ISOTOPE : ISOform-guided prediction of epiTOPEs in cancer

Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE

The use of sequencing technologies for enhanced understanding of molecular determinants in renal diseases

Les maladies rénales ont un impact important sur l'économie de tout système de santé dans le monde. En outre, le nombre de patients augmente régulièrement au cours des dernières décennies avec une prévalence de plus de 500 000 nouveaux cas de maladie rénale en phase terminale (ESRD) dans le monde entier chaque année. L'ESRD est l'étape finale de la maladie rénale chronique (CKD) qui a comme principales causes le diabète et l'hypertension, ainsi que la glomérulonéphrite, urolithiasis, la polykystose rénale autosomique dominante (ADPKD) et la progression de la lésion rénale aiguë (LRA), entre autres. Cependant, dans de nombreux cas, les mécanismes de ces maladies affectant le rein et sa fonction sont mal connus ou difficiles à diagnostiquer. Dans le cadre de cette étude, nous avons utilisé des technologies plus récentes, des méthodologies et des approches d'analyse de données pour jeter un peu de lumière dans les pathomécanismes de la CKD et de l'AKI. En outre, l'amélioration potentielle de la valeur diagnostique des tests diagnostiques déjà existants (par exemple ADPKD). Au cours des dernières années, les progrès dans les technologies de séquençage de l'ADN ont révolutionné le domaine de la recherche clinique et du diagnostic. Le séquençage à haut débit tel que le séquençage de prochaine génération (NG) est utilisé en raison de sa haute qualité et de précision lors que l'analyse des échantillons d'ADN. D'autres technologies de séquençage ont également montré leur valeur, comme le séquençage à longue lecture qui est utilisé en raison de ses longues lectures de séquençage et de la précision de résolution de séquençages de faible complexité, telles que les régions répétitives ou des régions de GC-pourcentage élevé. Dans le cadre de cette thèse, nous avons utilisé plusieurs méthodes de pointe de séquençage appliquées à la recherche clinique sur la maladie rénale afin de: 1. Améliorer la valeur diagnostique des tests diagnostiques déjà existants pour l'ADPKD. ADPKD est une maladie héréditaire qui représente de 5% à 10% de l'ESRD. Cependant, le criblage du principal gène ADPKD PKD1 est difficile en raison de sa structure multi-exon, de son hétérogénéité allélique et de son homologie élevée avec six pseudogènes PKD1, ainsi que d'une teneur en GC extrêmement élevée. En utilisant le séquençage direct à longue lecture, nous avons montré que le diagnostic ADPKD sans interférence des séquences homologues PKD1 est possible. 2. Caractériser le profil d'expression de l'IRA et des mécanismes sous-jacents en utilisant le séquençage de l'ARN. Les patients qui subissent une chirurgie majeure peuvent développer une IRA qui a été associée à un risque de mortalité plus élevé et une fonctionnalité rénale réduite, et un risque élevé de progression de la CKD. Certaines preuves indiquent que le système tubulaire est au milieu de cette pathophysiologie et de la récupération ultérieure. Cependant, les facteurs impliqués dans cette reprise sont encore mal compris. Dans ce contexte, nous avons caractérisé les profils d'expression de l'ARN messager rénal d'AKI dans des constructions de type sauvage et knock-out Gdf15. Gdf15 a été identifié comme étant associé à des lésions tubulaires inférieures suggérant un rôle protecteur du rein. Nous avons identifié 89 facteurs de transcription qui sont potentiellement moteurs des mécanismes de réponse dans l'IRA, ainsi que d'autres facteurs de transcription 13 éventuellement liés avec les mécanismes de protection de Gdf15. 3. Évaluer les limites pratiques du séquençage de l'ARN pour caractériser les maladies glomérulaires pour la CKD. L'analyse des biopsies rénales est très informative pour déterminer le stade de la maladie glomérulaire du patient et les taux de progression. Cependant, les biopsies congelées fraîches sont limitées ou inexistantes par rapport aux biopsies rénales fixées au formol et à la paraffine (FFPE) plus abondantes. Les tissus FFPE peuvent être facilement stockés pendant de longues périodes, ce qui permet de disposer d'importantes archives d'échantillons avec de nombreuses années de collecte de données cliniques et de suivi. Nous avons montré que la caractérisation des profils d'expression de la maladie glomérulaire par séquençage d'ARN à partir d'échantillons de FFPE est possible. Cependant, nos données suggèrent que le nombre requis de glomérules dans les coupes transversales peut être supérieur au nombre de glomérules présents dans une biopsie rénale habituelle. Enfin, nous avons développé l'impact futur des résultats obtenus dans le cadre de la recherche clinique et leur valeur pour la compréhension ou le diagnostic des maladies rénales.Renal diseases have a high impact on the economy of any health care system worldwide. In addition, patient numbers are steadily increasing over the past decades with a prevalence of over 500.000 new end stage renal disease (ESRD) worldwide cases every year. ESRD is the final stage of chronic kidney disease (CKD) that has as the leading causes diabetes and hypertension, as well as glomerulonephritis, urolithiasis, autosomal dominant polycystic kidney disease (ADPKD), and progression of acute kidney injury (AKI), among others. However, in many cases, the mechanisms of these diseases affecting kidney and its function are poorly understood, or difficult to diagnose. Within this study, we used newer technologies, methodologies, and data analysis approaches to throw some light into the pathomechanisms of CKD and AKI. Moreover, potentially improving the diagnostic value for already existing diagnostic assays (e.g. ADPKD). In the past years, advances in DNA sequencing technologies have revolutionized the field of clinical research and diagnostics. High throughput sequencing such as next-generation sequencing (NGs) is being used because of its high quality and accuracy when analysing DNA samples. Other sequencing technologies have also shown their value such as long-read sequencing which is used because of its longer sequencing reads and accuracy resolving low-complexity sequences, such as repetitive regions or high GC-percent regions. Within the scope of this thesis we used several cutting-edge sequencing approaches applied to renal disease's clinical research to: 1. Improve the diagnostic value of already existing diagnostic assays for ADPKD. ADPKD is an inherited disease that accounts for 5% to 10% of ESRD. However, the screening of the main ADPKD gene PKD1 is challenging because of its multi-exon structure, allelic heterogeneity, and high homology with six PKD1 pseudogenes, as well as extremely high GC content. Using direct long-read sequencing we showed that ADPKD diagnostics without interference of PKD1 homologous sequences is possible. 2. Characterize the expression profile of AKI and the underlying mechanisms using RNA sequencing. Patients undergoing major surgery may develop AKI which has been associated with higher mortality risk and reduced renal functionality, and high risk of progression of CKD. Some evidences pointed out to the tubular system being at the middle of this pathophysiology and further recovery. However, the factors involved in this recovery are still poorly understood. In this context, we characterized renal messenger RNA expression profiles of AKI in wild type and Gdf15 knock out constructs. Gdf15 was identified to be associated with lower tubular damage suggesting a protective role of the kidney. We identified 89 transcription factors that are potentially driving the response mechanisms in AKI, as well as other 13 transcription factors possibly linked with the protective mechanisms of Gdf15. 3. Evaluate the practical boundaries of RNA sequencing to characterize glomerular diseases for CKD. The analysis of renal biopsies is very informative to determine patient's glomerular disease stage and progression rates. However, fresh frozen biopsies are limited or non-existent compared to the more abundant formalin-fixed, paraffin-embedded (FFPE) renal biopsies. FFPE tissue can be easily stored for long periods of time, allowing large sample archives with many years of clinical data collection and follow-up. We showed that characterizing glomerular disease expression profiles by RNA sequencing from FFPE samples is possible. However, our data suggests that the required number of glomeruli in cross sections may be higher than the number glomeruli present in a usual renal biopsy. Finally, we elaborated about the future impact of the results obtained in the context of clinical research, and their value for the understanding or diagnostics of renal diseases

The role of epigenetics in renal ageing

An ability to separate natural ageing processes from processes specific to morbidities is required to understand the heterogeneity of age-related organ dysfunction. Mechanistic insight into how epigenetic factors regulate ageing throughout the life course, linked to a decline in renal function with ageing, is already proving to be of value in the analyses of clinical and epidemiological cohorts. Noncoding RNAs provide epigenetic regulatory circuits within the kidney, which reciprocally interact with DNA methylation processes, histone modification and chromatin. These interactions have been demonstrated to reflect the biological age and function of renal allografts. Epigenetic factors control gene expression and activity in response to environmental perturbations. They also have roles in highly conserved signalling pathways that modulate ageing, including the mTOR and insulin/insulin-like growth factor signalling pathways, and regulation of sirtuin activity. Nutrition, the gut microbiota, inflammation and environmental factors, including psychosocial and lifestyle stresses, provide potential mechanistic links between the epigenetic landscape of ageing and renal dysfunction. Approaches to modify the renal epigenome via nutritional intervention, targeting the methylome or targeting chromatin seem eminently feasible, although caution is merited owing to the potential for intergenerational and transgenerational effects

Deep analysis of neuroblastoma core regulatory circuitries using online databases and integrated bioinformatics shows their pan-cancer roles as prognostic predictors.

AIM: Neuroblastoma is a heterogeneous childhood cancer derived from the neural crest. The dual cell identities of neuroblastoma include Mesenchymal (MES) and Adrenergic (ADRN). These identities are conferred by a small set of tightly-regulated transcription factors (TFs) binding super enhancers, collectively forming core regulatory circuitries (CRCs). The purpose of this study was to gain a deep understanding of the role of MES and ADRN TFs in neuroblastoma and other cancers as potential indicators of disease prognosis, progression, and relapse. METHODS: To that end, we first investigated the expression and mutational profile of MES and ADRN TFs in neuroblastoma. Moreover, we established their correlation with neuroblastoma risk groups and overall survival while establishing their extended networks with long non-coding RNAs (lncRNAs). Furthermore, we analysed the pan-cancer expression and mutational profile of these TFs and their correlation with patient survival and finally their network connectivity, using a panel of bioinformatic tools including GEPIA2, human pathology atlas, TIMER2, Omicsnet, and Cytoscape. RESULTS: We show the association of multiple MES and ADRN TFs with neuroblastoma risk groups and overall survival and find significantly higher expression of various MES and ADRN TFs compared to normal tissues and their association with overall survival and disease-free survival in multiple cancers. Moreover, we report the strong correlation of the expression of these TFs with the infiltration of stromal and immune cells in the tumour microenvironment and with stemness and metastasis-related genes. Furthermore, we reveal extended pan-cancer networks comprising these TFs that influence the tumour microenvironment and metastasis and may be useful indicators of cancer prognosis and patient survival. CONCLUSION: Our meta-analysis shows the significance of MES and ADRN TFs as indicators of patient prognosis and the putative utility of these TFs as potential novel biomarkers