of the chromatin-localized Rx1 complex are largely unknown. Here, we report a physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP accumulates in the nucleoplasm and nucleolus, interacts with chromatin, and redistributes Rx1 to the nucleolus in a subpopulation of imaged cells. Rx1 overexpression reduces the interaction between NbDBCP and chromatin. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X (PVX), and this activity requires an intact bromodomain. Previously, Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor, NbGlk1. Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding, suggesting a mode of action for NbDBCP's inhibitory effect on immunity. This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception.