Serial Postoperative Circulating Tumor DNA Assessment Has Strong Prognostic Value During Long-Term Follow-Up in Patients With Breast Cancer.

Purpose: Here, we report the sensitivity of a personalized, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera) for detection of molecular relapse during long-term follow-up of patients with breast cancer.

Methods: A total of 156 patients with primary breast cancer were monitored clinically for up to 12 years after surgery and adjuvant chemotherapy. Semiannual blood samples were prospectively collected, and analyzed retrospectively to detect residual disease by ultradeep sequencing using ctDNA assays, developed from primary tumor whole-exome sequencing data.

Circulating tumour DNA dynamics during alternating chemotherapy and hormonal therapy in metastatic breast cancer: the ALERT study.

Purpose: Although changes in circulating tumour DNA (ctDNA) in breast cancer are well described, the kinetics of their fluctuations has not been described over short timescales. We investigated ctDNA dynamics during alternating cycles of chemotherapy and hormonal treatment in pre-treated patients with oestrogen receptor-positive metastatic breast cancer.

Methods: Patients received alternating, 9-week cycles of eribulin and aromatase inhibitors (AIs).

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

Objectives: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful.

Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer.

Background: Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients.

Method: A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients.

The Evolution of Affordable Technologies in Liquid Biopsy Diagnostics: The Key to Clinical Implementation.

Cancer remains a leading cause of death worldwide, despite many advances in diagnosis and treatment. Precision medicine has been a key area of focus, with research providing insights and progress in helping to lower cancer mortality through better patient stratification for therapies and more precise diagnostic techniques. However, unequal access to cancer care is still a global concern, with many patients having limited access to diagnostic tests and treatment regimens.

A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA.

Background: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs).

Methods: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™.

How to design and implement a university-based COVID-19 testing programme? An evaluation of a novel RT-LAMP COVID-19 testing programme in a UK university.

Background: Little is known about how asymptomatic testing as a method to control transmission of COVID-19 can be implemented, and the prevalence of asymptomatic infection within university populations. The objective of this study was to investigate how to effectively set-up and implement a COVID-19 testing programme using novel reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology and to quantify the scale of asymptomatic infection on a university campus.

Methods: An observational study to describe the set-up and implementation of a novel COVID-19 testing programme on a UK university campus between September and December 2020.

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer.

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines.

Shallow WGS of individual CTCs identifies actionable targets for informing treatment decisions in metastatic breast cancer.

Background: We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples.

Methods: Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher).

A rapid RT-LAMP SARS-CoV-2 screening assay for collapsing asymptomatic COVID-19 transmission.

Purpose: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population.

Methods: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples.

Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.

The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression.

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation.

Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of circulating cell-free DNA fragments in the patients' blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy.

Mapping the Allosteric Action of Antagonists A740003 and A438079 Reveals a Role for the Left Flipper in Ligand Sensitivity at P2X7 Receptors.

P2X7 receptor (P2X7R) activation requires ∼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site.

Unique residues in the ATP gated human P2X7 receptor define a novel allosteric binding pocket for the selective antagonist AZ10606120.

The P2X7 receptor (P2X7R) for ATP is a therapeutic target for pathophysiological states including inflammation, pain management and epilepsy. This is facilitated by the predicted low side effect profile as the high concentrations of ATP required to activate the receptor are usually only found following cell damage/disease and so P2X7Rs respond to a "danger" signal and are not normally active. AZ10606120 is a selective antagonist for P2X7Rs (IC of ~10 nM) and ineffective at the P2X1R (at 10 μM).

Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels.

P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components.

P2X receptor chimeras highlight roles of the amino terminus to partial agonist efficacy, the carboxyl terminus to recovery from desensitization, and independent regulation of channel transitions.

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.

Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.

P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor.

The intracellular amino terminus plays a dominant role in desensitization of ATP-gated P2X receptor ion channels.

P2X receptors show marked variations in the time-course of response to ATP application from rapidly desensitizing P2X1 receptors to relatively sustained P2X2 receptors. In this study we have used chimeras between human P2X1 and P2X2 receptors in combination with mutagenesis to address the contribution of the extracellular ligand binding loop, the transmembrane channel, and the intracellular regions to receptor time-course. Swapping either the extracellular loop or both transmembrane domains (TM1 and -2) between the P2X1 and P2X2 receptors had no effect on the time-course of ATP currents in the recipient receptor.

Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating.

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed.

Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors.

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100.

P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation.

P2X1 receptors for ATP contribute to signalling in a variety of cell types and following stimulation undergo rapid desensitisation (within 1 s), and require approximately 5 min to recover. In HEK293 cells P2X1 receptors C-terminally tagged with enhanced green fluorescent protein (P2X1-eGFP) were predominantly expressed at the cell surface. Following > 90% photo-bleaching of P2X1-eGFP within a 6 microm(2) circle at the cell surface fluorescence recovery after photo-bleaching (FRAP) was fit with a time constant of approximately 60 s and recovered to approximately 75% of pre-bleach levels.

Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis.

At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrPhiPhixLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by approximately 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced.