An Integrated Immunopeptidomics and Proteogenomics Framework to Discover Non-Canonical Targets for Cancer Immunotherapy

Un élément essentiel de l’immunothérapie appliquée au cancer est l’identification de peptides liant les antigènes des leucocytes humains (HLA) et capables d’induire une puissante réponse T anti-tumorale. La spectrométrie de masse (MS) constitue actuellement la seule méthode non-biaisée permettant une analyse détaillée du panel d’antigènes susceptibles d’être présentés aux lymphocytes T in vivo. L’utilisation de cette méthode en clinique requiert toutefois des améliorations significatives de la méthodologie utilisée lors de l’identification des peptides HLA. Un consortium multidisciplinaire de chercheurs a récemment mis en lumière les problèmes actuellement liés à l’utilisation de la MS en immunopeptidomique, soulignant le besoin de développer de nouvelles méthodes et mettant en évidence le défi que représente la standardisation de l’immuno-purification des molécules HLA. La première partie de cette thèse vise à optimiser les méthodes expérimentales permettant l’extraction des peptides apprêtés aux HLA. L’optimisation de la méthodologie de base a permis des améliorations notables en terme de débit, de reproductibilité, de sensibilité et a permis une purification séquentielle des molécules de HLA de classe I de classe II ainsi que de leurs peptides, à partir de lignées cellulaires ou de tissus. En comparaison avec les méthodes existantes, ce protocole comprend moins d’étapes et permet de limiter la manipulation des échantillons ainsi que le temps de purification. Cette méthode, pour les peptides HLA extraits, a permis d’obtenir des taux de reproductibilité et de sensibilité sans précédents (corrélations de Pearson jusqu'à 0,98 et 0,97 pour les HLA de classe I et de classe II, respectivement). De plus, la faisabilité d’études comparatives robustes a été démontrée à partir d’une lignée cellulaire de cancer de l’ovaire, traitée à l'interféron gamma. En effet, cette nouvelle méthode a mis en évidence des changements quantitatifs et qualitatifs du catalogue de peptides présentés aux HLA. Les résultats obtenus ont mis en avant une augmentation de la présentation de longs ligands chymotryptiques de classe I. Ce phénomène est probablement lié à la modulation de la machinerie de traitement et de présentation des antigènes. Dans cette première partie de thèse, nous avons développé une méthodologie robuste et rationalisée, facilitant la purification des HLA et pouvant être appliquée en recherche fondamentale et translationnelle. Bien que les néoantigènes représentent une cible attractive, des études récentes ont mis en évidence l’existence des antigènes non canoniques. Ces antigènes tumoraux, bien que non mutés, sont aussi spécifiques aux cellules cancéreuses et semblent jouer un rôle important dans l’immunité anti-tumorale. La seconde partie de cette thèse a pour objectif le développement d’une méthodologie d’analyse permettant l’identification ainsi que la validation de ces antigènes particuliers. Les antigènes non canoniques sont d'origine présumée non codante et ne sont, par conséquent, que rarement inclus dans les bases de données des séquences de protéines de référence. De ce fait, ils ne sont généralement pas pris en compte lors des recherches de MS utilisant de telles bases de données. Afin de palier ce problème et de permettre leur identification par MS, le séquençage de l'exome entier, le séquençage de l'ARN sur une population de cellules et sur des cellules uniques, ainsi que le profilage des ribosomes ont été intégrés aux données d’immunopeptidomique. Ainsi, NewAnce, un programme informatique permettant de combiner les données de deux outils de recherche MS en tandem, a été développé afin de calculer le taux d’antigènes non canoniques identifiés comme faux positifs. L’utilisation de NewAnce sur des lignées cellulaires provenant de patients atteints de mélanomes ainsi que sur des biopsies de cancer du poumon a permis l’identification précise de centaines de peptides HLA non classiques, spécifiques aux cellules tumorales et communs à plusieurs patients. Le niveau de confirmation des peptides non canoniques a ensuite été testé à l’aide d’une approche de MS ciblée. Les peptides résultant de ces analyses ont été minutieusement validés pour un des échantillons de mélanome disponibles. De plus, le profilage des ribosomes a révélé que les nouveaux cadres de lecture ouverts, desquels résultent certains de ces peptides non classiques, sont activement traduits. L’évaluation de l’immunogenicité de ces peptides a été évaluée avec des cellules immunitaires autologues et a révélé un épitope immunogène non canonique, provenant d'un cadre de lecture ouvert alternatif du gène ABCB5, un marqueur des cellules souches du mélanome. De manière globale, les résultats obtenus au cours de cette thèse soulignent la possibilité d’inclure ce type d’analyse de proteogénomique dans un protocole d’identification de néoantigènes existant. Cela permettrait d’inclure et prioriser des antigènes tumoraux non classiques et de proposer aux patients en impasse thérapeutique des immunothérapies anti-tumorales personnalisées. -- A central factor to the development of cancer immunotherapy is the identification of clinically relevant human leukocyte antigen (HLA)-bound peptides that elicit potent anti-tumor T cell responses. Mass spectrometry (MS) is the only unbiased technique that captures the in vivo presented HLA repertoire. However, significant improvements in MS-based HLA peptide discovery methodologies are necessary to enable the smooth transition to the clinic. Recently, a consortium of multidisciplinary researchers presented current issues in clinical MS-based immunopeptidomics, highlighting method development and standardization challenges in HLA immunoaffinity purification. The first part of this thesis addresses improvements to the experimental method for HLA peptide extraction. The approach was optimized with several new