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IntroductionMammographic screening identifies many women with small breast cancers with favourable biological features, which have an excellent prognosis. Some of these may never have become clinically apparent without screening and are commonly described as ‘overdiagnosed’ cancers. Despite this, all patients with screen-detected cancers are currently treated with surgical excision and sentinel lymph node biopsy, although this may represent overtreatment. There is, therefore, a need for less invasive approaches to reduce treatment burden for patients while maintaining current excellent oncological outcomes. Vacuum-assisted excision (VAE) may represent such an alternative treatment approach, and the SMALL (Open Surgery versus Minimally invasive-vacuum Assisted excision for smaLL screen-detected breast cancer) trial aims to investigate the use of VAE for the safe de-escalation of surgical treatment for such excellent prognosis invasive breast cancers.MethodsSMALL is a prospective, multicentre, randomised phase III trial of VAE versus surgery in patients with small, biologically favourable screen-detected invasive breast cancer. SMALL has an innovative hybrid design with coprimary endpoints. These include a randomised non-inferiority comparison of surgical re-excision rates following initial treatment, and a single-arm analysis of local recurrence at 5 years following VAE. Secondary outcomes include complication rates, overall survival, quality of life and a health economic analysis. The trial includes a QuinteT Recruitment Intervention to support recruitment.Ethics and disseminationEthical approval was obtained from the Office for Research Ethics (Northern Ireland) for all UK sites. Results will be submitted for publication in a peer-reviewed journal, presented, shared with patient partners and with relevant professional organisations to inform future guideline development for the management of screen-detected breast cancer.Trial registration numberISRCTN12240119.

Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. Long term survival of high-risk NB (HR-NB) currently remains less than 50% at 5 years despite intensive high-dose multimodal treatment. DNA damage response (DDR) defects are frequently observed in HR-NB including allelic deletion and loss of function mutations in key DDR genes, leading to cell cycle checkpoint dysfunction, and oncogene induced replication stress (RS). Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. The aim of this thesis is to identify determinants of sensitivity to ATR and PARP inhibition, alone and in combination, in preclinical models of NB. The baseline level of RS and activity of DDR kinases ATM and ATR was characterised in a panel NB cell lines as well the mutation and/or deletion status of genes previously reported to be synthetically lethal with PARP or ATR inhibition. MYCN-induced RS resulted in vulnerability to ATR and PARP inhibition, by VE821 and olaparib, respectively. ATM deficiency was also associated with sensitivity to ATR inhibition. VE-821 sensitised NB cell lines to olaparib cytotoxicity, which was synergistic in all cell lines. There was a significant positive correlation between fold sensitisation by VE-821 and MYCN protein expression. To obtain greater mechanistic understanding of the synergy between ATR and PARP inhibition, the effect of dual inhibition on markers of RS, cell cycle checkpoints and DNA repair were measured. VE-821 abrogated olaparib-induced S and G2 checkpoint arrest, increased olaparib-induced markers of RS and reduced homologous recombination repair foci. Taken together the work presented in this thesis provides strong rationale for the introduction of ATR and PARP inhibitors for the treatment of HR-NB exhibiting high levels of RS.

Chapter 8b discusses the considerable controversy as to the optimal management of localized, muscle invasive bladder cancer. Surgical removal of the bladder is considered the ‘gold standard’ in many countries but there is no randomized trial to underpin this statement, and many patients are unfit for surgery. Reported 5year survival rates with radiotherapy are remarkably similar to surgical series. Thus even if surgery is genuinely better than radiotherapy for patients fit for both approaches, there are many patients for whom radical surgery is simply not suitable and hence bladderpreserving techniques are appropriate.

Sociologists have long studied the social aspects of music. In the body of existing literature, sociologists have often used the terms musical genre and musical styles interchangeably, yet a distinction can be made between the two. Musical styles are shown to be subordinate to musical genres. The process of categorization of contemporary American popular music of the twentieth century is considered and an analysis of emergence, duration and demise of both styles and genre is made. Six methods used to label musical styles were discovered and are described. During this time frame, a shift in labeling is shown to occur due to commodification of music in the latter half of the twentieth century.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein 1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals. Chronic infection with SARS-CoV-2 leads to the emergence of viral variants that show reduced susceptibility to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies 1 – 3 . Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed 4 ) identified multiple new candidate PID-associated genes, including IVNS1ABP . We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of—and interplay between—novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans. Whole-genome sequencing analysis of individuals with primary immunodeficiency identifies new candidate disease-associated genes and shows how the interplay between genetic variants can explain the variable penetrance and complexity of the disease.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021. A study of the evolution of the SARS-CoV-2 virus in England between September 2020 and June 2021 finds that interventions capable of containing previous variants were insufficient to stop the more transmissible Alpha and Delta variants.

Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro . Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections. Fluoroquinolone antibiotics are widely used to treat serious bacterial infections, but resistance is an increasing problem. Here the authors describe the synthesis and characterization of novel deoxynybomycin derivatives that exhibit activity against fluoroquinolone-resistant infections in an in vivo model.

BackgroundThe molecular genetic basis of pulmonary arterial hypertension (PAH) is heterogeneous, with at least 26 genes displaying putative evidence for disease causality. Heterozygous variants in the ATP13A3 gene were recently identified as a new cause of adult-onset PAH. However, the contribution of ATP13A3 risk alleles to child-onset PAH remains largely unexplored.Methods and resultsWe report three families with a novel, autosomal recessive form of childhood-onset PAH due to biallelic ATP13A3 variants. Disease onset ranged from birth to 2.5 years and was characterised by high mortality. Using genome sequencing of parent–offspring trios, we identified a homozygous missense variant in one case, which was subsequently confirmed to cosegregate with disease in an affected sibling. Independently, compound heterozygous variants in ATP13A3 were identified in two affected siblings and in an unrelated third family. The variants included three loss of function variants (two frameshift, one nonsense) and two highly conserved missense substitutions located in the catalytic phosphorylation domain. The children were largely refractory to treatment and four died in early childhood. All parents were heterozygous for the variants and asymptomatic.ConclusionOur findings support biallelic predicted deleterious ATP13A3 variants in autosomal recessive, childhood-onset PAH, indicating likely semidominant dose-dependent inheritance for this gene.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype.