Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Miscellaneous Rheumatic Diseases [73-83]: 73. Is There a Delay in Specialist Referral of Hot Swollen Joint?

Background: Patients with acute, hot, swollen joints commonly present to general practitioners, emergency departments and/or acute admitting teams rather than directly to rheumatology. It is imperative to consider septic arthritis in the differential diagnosis of these patients. The British Society of Rheumatology (BSR) has produced guidelines for the management of this condition, which include recommendations for early specialist referral and joint aspiration of all patients with suspected septic arthritis. We examined whether the initial management of patients with acute hot swollen joint(s) at University College London Hospital (UCLH) follows BSR guidelines. Methods: For the period Feb to Nov 2009, appropriate patients were identified by searching the UCLH database using the diagnostic terms, "pyogenic arthritis”, "septic arthritis” and "gout”; and from all joint aspirate requests sent to microbiology. Medical notes were obtained and any patients who had elective arthroscopies or chronic (> 6 weeks) symptoms were excluded. Data were collected on the time taken from the onset of symptoms to specialist (orthopaedic/rheumatology) referral and joint aspiration, collection of blood cultures and antibiotic treatment with or without microbiology advice. Results: Twenty patients were identified with hot swollen (18 monoarticular, 3 prosthetic) joint(s) of < 2 weeks duration. Of whom, 3/20 (15%) were admitted directly to rheumatology, 7/20 (35%) to the acute admissions unit, 3/20 (15%) to orthopaedic, 4/20 (20%) to a medical team and 1/20 (5%) to general surgery. In 19 (95%) cases, specialist (rheumatology/orthopaedic) advice was sought. Of 14 cases not seen directly by specialists 9 (64%) were referred at 24-48 h and 5 (36%) at 48-192 h. All 20 patients had joint aspiration. In 9/20 (45%) of cases, joint aspiration was performed in less than 6 h, 3/20 (15%) cases at 6-24h and 6/20 (30%) cases at 24-192 h and was not recorded in two patients. Of these, crystals were identified in two and one was culture positive. Blood cultures were received for only 6/20 (30%) of cases and only clearly documented to have been taken prior to antibiotic therapy and none were positive. Of 14/20 (70%) started on antibiotic treatment empirically, only 6 (42%) were preceded by joint aspiration. In the 6 patients not treated with antibiotics due to low index of suspicion of septic arthritis, synovial fluid and blood cultures were negative. Microbiology advice was sought in 10/20 (50%) of cases by the admitting teams but the timing of this advice is unclear. Conclusions: Despite the provision of 24 h rheumatology and orthopaedic cover at UCLH, we found a significant delay in acute medical firms seeking specialist advice on the management of patients with acute, hot swollen joints with subsequent deviation from BSR guidelines. Consequently, we plan to increase awareness of these guidelines amongst medical firms at UCLH. Disclosure statement: All authors have declared no conflicts of interes

Evaluation of a COVID ‐19 fundamental nursing care guideline versus usual care: The COVID‐NURSE cluster randomized controlled trial

Aim: To evaluate the impact of usual care plus a fundamental nursing care guideline compared to usual care only for patients in hospital with COVID‐19 on patient experience, care quality, functional ability, treatment outcomes, nurses' moral distress, patient health‐related quality of life and cost‐effectiveness. Design: Parallel two‐arm, cluster‐level randomized controlled trial. Methods: Between 18th January and 20th December 2021, we recruited (i) adults aged 18 years and over with COVID‐19, excluding those invasively ventilated, admitted for at least three days or nights in UK Hospital Trusts; (ii) nurses caring for them. We randomly assigned hospitals to use a fundamental nursing care guideline and usual care or usual care only. Our patient‐reported co‐primary outcomes were the Relational Aspects of Care Questionnaire and four scales from the Quality from the Patient Perspective Questionnaire. We undertook intention‐to‐treat analyses. Results: We randomized 15 clusters and recruited 581 patient and 418 nurse participants. Primary outcome data were available for 570–572 (98.1%–98.5%) patient participants in 14 clusters. We found no evidence of between‐group differences on any patient, nurse or economic outcomes. We found between‐group differences over time, in favour of the intervention, for three of our five co‐primary outcomes, and a significant interaction on one primary patient outcome for ethnicity (white British vs. other) and allocated group in favour of the intervention for the ‘other’ ethnicity subgroup. Conclusion: We did not detect an overall difference in patient experience for a fundamental nursing care guideline compared to usual care. We have indications the guideline may have aided sustaining good practice over time and had a more positive impact on non‐white British patients' experience of care. Implications for the Profession and/or Patient Care: We cannot recommend the wholescale implementation of our guideline into routine nursing practice. Further intervention development, feasibility, pilot and evaluation studies are required. Impact: Fundamental nursing care drives patient experience but is severely impacted in pandemics. Our guideline was not superior to usual care, albeit it may sustain good practice and have a positive impact on non‐white British patients' experience of care. Reporting Method: CONSORT and CONSERVE. Patient or Public Contribution: Patients with experience of hospitalization with COVID‐19 were involved in guideline development and writing, trial management and interpretation of findings

Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers

REST:a preoperative tailored sleep intervention for patients undergoing total knee replacement – feasibility study for a randomised controlled trial

Objectives To test the feasibility of a randomised controlled trial (RCT) of a novel preoperative tailored sleep intervention for patients undergoing total knee replacement.Design Feasibility two-arm two-centre RCT using 1:1 randomisation with an embedded qualitative study.Setting Two National Health Service (NHS) secondary care hospitals in England and Wales.Participants Preoperative adult patients identified from total knee replacement waiting lists with disturbed sleep, defined as a score of 0–28 on the Sleep Condition Indicator questionnaire.Intervention The REST intervention is a preoperative tailored sleep assessment and behavioural intervention package delivered by an Extended Scope Practitioner (ESP), with a follow-up phone call 4 weeks postintervention. All participants received usual care as provided by the participating NHS hospitals.Outcome measures The primary aim was to assess the feasibility of conducting a full trial. Patient-reported outcomes were assessed at baseline, 1-week presurgery, and 3 months postsurgery. Data collected to determine feasibility included the number of eligible patients, recruitment rates and intervention adherence. Qualitative work explored the acceptability of the study processes and intervention delivery through interviews with ESPs and patients.Results Screening packs were posted to 378 patients and 57 patients were randomised. Of those randomised, 20 had surgery within the study timelines. An appointment was attended by 25/28 (89%) of participants randomised to the intervention. Follow-up outcomes measures were completed by 40/57 (70%) of participants presurgery and 15/57 (26%) postsurgery. Where outcome measures were completed, data completion rates were 80% or higher for outcomes at all time points, apart from the painDETECT: 86% complete at baseline, 72% at presurgery and 67% postsurgery. Interviews indicated that most participants found the study processes and intervention acceptable.Conclusions This feasibility study has demonstrated that with some amendments to processes and design, an RCT to evaluate the clinical and cost-effectiveness of the REST intervention is feasible

TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

ABSTRACTTrYbe® is an Fc-free therapeutic antibody format, capable of engaging up to three targets simultaneously, with long in vivo half-life conferred by albumin binding. This format is shown by small-angle X-ray scattering to be conformationally flexible with favorable ‘reach’ properties. We demonstrate the format’s broad functionality by co-targeting of soluble and cell surface antigens. The benefit of monovalent target binding is illustrated by the lack of formation of large immune complexes when co-targeting multivalent antigens. TrYbes® are manufactured using standard mammalian cell culture and protein A affinity capture processes. TrYbes® have been formulated at high concentrations and have favorable drug-like properties, including stability, solubility, and low viscosity. The unique functionality and inherent developability of the TrYbe® makes it a promising multi-specific antibody fragment format for antibody therapy

Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame

An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.

Funder: Quadram Institute Bioscience BBSRC Strategic Programme: Microbes in the Food Chain; Grant(s): BB/R012504/1) and its constituent projects BBS/E/F/000PR10348 and BBS/E/F/000PR10349Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results

2015 White Paper on Recent Issues in Bioanalysis: Focus on New Technologies & Biomarkers (Part 1 – Small Molecule by LCMS)

The 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) was held in Miami, Florida on April 13-17, 2015 with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The workshop included three sequential core workshop days and six specialized training sessions that together spanned an entire week in order to allow exhaustive and thorough coverage of major issues in bioanalysis, biomarkers and immunogenicity. Like the previous workshops, the 9th WRIB was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in both small and large molecule bioanalysis using LCMS, hybrid LBA/LCMS, and LBA approaches. An in depth focus was on biomarkers, immunogenicity and emerging technologies. The actively contributing chairs in the 2015 edition of the WRIB were Dr. Eric Fluhler (Pfizer), Dr. Jan Welink (EMA / Dutch MEB), Dr. Brad Ackermann (Eli Lilly), Dr. Fabio Garofolo (Angelini Pharma), Dr. An Song (Genentech), Dr. Theingi Thway (Amgen), Dr. Lakshmi Amaravadi (Biogen Idec) and Dr. Heather Myler (Bristol-Myers Squibb). The numerous regulatory agency representatives who contributed to the 9th WRIB included Dr. Sam Haidar (US FDA), Dr. Susan Kirshner (US FDA), Dr. Brian Booth (US FDA), Dr. Michael Skelly (US FDA), Dr. Nilufer Tampal (US FDA), Dr. Jan Welink (EMA/Dutch MEB), Dr. Olivier Le Blaye (France ANSM), Ms. Emma Whale (UK MHRA), Mr. Stephen Vinter (UK MHRA), Dr. Bärbel Witte (German BfArM), Dr. Mark Bustard (Health Canada), Mr. Gustavo Mendes Lima Santos (Brazil ANVISA) and Dr. Noriko Katori (Japan MHLW-NIHS). Each of the three Sequential Core Workshop days was designed to cover a wide-range of bioanalytical topics suggested by members of the community, and included presentations from industry leaders and regulatory representatives, culminating in an open panel discussion between the presenters, regulators and attendees in order to determine the consensus items presented in this white paper