developments, facilitating high-throughput, reproducible, scalable, and sensitive sequential immunoaffinity purification of HLA class I and class II peptides from cell lines and tissue samples. The method showed increased speed, and reduced sample handling when compared to previous methods. Unprecedented depth and high reproducibility were achieved for the obtained HLA peptides (Pearson correlations up to 0.98 and 0.97 for HLA class I and HLA class II, respectively). Additionally, the feasibility of performing robust comparative studies was demonstrated on an ovarian cancer cell line treated with interferon gamma. Both quantitative and qualitative changes were detected in the cancer HLA repertoire upon treatment. Specifically, a yet unreported and interesting phenomenon was the upregulated presentation of longer and chymotryptic-like HLA class I ligands, likely related to the modulation of the antigen processing and presentation machinery. Taken together, a robust and streamlined framework was built that facilitates peptide purification and its application in basic and translational research. Furthermore, recent studies have shed light that, along with the highly attractive mutated neoantigens, other non-mutated, yet tumor-specific, non-canonical antigens may also play an important role in anti-tumor immunity. Non-canonical antigens are of presumed non-coding origin and not commonly included in protein reference databases, and are therefore typically disregarded in database-dependent MS searches. The second part of this thesis develops an analytical workflow enabling the confident identification and validation of non- canonical tumor antigens. For this purpose, whole exome sequencing, bulk and single-cell RNA sequencing and ribosome profiling were integrated with MS-based immunopeptidomics for personalized non-canonical HLA peptide discovery. A computational module called NewAnce was designed, which combines the results of two tandem MS search tools and implements group-specific false discovery rate calculations to control the error specifically for the non-canonical peptide group. When applied to patient-derived melanoma cell lines and paired lung cancer and normal tissues, NewAnce resulted in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides. Next, the level of non-canonical peptide confirmation was tested in a targeted MS-based approach, and selected non-canonical peptides were extensively validated for one melanoma sample. Furthermore, the novel open reading frames that generate a selection of these non- canonical peptides were found to be actively translated by ribosome profiling. Importantly, these peptides were assessed with autologous immune cells and a non-canonical immunogenic epitope was discovered from an alternative open reading frame of melanoma stem cell marker gene ABCB5. This thesis concludes by highlighting the possibility of incorporating the proteogenomics pipeline into existing neoantigen discovery engines in order to prioritize tumor-specific non-canonical peptides for cancer immunotherapy. -- Maladie très hétérogène et multifactorielle, le cancer représente à ce jour la seconde cause de décès dans le monde. Bien que le système immunitaire soit capable de reconnaître puis d’éliminer les cellules cancéreuses, ces dernières peuvent à leur tour s’adapter et accumuler des mutations leur permettant d’échapper à cette reconnaissance. L’immunothérapie anti-tumorale démontre le rôle clé de l’immunité dans l’éradication des tumeurs. Cependant, ces thérapies prometteuses ne sont efficaces que chez une petite proportion des patients traités. Une étape majeure dans l’établissement d’une réponse immunitaire anti-tumorale est la reconnaissance d’antigènes associés aux tumeurs. Des études récentes ont montré que les antigènes tumoraux issus de régions non-codantes du génome (antigènes non-canoniques) peuvent jouer un rôle clé dans l’induction de réponses immunitaires. Ainsi, l’identification de ces antigènes tumoraux particuliers permettrait de guider le développement d’immunothérapies anti-cancéreuses personnalisées telles que la vaccination ou encore le transfert adoptif de lymphocytes T reconnaissant ces cibles. La spectrométrie de masse (MS) est une technique non biaisée permettant l’identification et l’analyse du répertoire des antigènes présentés in vivo. Cependant, cette technique nécessite d’être optimisée et standardisée afin d’être utilisée en clinique. Ainsi, la première partie de ces travaux de thèse a été dédiée à l’optimisation expérimentale de cette méthode à partir d’échantillons de tissus et de lignées cellulaires. En comparaison avec les protocoles standards, cette technique permet une couverture plus complète, rapide et reproductible du répertoire de peptides apprêtés aux HLA. La seconde partie de cette thèse a été consacrée au développement d’une méthode permettant l’identification d’antigènes tumoraux non-canoniques via le séquençage d’ARN cellulaire, ribosomique et l’utilisation de notre méthode d’immunopeptidomique optimisée. Afin de contrôler l’identification de faux positifs, nous avons élaboré un nouveau module computationnel. Ce module a permis l’identification de plusieurs centaines de peptides-HLA non-canoniques, partagés et spécifiques au mélanome et au cancer du poumon. Le séquençage des ARN ribosomiques a mis en évidence la traduction de nouveaux cadre ouverts de lecture desquels sont traduits de nouveaux peptides non-canoniques. Cette technique nous a permis de mettre en évidence un épitope immunogène issu du gène ABCB5, un marqueur de cellules souches cancéreuses préalablement identifié dans le mélanome. De manière globale, ces travaux de thèse, alliant immunopeptidomique et protéogénomique, ont permis la mise au point d’une méthode expérimentale permettant une meilleure identification d’antigènes tumoraux. Nous espérons que ces résultats amélioreront l’identification et la priorisation de cibles pertinentes pour l’immunothérapie anti-cancéreuse en clinique

A novel approach to increasing community capacity for weight management a volunteer-delivered programme (ActWELL) initiated within breast screening clinics:a randomised controlled trial

Background: It is estimated that around 30% of breast cancers in post-menopausal women are related to lifestyle. The breast cancer-pooling project demonstrated that sustained weight loss of 2 to 4.5 kg is associated with an 18% lower risk of breast cancer, highlighting the importance of small changes in body weight. Our study aimed to assess the effectiveness a volunteer-delivered, community based, weight management programme (ActWELL) for women with a BMI > 25 kg/m2 attending NHS Scotland Breast Screening clinics. Methods: A multicentre, 1:1 parallel group, randomised controlled trial was undertaken in 560 women aged 50 to 70 years with BMI > 25 kg/m2. On completion of baseline measures, all participants received a breast cancer prevention leaflet. Intervention group participants received the ActWELL intervention which focussed on personalised diet advice and pedometer walking plans. The programme was delivered in leisure centres by (the charity) Breast Cancer Now volunteer coaches. Primary outcomes were changes between groups at 12 months in body weight (kg) and physical activity (accelerometer measured step count). Results: Two hundred seventy-nine women were allocated to the intervention group and 281 to the comparison group. Twelve-month data were available from 240 (81%) intervention and 227 (85%) comparison group participants. Coaches delivered 523 coaching sessions and 1915 support calls to 279 intervention participants. Mean weight change was − 2.5 kg (95% CI − 3.1 to − 1.9) in the intervention group and − 1.2 kg (− 1.8 to 0.6) in the comparison group. The adjusted mean difference was − 1.3 kg (95% CI − 2.2 to − 0.4, P = 0.003). The odds ratio for losing 5% weight was 2.20 (95% CI 1.4 to 3.4, p = 0.0005) in favour of the intervention. The adjusted mean difference in step counts between groups was 483 steps/day (95% CI − 635 to 1602) (NS). Conclusions: A community weight management intervention initiated at breast screening clinics and delivered by volunteer coaches doubled the likelihood of clinically significant weight loss at 12 months (compared with usual care) offering significant potential to decrease breast cancer risk

Correction to:A novel approach to increasing community capacity for weight management a volunteer-delivered programme (ActWELL) initiated within breast screening clinics: a randomised controlled trial

Acknowledgements Our thanks to Elizabeth Banks who advised and assisted with many aspects of the study and also to the many women who commented on the development and design of this study including those on our Public Advisory Team (Pamela Deponio, Maggie Taylor and Mary Wotherspoon). Funding This work was supported by The Scottish Government, grant number BC/Screening/17/01. The funders provided independent referee reports which guided the final study design. The funders have read this manuscript. In-kind support was given by Breast Cancer Now for facilitating this study.Peer reviewedPublisher PD

CowN Sustains Nitrogenase Turnover in the Presence of the Inhibitor Carbon Monoxide

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase

Painting the Nation:Examining the Intersection Between Politics and the Visual Arts Market in Emerging Economies

Politics and art have throughout history, intersected in diverse and complex ways. Ideologies and political systems have used the arts to create a certain image and, depending on the form of government this has varied from clear-cut state propaganda, to patronage, to more indirect arms-length funding procedures. Therefore, artists working within the macro-level socio-political context cannot help but be influenced, inspired and sometimes restricted by these policies and political influences. This article examines the contemporary art markets of two emerging, Socialist economies to investigate the relationship between state pol-itics and the contemporary visual arts market. We argue that the respective governments and art worlds are trying to construct a brand narrative for their nations, but that these discourses are often at cross-purposes. In doing so, we illustrate that it is impos-sible to separate a consideration of the artwork from the macro-level context in which it is produced, distributed, and consumed

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Functional materials discovery using energy–structure–function maps

Molecular crystals cannot be designed in the same manner as macroscopic objects, because they do not assemble according to simple, intuitive rules. Their structures result from the balance of many weak interactions, rather than from the strong and predictable bonding patterns found in metal–organic frameworks and covalent organic frameworks. Hence, design strategies that assume a topology or other structural blueprint will often fail. Here we combine computational crystal structure prediction and property prediction to build energy–structure–function maps that describe the possible structures and properties that are available to a candidate molecule. Using these maps, we identify a highly porous solid, which has the lowest density reported for a molecular crystal so far. Both the structure of the crystal and its physical properties, such as methane storage capacity and guest-molecule selectivity, are predicted using the molecular structure as the only input. More generally, energy–structure–function maps could be used to guide the experimental discovery of materials with any target function that can be calculated from predicted crystal structures, such as electronic structure or mechanical properties

Breast cancer management pathways during the COVID-19 pandemic: outcomes from the UK ‘Alert Level 4’ phase of the B-MaP-C study

Abstract: Background: The B-MaP-C study aimed to determine alterations to breast cancer (BC) management during the peak transmission period of the UK COVID-19 pandemic and the potential impact of these treatment decisions. Methods: This was a national cohort study of patients with early BC undergoing multidisciplinary team (MDT)-guided treatment recommendations during the pandemic, designated ‘standard’ or ‘COVID-altered’, in the preoperative, operative and post-operative setting. Findings: Of 3776 patients (from 64 UK units) in the study, 2246 (59%) had ‘COVID-altered’ management. ‘Bridging’ endocrine therapy was used (n = 951) where theatre capacity was reduced. There was increasing access to COVID-19 low-risk theatres during the study period (59%). In line with national guidance, immediate breast reconstruction was avoided (n = 299). Where adjuvant chemotherapy was omitted (n = 81), the median benefit was only 3% (IQR 2–9%) using ‘NHS Predict’. There was the rapid adoption of new evidence-based hypofractionated radiotherapy (n = 781, from 46 units). Only 14 patients (1%) tested positive for SARS-CoV-2 during their treatment journey. Conclusions: The majority of ‘COVID-altered’ management decisions were largely in line with pre-COVID evidence-based guidelines, implying that breast cancer survival outcomes are unlikely to be negatively impacted by the pandemic. However, in this study, the potential impact of delays to BC presentation or diagnosis remains unknown